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Scientific References on Diindolylmethane

This section is updated regularly. Paper abstracts are presented in chronological order from most recent.

Invest New Drugs. 2013 May 25. [Epub ahead of print]

Ring-substituted analogs of 3,3'-diindolylmethane (DIM) induce apoptosis and
necrosis in androgen-dependent and -independent prostate cancer cells.

Goldberg AA, Titorenko VI, Beach A, Abdelbaqi K, Safe S, Sanderson JT.

INRS - Institut Armand-Frappier, Université du Québec, 531 boulevard des
Prairies, Laval, QC, Canada.

We recently reported that novel ring-substituted analogs of 3,3'-diindolylmethane
(ring-DIMs) have anti-androgenic and growth inhibitory effects in
androgen-dependent prostate cancer cells. The objectives of this study were to
confirm the ability of 4,4'- and 7,7'-dibromo- and dichloro-substituted ring-DIMs
to inhibit androgen-stimulated proliferation of androgen-dependent LNCaP human
prostate cancer cells using a non-invasive, real-time monitoring technique. In
addition, their ability to induce apoptotic and necrotic cell death in
androgen-dependent as well as -independent (PC-3) prostate cancer cells was
studied. Prostate cancer cells were treated with increasing concentrations of DIM
and ring-DIMs (0.3-30 μM) and effects on cell proliferation were measured in
real-time using an xCELLigence cellular analysis system. Chromatin condensation
and loss of membrane integrity were determined by Hoechst and propidium iodide
staining, respectively. Apoptotic protein markers were measured by immunoblotting
and activation of caspases determined using selective fluorogenic substrates.
Intra- and extracellular concentrations of DIM and ring-DIMs were assessed by
electrospray ionization tandem mass spectrometry. Ring-DIMs inhibited
androgen-stimulated LNCaP cell proliferation and induced apoptosis and necrosis
in LNCaP and PC-3 cells with 2-4 fold greater potencies than DIM. DIM and the
ring-DIMs increased caspases -3, -8 and -9 activity, elevated expression of Fas,
FasL, DR4 and DR5 protein, and induced PARP cleavage in both cell lines. The
cytotoxicity of the most potent ring-DIM, 4,4'-dibromoDIM, but not the other
compounds was decreased by an inhibitor of caspase -3. The 4,4'-dibromoDIM was
primarily found in the extracellular medium, whereas all other compounds were
present to a much larger extent in the cell. In conclusion, ring-DIMs inhibited
prostate cancer cell growth and induced cell death in LNCaP and PC-3 cells with
greater potencies than DIM; they also structure-dependently activated different
cell death pathways suggesting that these compounds have clinical potential as
chemopreventive and chemotherapeutic agents in prostate cancer, regardless of
hormone-dependency.

PMID: 23709189  [PubMed - as supplied by publisher]

AAPS J. 2013 May 9. [Epub ahead of print]

Epigenetic Modifications of Nrf2 by 3,3'-diindolylmethane In Vitro in TRAMP C1
Cell Line and In Vivo TRAMP Prostate Tumors.

Wu TY, Khor TO, Su ZY, Saw CL, Shu L, Cheung KL, Huang Y, Yu S, Kong AN.

Center for Cancer Prevention Research, Department of Pharmaceutics, Ernest Mario
School of Pharmacy, Rutgers, The State University of New Jersey, Room 228, 160
Frelinghuysen Road, Piscataway, New Jersey, 08854, USA.

3,3'-diindolylmethane (DIM) is currently being investigated in many clinical
trials including prostate, breast, and cervical cancers and has been shown to
possess anticancer effects in several in vivo and in vitro models. Previously,
DIM has been reported to possess cancer chemopreventive effects in prostate
carcinogenesis in TRAMP mice; however, the in vivo mechanism is unclear. The
present study aims to investigate the in vitro and in vivo epigenetics modulation
of DIM in TRAMP-C1 cells and in TRAMP mouse model. In vitro study utilizing
TRAMP-C1 cells showed that DIM suppressed DNMT expression and reversed CpG
methylation status of Nrf2 resulting in enhanced expression of Nrf2 and
Nrf2-target gene NQO1. In vivo study, TRAMP mice fed with DIM-supplemented diet
showed much lower incidence of tumorigenesis and metastasis than the untreated
control group similar to what was reported previously. DIM increased apoptosis,
decreased cell proliferation and enhanced Nrf2 and Nrf2-target gene NQO1
expression in prostate tissues. Importantly, immunohistochemical analysis showed
that DIM reduced the global CpG 5-methylcytosine methylation. Focusing on one of
the early cancer chemopreventive target gene Nrf2, bisulfite genomic sequencing
showed that DIM decreased the methylation status of the first five CpGs of the
Nrf2 promoter region, corroborating with the results of in vitro TRAMP-C1 cells.
In summary, our current study shows that DIM is a potent cancer chemopreventive
agent for prostate cancer and epigenetic modifications of the CpG including Nrf2
could be a potential mechanism by which DIM exerts its chemopreventive effects.

PMID: 23658110

Hum Exp Toxicol. 2013 Apr;32(4):344-53. doi: 10.1177/0960327112462727.

Effect of diindolylmethane on Ca(2+) homeostasis and viability in MDCK renal
tubular cells.

Fang YC, Chou CT, Chi CC, Lin KL, Li YD, Cheng HH, Lu YC, Cheng JS, Kuo CC, Jan
CR.

Department of Laboratory Medicine, Zuoying Armed Forces General Hospital,
Kaohsiung, Taiwan.

The effect of the natural product diindolylmethane (DIM) on cytosolic Ca(2+)
concentrations ([Ca(2+)]i) and viability in MDCK renal tubular cells was
explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure
[Ca(2+)]i. DIM at concentrations 1-50 μM induced a [Ca(2+)]i rise in a
concentration-dependent manner. The response was reduced partly by removing
Ca(2+). DIM induced Mn(2+) influx leading to quenching of fura-2 fluorescence.
DIM-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365 and
protein kinase C modulators. In the absence of extracellular Ca(2+), incubation
with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or
2,5-di-tert-butylhydroquinone (BHQ) greatly inhibited DIM-induced [Ca(2+)]i rise.
Incubation with DIM abolished TG or BHQ-induced [Ca(2+)]i rise. Inhibition of
phospholipase C with U73122 reduced DIM-induced [Ca(2+)]i rise by 50%. At 1, 10,
40 and 50 μM, DIM slightly enhanced cell proliferation. The effect of 50 μM DIM
was reversed by chelating cytosolic Ca(2+) with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In sum, in MDCK cells,
DIM induced a [Ca(2+)]i rise by evoking phospholipase C-dependent Ca(2+) release
from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive
store-operated Ca(2+) channels. DIM did not induce cell death.

PMID: 23613483  [PubMed - in process]

In Vivo. 2013 May-Jun;27(3):299-304.

Evaluation of 3,3'-diindolylmethane with gardasil quadrivalent HPV vaccine in
K14-HPV16-transgenic mice cervical histology.

Sepkovic DW, Pagan DV, Stein J, Carlisle AD, Ksieski HB, Auborn K, Nyirenda T,
Bradlow HL.

Ph.D. The David and Alice Jurist Institute for Research, Hackensack University
Medical Center. Hackensack, NJ, U.S.A. dsepkovic@HackensackUMC.org.

BACKGROUND: The effects of 3,3'-diindolylmethane (DIM) together with the Gardasil
vaccine on cervical histology were evaluated using the K14-HPV16-transgenic mouse
model. The possibility that DIM could enhance the efficacy of this preventive
vaccine in this model was explored.
MATERIALS AND METHODS: Transgenic mice were given 1000 mg/kg of DIM in the diet
for 28 weeks. The mice were injected with Gardasil Quadrivalent HPV vaccine. Some
mice were sacrificed at 28 weeks. Other groups were removed from the DIM diet
after 28 weeks to a diet with no DIM for either 4 or 8 weeks.
RESULTS: Cervical histology indicated that a high percentage of transgenic mice
fed DIM and vaccinated with Gardasil manifested normal cervical epitheliums at 4
weeks after DIM discontinuation.
CONCLUSION: Vaccination pre-supplemented with DIM may provide with a window of
protection of at least four weeks in this transgenic model. However,
extrapolation to the effect in humans is beyond the limited scope of the
histological data presented here.

PMID: 23606684 

Br J Pharmacol. 2013 Apr 16. doi: 10.1111/bph.12205. [Epub ahead of print]

Indoles mitigate the development of experimental autoimmune encephalomyelitis by
induction of reciprocal differentiation of regulatory T cells and Th17 cells.

Rouse M, Singh NP, Nagarkatti PS, Nagarkatti M.

Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, SC 29208, USA.

BACKGROUND AND PURPOSE: Dietary indole derivatives, indole-3-carbinol (I3C), and
diindolylmethane (DIM), possess anti-cancer properties and exhibit the
characteristics of aryl hydrocarbon receptor (AhR) ligands. Because AhR
activation has recently been shown to regulate T cell differentiation, we tested
the hypothesis that I3C and DIM may mediate anti-inflammatory properties by
promoting regulatory T cell (T-regs) differentiation while inhibiting Th17 cells.
EXPERIMENTAL APPROACH: We investigated the therapeutic efficacy of I3C and DIM
against experimental autoimmune encephalomyelitis (EAE), a murine model of
multiple sclerosis (MS). The efficacy was evaluated based on clinical scores of
paralysis, histopathology, serum cytokines, and infiltration of T cells in the
CNS. We next studied the mechanism of induction of T cells against myelin
oligodendrocyte glycoprotein (MOG35-55 ) peptide, both in vivo and in vitro,
specifically investigating the differentiation of T-regs and Th17 cells, and
determined if indoles were acting through AhR. KEY RESULTS: Pre-treatment of EAE
mice with I3C or DIM completely prevented the clinical symptoms and cellular
infiltration into the CNS. Also, post-treatment of EAE with I3C or DIM also
proved highly effective in curtailing the overall severity of the disease. In
addition, I3C or DIM promoted the generation of T-regs, while downregulating the
induction of MOG-specific Th17 cells. The regulation of FoxP3 induction and
suppression of Th17 cells by indoles in vivo and in vitro were found to be
AhR-dependent. CONCLUSIONS AND IMPLICATIONS: Together, our studies demonstrate
for the first time that I3C and DIM may serve as novel therapeutics to suppress
neuroinflammation seen during MS through activation of AhR.

PMID: 23586923 

Cancer Prev Res (Phila). 2013 Jun;6(6):519-29. doi:
10.1158/1940-6207.CAPR-12-0419. Epub 2013 Apr 12.

Indole-3-Carbinol and 3',3'-Diindolylmethane Modulate Androgen's Effect on C-C
Chemokine Ligand 2 and Monocyte Attraction to Prostate Cancer Cells.

Kim EK, Kim YS, Milner JA, Wang TT.

U.S. Department of Agriculture, Building 307C, Room 132, BARC-EAST, 10300
Baltimore Ave., Beltsville, MD 20705. tom.wang@ars.usda.gov.

Inflammation has a role in prostate tumorigenesis. Recruitment of inflammatory
monocytes to the tumor site is mediated by C-C chemokine ligand 2 (CCL2) through
binding to its receptor CCR2. We hypothesized that androgen could modulate CCL2
expression in hormone-responsive prostate cancer cells and thereby promote
recruitment of monocytes. Given the inhibitory effect of broccoli-derived
compounds indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) on
androgen-dependent pathways, we also reasoned that I3C and DIM could modulate the
effect of androgen on CCL2-mediated pathways. Dihydrotestosterone was found to
induce a time-dependent (0-72 hours) and concentration-dependent (0-1 nmol/L)
increase in CCL2 mRNA levels in androgen-responsive human prostate cancer cells
(LNCaP). This increase in CCL2 mRNA corresponded with increased secretion of CCL2
protein. The effect of dihydrotestosterone was mediated through an androgen
receptor (AR)-dependent pathway as small inhibitor RNA against AR negated the
induction of CCL2. Although dihydrotestosterone also induced TWIST1 mRNA, an
epithelial-mesenchymal transition-related factor, and purported inducer of CCL2,
blocking its expression with small inhibitor RNA did not inhibit
dihydrotestosterone induction of CCL2 mRNA. Moreover, conditioned media from
androgen-treated cells promoted human monocyte THP-1 cell migration and this
effect was blocked by antibody against CCL-2. Both I3C and DIM inhibited
promotional effects of dihydrotestosterone on CCL2 and migration. These results
show that androgen may regulate CCL2 and promote inflammatory microenvironment in
prostate tumors and that this process can be blocked by broccoli-derived
compounds. Cancer Prev Res; 6(6); 519-29. ©2013 AACR.

PMID: 23585426  [PubMed - in process]

Toxicol Appl Pharmacol. 2013 Jul 15;270(2):139-48. doi:
10.1016/j.taap.2013.03.029. Epub 2013 Apr 11.

The aryl hydrocarbon receptor and estrogen receptor alpha differentially modulate
nuclear factor erythroid-2-related factor 2 transactivation in MCF-7 breast
cancer cells.

Lo R, Matthews J.

Department of Pharmacology and Toxicology, University of Toronto, Toronto,
Ontario, Canada.

Nuclear factor erythroid-2-related factor 2 (NRF2; NFE2L2) plays an important
role in mediating cellular protection against reactive oxygen species. NRF2
signaling is positively modulated by the aryl hydrocarbon receptor (AHR) but
inhibited by estrogen receptor alpha (ERα). In this study we investigated the
crosstalk among NRF2, AHR and ERα in MCF-7 breast cancer cells treated with the
NRF2 activator sulforaphane (SFN), a dual AHR and ERα activator,
3,3'-diindolylmethane (DIM), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or
17β-estradiol (E2). SFN-dependent increases in NADPH-dependent oxidoreductase 1
(NQO1) and heme oxygenase I (HMOX1) mRNA levels were significantly reduced after
co-treatment with E2. E2-dependent repression of NQO1 and HMOX1 was associated
with increased ERα but reduced p300 recruitment and reduced histone H3
acetylation at both genes. In contrast, DIM+SFN or TCDD+SFN induced NQO1 and
HMOX1 mRNA expression to levels higher than SFN alone, which was prevented by
RNAi-mediated knockdown of AHR. DIM+SFN but not TCDD+SFN also induced recruitment
of ERα to NQO1 and HMOX1. However, the presence of AHR at NQO1 and HMOX1 restored
p300 recruitment and histone H3 acetylation, thereby reversing the ERα-dependent
repression of NRF2. Taken together, our study provides further evidence of
functional interplay among NRF2, AHR and ERα signaling pathways through altered
p300 recruitment to NRF2-regulated target genes.

PMID: 23583297  [PubMed - in process]

Carcinogenesis. 2013 Apr 8. [Epub ahead of print]

In vivo and in vitro study on the role of 3, 3'-Diindolylmethane (DIM) in
treatment and prevention of nasopharyngeal carcinoma.

Chen C, Chen SM, Xu B, Chen Z, Wang F, Ren J, Xu Y, Wang Y, Xiao BK, Tao ZZ.

Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan
University, Wuhan 430060, China.

Nasopharyngeal carcinoma (NPC) is characterized by insidious progression and
atypical early symptoms, and mostly diagnosed in middle or late stages. The
long-term prognosis is not very good after conventional radiotherapy or
chemotherapy, and the therapy often has strong toxic effects on normal tissue and
organs. There were in-vitro and in-vivo pre-clinical evidences to support
chemotherapeutic and chemopreventive effect of 3, 3'-Diindolylmethane (DIM),
which was a natural compound extracted from cruciferous plants. The in-vitro
experiments showed that 100µM DIM DIM could induce remarkable apoptosis of NPC
cells, and no obvious damage was observed in normal human bronchial epithelial
cells and human liver cells(P <0.01). DIM could simultaneously regulate several
signaling pathways directly related to NPC, such as PI3K, MAPK, Akt and NF-κB. In
animal model of nasopharyngeal heterotopic transplanted tumors, the volume of
transplanted tumor in animal raised by feed containing DIM was significantly less
than that of control group (P<0.01). The animals with 2 weeks of preventive
feeding with feed containing DIM before inoculation had the smallest volume of
transplanted tumor (P<0.01). Intake of DIM had no toxic effects on vital organs
such as heart, liver and kidney of experimental animals. In summary, the results
of this study confirmed that the DIM effectively induced apoptosis of
nasopharyngeal carcinoma cells, and had a preventive and curative role in the
development and progression of nasopharyngeal carcinoma. The drug was safe and
had no toxic effects on normal tissues and organs.

PMID: 23568953  [PubMed - as supplied by publisher]

PLoS One. 2013;8(3):e59798. doi: 10.1371/journal.pone.0059798. Epub 2013 Mar 28.

2,2'-diphenyl-3,3'-diindolylmethane: a potent compound induces apoptosis in
breast cancer cells by inhibiting EGFR pathway.

Bhowmik A, Das N, Pal U, Mandal M, Bhattacharya S, Sarkar M, Jaisankar P, Maiti
NC, Ghosh MK.

Division of Cancer Biology and Inflammatory Disorder, Council of Scientific and
Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), Kolkata,
West Bengal, India.

Despite recent advances in medicine, 30-40% of patients with breast cancer show
recurrence underscoring the need for improved effective therapy. In this study,
by in vitro screening we have selected a novel synthetic indole derivative
2,2'-diphenyl-3,3'-diindolylmethane (DPDIM) as a potential anti- breast cancer
agent. DPDIM induces apoptosis both in vitro in breast cancer cells MCF7, MDA-MB
231 and MDA-MB 468 and in vivo in 7,12-dimethylbenz[α]anthracene (DMBA) induced
Sprague-Dawley (SD) rat mammary tumor. Our in vitro studies show that DPDIM
exerts apoptotic effect by negatively regulating the activity of EGFR and its
downstream molecules like STAT3, AKT and ERK1/2 which are involved in the
proliferation and survival of these cancer cells. In silico predictions also
suggest that DPDIM may bind to EGFR at its ATP binding site. DPDIM furthermore
inhibits EGF induced increased cell viability. We have also shown decreased
expression of pro-survival factor Bcl-XL as well as increase in the level of
pro-apoptotic proteins like Bax, Bad, Bim in DPDIM treated cells in vitro and in
vivo. Our results further indicate that the DPDIM induced apoptosis is mediated
through mitochondrial apoptotic pathway involving the caspase-cascade. To the
best of our knowledge this is the first report of DPDIM for its anticancer
activity. Altogether this report suggests that DPDIM could be an effective
therapeutic agent for breast cancer.

PMCID: PMC3610887
PMID: 23555785  [PubMed - in process]

Aging Cell. 2013 Mar 23. doi: 10.1111/acel.12077. [Epub ahead of print]

Chemical genetic screen in fission yeast reveals roles for vacuolar
acidification, mitochondrial fission, and cellular GMP levels in lifespan
extension.

Stephan J, Franke J, Ehrenhofer-Murray AE.

Zentrum für Medizinische Biotechnologie, Universität Duisburg-Essen, Essen,
Germany.

The discovery that genetic mutations in several cellular pathways can increase
lifespan has lent support to the notion that pharmacological inhibition of aging
pathways can be used to extend lifespan and to slow the onset of age-related
diseases. However, so far, only few compounds with such activities have been
described. Here, we have conducted a chemical genetic screen for compounds that
cause the extension of chronological lifespan of Schizosaccharomyces pombe. We
have characterized eight natural products with such activities, which has allowed
us to uncover so far unknown anti-aging pathways in S. pombe. The ionophores
monensin and nigericin extended lifespan by affecting vacuolar acidification, and
this effect depended on the presence of the vacuolar ATPase (V-ATPase) subunits
Vma1 and Vma3. Furthermore, prostaglandin J2 displayed anti-aging properties due
to the inhibition of mitochondrial fission, and its effect on longevity required
the mitochondrial fission protein Dnm1 as well as the G-protein-coupled glucose
receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP)
synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension,
indicating that an imbalance in guanine nucleotide levels impinges upon
longevity. We furthermore have identified diindolylmethane (DIM), tschimganine,
and the compound mixture mangosteen as inhibiting aging. Taken together, these
results reveal unanticipated anti-aging activities for several phytochemicals and
open up opportunities for the development of novel anti-aging therapies.

PMID: 23521895  [PubMed - as supplied by publisher]

Eur J Med Chem. 2013 May;63:435-43. doi: 10.1016/j.ejmech.2013.02.024. Epub 2013
Feb 28.

Discovery of 3,3'-diindolylmethanes as potent antileishmanial agents.

Bharate SB, Bharate JB, Khan SI, Tekwani BL, Jacob MR, Mudududdla R, Yadav RR,
Singh B, Sharma PR, Maity S, Singh B, Khan IA, Vishwakarma RA.

Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR),
Canal Road, Jammu 180001, India. Electronic address: sbharate@iiim.ac.in.

An efficient protocol for synthesis of 3,3'-diindolylmethanes using recyclable
Fe-pillared interlayered clay (Fe-PILC) catalyst under aqueous medium has been
developed. All synthesized 3,3'-diindolylmethanes showed promising
antileishmanial activity against Leishmania donovani promastigotes as well as
axenic amastigotes. Structure-activity relationship analysis revealed that
nitroaryl substituted diindolylmethanes showed potent antileishmanial activity.
The 4-nitrophenyl linked 3,3'-diindolylmethane 8g was found to be the most potent
antileishmanial analog showing IC50 values of 7.88 and 8.37 μM against both
L. donovani promastigotes and amastigotes, respectively. Further, a pharmacophore
based QSAR model was established to understand the crucial molecular features of
3,3'-diindolylmethanes essential for potent antileishmanial activity. These
compounds also exhibited promising antifungal activity against Cryptococcus
neoformans, wherein fluorophenyl substituted 3,3'-diindolylmethanes were found to
be most potent antifungal agents. Developed synthetic protocol will be useful for
economical and eco-friendly synthesis of potent antileishmanial and antifungal
3,3'-diindolylmethane class of compounds.

PMCID: PMC3674182 [Available on 2014/5/1]
PMID: 23517732  [PubMed - in process]

Oncol Lett. 2013 Feb;5(2):655-662. Epub 2012 Dec 5.

Anticancer effects of 3,3'-diindolylmethane are associated with G1 arrest and
mitochondria-dependent apoptosis in human nasopharyngeal carcinoma cells.

Xu Y, Zhang J, Shi W, Liu Y.

Departments of Otolaryngology, Wuhan University, Wuhan 430060, Hubei, P.R. China.

The antitumor effects of 3,3'-diindolylmethane (DIM) are exhibited in a number of
human cancer cells. However, there have been few studies performed concerning the
effect of DIM on nasopharyngeal cancer (NPC) cells. In the present study, we
examined the in vitro antitumor activity of DIM on the poorly differentiated NPC
cell line CNE-2. The potential molecular mechanisms of the activity were also
explored. CNE-2 cells were treated with varying concentrations of DIM for
different times. Cell proliferation and apoptosis were detected and the molecular
mechanisms involved in these effects were characterized. The results demonstrated
that DIM at concentrations of 15-100 μM caused dose- and time-dependent
inhibition of CNE-2 cell proliferation. Flow cytometry analysis revealed a high
sub-G1 cell peak following treatment with DIM, and the rate of apoptosis
increased. DIM may elevate the levels of cleaved Bid and Bax and enhance
mitochondrial membrane depolarization, allowing the efflux of cytochrome c, Smac
and Omi into the cytosol. The levels of caspases-3, -8 and -9 and cleaved poly
(ADP-ribose) polymerase (PARP) were upregulated following DIM treatment in a
dose-dependent manner. DIM also inhibits the phosphorylation of IκB-α, and showed
dose-dependent inhibition of Bcl-2, XIAP and NF-κB in CNE-2 cells in vitro. These
results indicate that DIM inhibits cell proliferation by inducing cell cycle
arrest at G0/G1 phase and induces the apoptosis of CNE-2 cells by regulating
multiple molecules in a mitochondria-dependent pathway. DIM may be a preventive
and therapeutic agent against NPC.

PMCID: PMC3573071
PMID: 23420395  [PubMed]

Antioxid Redox Signal. 2013 Mar 14. [Epub ahead of print]

Antioxidant Function of Isoflavone and 3,3'-Diindolylmethane: Are They Important
for Cancer Prevention and Therapy?

Li Y, Kong D, Ahmad A, Bao B, Sarkar FH.

1 Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine , Detroit, Michigan.

Abstract Significance: Oxidative stress has been mechanistically linked with
aging and chronic diseases, including cancer. In fact, oxidative stress status,
chronic disease-related inflammation, and cancer occurred in the aging population
are tightly correlated. It is well known that the activation of nuclear factor
kappa B (NF-κB) plays important roles in oxidative stress, inflammation, and
carcinogenesis. Therefore, targeting NF-κB is an important preventive or
therapeutic strategy against oxidative stress, inflammation, and cancer. Recent
Advances: A variety of natural compounds has been found to reduce oxidative
stress through their antioxidant activity. Among them, isoflavone,
indole-3-carbinol (I3C), and its in vivo dimeric compound 3,3'-diindolylmethane
(DIM) have shown their promising effects on the inhibition of NF-κB with
corresponding reduction of oxidative stress. Critical Issues: It has been found
that isoflavone, I3C, and DIM could inhibit cancer development and progression by
regulating multiple cellular signaling pathways that are related to oxidative
stress and significantly deregulated in cancer. Future Directions: The
antioxidative and anticancer effects of these natural agents make them strong
candidates for chemoprevention and/or therapy against human malignancies.

PMID: 23391445 

PLoS One. 2013;8(1):e53427. doi: 10.1371/journal.pone.0053427. Epub 2013 Jan 9.

3,3'-Diindolylmethane protects against cardiac hypertrophy via 5'-adenosine
monophosphate-activated protein kinase-α2.

Zong J, Deng W, Zhou H, Bian ZY, Dai J, Yuan Y, Zhang JY, Zhang R, Zhang Y, Wu
QQ, Guo HP, Li HL, Tang QZ.

Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, China.

PURPOSE: 3,3'-Diindolylmethane (DIM) is a natural component of cruciferous
plants. It has strong antioxidant and anti-angiogenic effects and promotes the
apoptosis of a variety of tumor cells. However, little is known about the
critical role of DIM on cardiac hypertrophy. In the present study, we
investigated the effects of DIM on cardiac hypertrophy.
METHODS: Multiple molecular techniques such as Western blot analysis, real-time
PCR to determine RNA expression levels of hypertrophic, fibrotic and oxidative
stress markers, and histological analysis including H&E for histopathology, PSR
for collagen deposition, WGA for myocyte cross-sectional area, and
immunohistochemical staining for protein expression were used.
RESULTS: In pre-treatment and reverse experiments, C57/BL6 mouse chow containing
0.05% DIM (dose 100 mg/kg/d DIM) was administered one week prior to surgery or
one week after surgery, respectively, and continued for 8 weeks after surgery. In
both experiments, DIM reduced to cardiac hypertrophy and fibrosis induced by
aortic banding through the activation of 5'-adenosine monophosphate-activated
protein kinase-α2 (AMPKα2) and inhibition of mammalian target of the rapamycin
(mTOR) signaling pathway. Furthermore, DIM protected against cardiac oxidative
stress by regulating expression of estrogen-related receptor-alpha (ERRα) and
NRF2 etc. The cardioprotective effects of DIM were ablated in mice lacking
functional AMPKα2.
CONCLUSION: DIM significantly improves left ventricular function via the
activation of AMPKα2 in a murine model of cardiac hypertrophy.

PMCID: PMC3541184
PMID: 23326427  [PubMed - in process]

J Pharmacol Exp Ther. 2013 Apr;345(1):125-38. doi: 10.1124/jpet.112.201558. Epub
2013 Jan 14.

Neuroprotective efficacy and pharmacokinetic behavior of novel anti-inflammatory
para-phenyl substituted diindolylmethanes in a mouse model of Parkinson's
disease.

De Miranda BR, Miller JA, Hansen RJ, Lunghofer PJ, Safe S, Gustafson DL,
Colagiovanni D, Tjalkens RB.

Center for Environmental Medicine, Department of Environmental and Radiological
Health Sciences, Animal Cancer Center, Colorado State University, Fort Collins,
Colorado 80523-1680, USA.

There are currently no registered drugs that slow the progression of
neurodegenerative diseases, in part because translation from animal models to the
clinic has been hampered by poor distribution to the brain. The present studies
examined a selected series of para-phenyl-substituted diindolylmethane (C-DIM)
compounds that display anti-inflammatory and neuroprotective efficacy in vitro.
We postulated that the pharmacokinetic behavior of C-DIM compounds after oral
administration would correlate with neuroprotective efficacy in vivo in a mouse
model of Parkinson's disease. Pharmacokinetics and metabolism of
1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (C-DIM5),
1,1-bis(3'-indolyl)-1-(phenyl)methane,
1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (C-DIM8), and
1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (C-DIM12) were determined in plasma
and brain of C57Bl/6 mice after oral and intravenous administration at 10 and 1
mg/Kg, respectively. Putative metabolites were measured in plasma, liver, and
urine. C-DIM compounds given orally displayed the highest area under the curve,
Cmax, and Tmax levels, and C-DIM12 exhibited the most favorable pharmacokinetics
of the compounds tested. Oral bioavailability of each compound ranged from 6%
(C-DIM8) to 42% (C-DIM12). After pharmacokinetic studies, the neuroprotective
efficacy of C-DIM5, C-DIM8, and C-DIM12 (50 mg/Kg per oral) was examined in mice
exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid for
14 days, a model of progressive neurodegeneration with a strong neuroinflammatory
component. C-DIM5 and C-DIM12 given orally once daily after one week of exposure
to MPTP and probenecid prevented further loss of dopaminergic neurons in the
substantia nigra pars compacta and striatal dopamine terminals, indicating that
these compounds could be effective therapeutic agents to prevent
neurodegeneration.

PMID: 23318470 

Chemistry. 2013 Feb 4;19(6):2180-4. doi: 10.1002/chem.201202647. Epub 2012 Dec
20.

Carbophilic 3-component cascades: access to complex bioactive cyclopropyl
diindolylmethanes.

Groome NM, Elboray EE, Inman MW, Dondas HA, Phillips RM, Kilner C, Grigg R.

School of Chemistry, University of Leeds, Leeds, LS2 9 JT, UK.

Naturally occurring indole-3-carbinol and 3,3-diindolylmethane show bioactivity
in a number of disparate disease areas, including cancer, prompting substantial
synthetic analogue activity. We describe a new approach to highly functionalised
derivatives that starts from allene gas and proceeds via the combination of a
three-component Pd(0)-catalysed cascade with a one-pot, three-component
carbophilic Pt(II) cascade linked to a stereoselective acid-catalysed
Mannich-Michael reaction that generates complex cyclopropyl diindolylmethanes
which show selective activity against prostate cancer cell lines.

PMID: 23280956  [PubMed - in process]

Anticancer Agents Med Chem. 2012 Dec 11. [Epub ahead of print]

Targeted Regulation of PI3K/Akt/mTOR/NF-κB Signaling by Indole Compounds and
their Derivatives: Mechanistic Details and Biological Implications for Cancer
Therapy.

Ahmad A, Biersack B, Li Y, Kong D, Bao B, Schobert R, Padhye SB, Sarkar FH.

Department of Pathology and Oncology, Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, MI 48201, USA. fsarkar@med.wayne.edu.

Indole compounds, found in cruciferous vegetables, are potent anti-cancer agents.
Studies with indole-3-carbinol (I3C) and its dimeric product,
3,3'-diindolylmethane (DIM) suggest that these compounds have the ability to
deregulate multiple cellular signaling pathways, including PI3K/Akt/mTOR
signaling pathway. These natural compounds are also effective modulators of
downstream transcription factor NF-κB signaling which might help explain their
ability to inhibit invasion and angiogenesis, and the reversal of
epithelial-to-mesenchymal transition (EMT) phenotype and drug resistance.
Signaling through PI3K/Akt/mTOR and NF-κB pathway is increasingly being realized
to play important role in EMT through the regulation of novel miRNAs which
further validates the importance of this signaling network and its regulations by
indole compounds. Here we will review the available literature on the modulation
of PI3K/Akt/mTOR/NF-κB signaling by both parental I3C and DIM, as well as their
analogs/derivatives, in an attempt to catalog their anticancer activity.

PMID: 23272910  [PubMed - as supplied by publisher]

Carcinogenesis. 2013 Apr;34(4):841-9. doi: 10.1093/carcin/bgs390. Epub 2012 Dec
13.

Chemoprevention of lung tumorigenesis by intranasally administered
diindolylmethane in A/J mice.

Qian X, Song JM, Melkamu T, Upadhyaya P, Kassie F.

Department of Veterinary Clinical Sciences Masonic Cancer Center, University of
Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455, USA.

The main reasons for the failure of most chemopreventive agents during clinical
trials are poor in vivo bioavailability and dose-limiting side effects. One
potential approach to surmount these problems in lung cancer chemoprevention
trials could be direct delivery of agents into the pulmonary tissue. In this
study, we assessed the efficacy of intranasally delivered Diindolylmethane (DIM) against
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK)-induced lung tumorigenesis in mice. Mice treated with NNK (two doses of
50mg/kg at an interval of a week, intraperitoneal) developed 16.3±2.9 lung tumors
per mouse. Post-carcinogen administration of DIM, via intranasal instillation,
for 24 weeks, twice a week, at a dose of 2mg per mouse (0.6mg pure
diindolylmethane per mouse) reduced the lung tumor multiplicity to 4.6±2.2 tumors
per mouse (72% reduction). Likewise, large tumors (>1mm) were almost completely
abolished and multiplicities of tumors with a size of 0.5-1mm were reduced by
74%. Tumor volume was also reduced by 82%. Further studies using an in vitro
model of lung tumorigenesis showed that DIM exhibited pronounced
antiproliferative and apoptotic effects in premalignant and malignant bronchial
cells but only minimal effects in parental immortalized cells through, at least
in part, suppression of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling
pathway. These results showed the potent lung tumor inhibitory activities of low
doses of DIM given via intranasal instillation and, therefore, intranasal
delivery of DIM holds a great promise for lung cancer chemoprevention in subjects
at high risk to develop lung cancer.

PMCID: PMC3616664 [Available on 2014/4/1]
PMID: 23239747  [PubMed - in process]

EMBO Mol Med. 2012 Dec;4(12):1308-19. doi: 10.1002/emmm.201201298. Epub 2012 Nov
9.

MicroRNA-146a-mediated downregulation of IRAK1 protects mouse and human small
intestine against ischemia/reperfusion injury.

Chassin C, Hempel C, Stockinger S, Dupont A, Kübler JF, Wedemeyer J, Vandewalle
A, Hornef MW.

Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical
School, Hannover, Germany. cecilia.chassin@gmail.com

Intestinal ischemia/reperfusion (I/R) injury causes inflammation and tissue
damage and is associated with high morbidity and mortality. Uncontrolled
activation of the innate immune system through toll-like receptors (Tlr) plays a
key role in I/R-mediated tissue damage but the underlying mechanisms have not
been fully resolved. Here, we identify post-transcriptional upregulation of the
essential Tlr signalling molecule interleukin 1 receptor-associated kinase (Irak)
1 as the causative mechanism for post-ischemic immune hyper-responsiveness of
intestinal epithelial cells. Increased Irak1 protein levels enhanced epithelial
ligand responsiveness, chemokine secretion, apoptosis and mucosal barrier
disruption in an experimental intestinal I/R model using wild-type, Irak1(-/-)
and Tlr4(-/-) mice and ischemic human intestinal tissue. Irak1 accumulation under
hypoxic conditions was associated with reduced K48 ubiquitination and enhanced
Senp1-mediated deSUMOylation of Irak1. Importantly, administration of microRNA
(miR)-146a or induction of miR-146a by the phytochemical diindolylmethane
controlled Irak1 upregulation and prevented immune hyper-responsiveness in mouse
and human tissue. These findings indicate that Irak1 accumulation triggers
I/R-induced epithelial immune hyper-responsiveness and suggest that the induction
of miR-146a offers a promising strategy to prevent I/R tissue injury.

PMCID: PMC3531605
PMID: 23143987  [PubMed - indexed for MEDLINE]

Curr Drug Targets. 2012 Dec;13(14):1869-75.

DIMming ovarian cancer growth.

Kandala PK, Srivastava SK.

Department of Biomedical Sciences and Cancer Biology Center, Texas Tech
University Health Sciences Center, Amarillo, Texas 79106, USA.

Ovarian cancer is the leading gynecologic malignancy with more than 22,000 new
cases and 15,000 deaths estimated each year. It is usually detected in late
stages with poor prognosis due to lack of sufficiently accurate screening tests.
Epidemiological studies continue to support the notion that consumption of
cruciferous vegetables reduces the risk of ovarian cancer. In the present review
article, we describe the anti-cancer effects of 3, 3'-diindolylmethane (DIM), a
compound present in cruciferous vegetables, against ovarian cancer. DIM targets
multiple aspects of cancer such as cellcycle regulation and survival, including
EGFR-JAK2-STAT3 signaling, checkpoint activation, caspase activation, endoplasmic
reticulum stress, autophagy and anoikis. This broad spectrum of anti-cancer
activities in conjunction with low systemic toxicity accentuates the
translational value of DIM in cancer therapy. Together, our pre-clinical studies
demonstrate that DIM has activity against ovarian cancer and hence should be
further investigated in clinical setting to exploit its therapeutic potential.

PMID: 23140296  [PubMed - in process]

Curr Drug Targets. 2012 Dec;13(14):1705-19.

Indole compounds against breast cancer: recent developments.

Biersack B, Schobert R.

Organic Chemistry Laboratory, University of Bayreuth, Universitaetsstrasse 30,
D-95440 Bayreuth, Germany. Bernhard.Biersack@uni-bayreuth.de

Breast cancer is still the leading cause of cancer deaths among women worldwide
and new therapies and drugs are continuously being conceived and explored to
better control or even cure this disease. Among the most efficacious
low-molecular drugs for the treatment of breast cancer are indole derivatives
such as 3,3'-diindolylmethane (DIM), the structurally complex antimitotic vinca
alkaloids, and the synthetic receptor tyrosine kinase inhibitor sunitinib. This
review is to give an insight into the latest developments in the field of indole
based drugs against breast cancers with a focus on those derived from natural
products and on their targets and modes of action. Pertinent literature is
covered from 2007 up to 2012.

PMID: 23140282  [PubMed - in process]

Eur J Nutr. 2012 Oct 23. [Epub ahead of print]

Modulation of CYP19 expression by cabbage juices and their active components:
indole-3-carbinol and 3,3'-diindolylmethene in human breast epithelial cell
lines.

Licznerska BE, Szaefer H, Murias M, Bartoszek A, Baer-Dubowska W.

Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences,
Poznan, Poland.

PURPOSE: The aim of this study was to evaluate the effect of white cabbage and
sauerkraut juices of different origin and indole-3-carbinol (I3C) and
diindolylmethane (DIM) on expression of CYP19 gene encoding aromatase, the key
enzyme of estrogen synthesis. METHODS: Human breast cell lines (MCF7, MDA-MB-231
and MCF10A) were examined to compare the action of cabbage juices versus their
active components (I3C, DIM). Real-time PCR and Western blot were used in order
to analyse CYP19 mRNA and protein, respectively. RESULTS: Remarkable differences
in the effect on CYP19 transcript and protein level were found between the
cabbage juices (in 2.5-25 mL/L concentrations) and indoles (in 2.5-50 μM doses)
in the three cell lines. While cabbage juices at the lower doses diminished the
aromatase expression in nontumorigenic/immortalized MCF10A breast cells
(0.25-0.86-fold change, P < 0.05), I3C and DIM were more efficient in decreasing
the aromatase expression in estrogen-dependant MCF7 breast cancer cells
(0.24-0.82-fold change, P < 0.05). Inhibition of aromatase by juice obtained from
cabbage grown on industrial farm was correlated with the induction of apoptosis
(1.7-1.8-fold change, P < 0.01) in MCF10A cells. In estrogen-independent
MDA-MB-231 cells, up-regulation of CYP19 expression by I3C and DIM (1.5-2.0-fold
change, P < 0.05) was observed. Similarly, in MCF7 cells juices increased
aromatase expression (1.1-2.2-fold change, P < 0.05). CONCLUSION: These results,
particularly that obtained in nontumorigenic/immortalized MCF10A cells, suggest
that chemopreventive activity of cabbage against breast cancer observed in
epidemiological studies may be partly explained by inhibition of the aromatase
expression.

PMID: 23090135  [PubMed - as supplied by publisher]

Mol Immunol. 2013 Apr;53(4):335-44. doi: 10.1016/j.molimm.2012.09.007. Epub 2012
Oct 17.

3,3'-Diindolylmethane alleviates oxazolone-induced colitis through Th2/Th17
suppression and Treg induction.

Huang Z, Jiang Y, Yang Y, Shao J, Sun X, Chen J, Dong L, Zhang J.

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing 210093, China.

The T cell is pivotal in orchestrating and promoting an immune response during
ulcerative colitis (UC). The aryl hydrocarbon receptor (AhR) is involved in the
regulation of T cell responses, and 3,3'-diindolylmethane (DIM) is a known ligand
of AhR. The aim of this study was to examine the therapeutic effects of DIM in
experimental colitis and to investigate the possible mechanisms underlying its
effects on mucosal T cell responses. The therapeutic effects of DIM were studied
in an oxazolone-induced colitis model. The pathologic markers of colitis were
measured, moreover, T-helper cell (Th)- and regulatory T cell (Treg)-related
transcription factor expression and associated colonic cytokine production were
determined. The impact of DIM on T cell differentiation was further investigated
in cultures of naive Th cells that were stimulated with anti-CD3/CD28 monoclonal
antibodies (mAbs). The administration of DIM attenuated experimental colitis, as
determined by pathological indices. DIM may affect signaling pathways downstream
of AhR, leading to decreased Th2/Th17 cells and increased Tregs. Ultimately, this
could result in the alleviation of experimental colitis. DIM has shown anti-UC
activity in animal models via inhibition of Th2/Th17 cells and promotion of Tregs
and may thus offer potential treatments for UC patients.

PMID: 23085552  [PubMed - indexed for MEDLINE]

PLoS One. 2012;7(10):e47186. doi: 10.1371/journal.pone.0047186. Epub 2012 Oct 9.

Activation of AMP-activated protein kinase by 3,3'-Diindolylmethane (DIM) is
associated with human prostate cancer cell death in vitro and in vivo.

Chen D, Banerjee S, Cui QC, Kong D, Sarkar FH, Dou QP.

Department of Oncology, Barbara Ann Karmanos Cancer Institute, Wayne State
University, School of Medicine, Detroit, Michigan, United States of America.

There is a large body of scientific evidence suggesting that
3,3'-Diindolylmethane (DIM), a compound derived from the digestion of
indole-3-carbinol, which is abundant in cruciferous vegetables, harbors
anti-tumor activity in vitro and in vivo. Accumulating evidence suggests that
AMP-activated protein kinase (AMPK) plays an essential role in cellular energy
homeostasis and tumor development and that targeting AMPK may be a promising
therapeutic option for cancer treatment in the clinic. We previously reported
that a formulated DIM (BR-DIM; hereafter referred as B-DIM) with higher
bioavailability was able to induce apoptosis and inhibit cell growth,
angiogenesis, and invasion of prostate cancer cells. However, the precise
molecular mechanism(s) for the anti-cancer effects of B-DIM have not been fully
elucidated. In the present study, we investigated whether AMP-activated protein
kinase (AMPK) is a molecular target of B-DIM in human prostate cancer cells. Our
results showed, for the first time, that B-DIM could activate the AMPK signaling
pathway, associated with suppression of the mammalian target of rapamycin (mTOR),
down-regulation of androgen receptor (AR) expression, and induction of apoptosis
in both androgen-sensitive LNCaP and androgen-insensitive C4-2B prostate cancer
cells. B-DIM also activates AMPK and down-regulates AR in androgen-independent
C4-2B prostate tumor xenografts in SCID mice. These results suggest that B-DIM
could be used as a potential anti-cancer agent in the clinic for prevention
and/or treatment of prostate cancer regardless of androgen responsiveness,
although functional AR may be required.

PMCID: PMC3467201
PMID: 23056607 

Top Curr Chem. 2012 Sep 6.

 

Cancer Chemoprevention and Nutri-Epigenetics: State of the Art and Future

Challenges.

 

Gerhauser C.

 

Division Epigenomics and Cancer Risk Factors, German Cancer Research Center, Im

Neuenheimer Feld 280, 69120, Heidelberg, Germany, c.gerhauser@dkfz.de.

 

The term "epigenetics" refers to modifications in gene expression caused by

heritable, but potentially reversible, changes in DNA methylation and chromatin

structure. Epigenetic alterations have been identified as promising new targets

for cancer prevention strategies as they occur early during carcinogenesis and

represent potentially initiating events for cancer development. Over the past few

years, nutri-epigenetics - the influence of dietary components on mechanisms

influencing the epigenome - has emerged as an exciting new field in current

epigenetic research. During carcinogenesis, major cellular functions and

pathways, including drug metabolism, cell cycle regulation, potential to repair

DNA damage or to induce apoptosis, response to inflammatory stimuli, cell

signalling, and cell growth control and differentiation become deregulated.

Recent evidence now indicates that epigenetic alterations contribute to these

cellular defects, for example epigenetic silencing of detoxifying enzymes, tumor

suppressor genes, cell cycle regulators, apoptosis-inducing and DNA repair genes,

nuclear receptors, signal transducers and transcription factors by promoter

methylation, and modifications of histones and non-histone proteins such as p53,

NF-κB, and the chaperone HSP90 by acetylation or methylation.The present review

will summarize the potential of natural chemopreventive agents to counteract

these cancer-related epigenetic alterations by influencing the activity or

expression of DNA methyltransferases and histone modifying enzymes.

Chemopreventive agents that target the epigenome include micronutrients (folate,

retinoic acid, and selenium compounds), butyrate, polyphenols from green tea,

apples, coffee, black raspberries, and other dietary sources, genistein and soy

isoflavones, curcumin, resveratrol, dihydrocoumarin, nordihydroguaiaretic acid

(NDGA), lycopene, anacardic acid, garcinol, constituents of Allium species and

cruciferous vegetables, including indol-3-carbinol (I3C), diindolylmethane (DIM),

sulforaphane, phenylethyl isothiocyanate (PEITC), phenylhexyl isothiocyanate

(PHI), diallyldisulfide (DADS) and its metabolite allyl mercaptan (AM), cambinol,

and relatively unexplored modulators of histone lysine methylation (chaetocin,

polyamine analogs). So far, data are still mainly derived from in vitro

investigations, and results of animal models or human intervention studies are

limited that demonstrate the functional relevance of epigenetic mechanisms for

health promoting or cancer preventive efficacy of natural products. Also, most

studies have focused on single candidate genes or mechanisms. With the emergence

of novel technologies such as next-generation sequencing, future research has the

potential to explore nutri-epigenomics at a genome-wide level to understand

better the importance of epigenetic mechanisms for gene regulation in cancer

chemoprevention.

 

PMID: 22955508 

 

Curr Cancer Drug Targets. 2012 Aug 16. [Epub ahead of print]

 

Inhibition of STAT Signalling in Bladder Cancer by Diindolylmethane - Relevance

to Cell Adhesion, Migration and Proliferation.

 

Sun Y, Cheng MK, Griffiths TR, Mellon JK, Kai B, Kriajevska M, Manson MM.

 

Department of Cancer Studies and Molecular Medicine, Biocentre, University of

Leicester, LE1 7RH, UK.

 

Effective treatments to prevent recurrence or progression of non-muscle-invasive

bladder cancer, or to inhibit metastasis of muscle-invasive forms of the disease,

would deliver significant patient benefit. Here the involvement of STAT

signalling and the chemopreventive potential of diindolylmethane (DIM) in human

bladder cancer were investigated. Muscle-invasive bladder cancer tissueswere

characterised by nuclear expression of phosphorylated STAT1, 3 and 5. In

E-cadherin positive tumour cell lines (RT112, RT4, HT1376), STAT5 was

constitutively phosphorylated, while E-cadherin negative lines (J82, T24, UMUC3)

contained phosphoSTAT3. Knockdown of STAT3induced G0/G1 arrest and inhibited

adhesion in J82cells, Knockdown of STAT1inhibited migration in J82 and RT112

lines. No significant increase in apoptosis was observed. In response to the

Janus kinase inhibitor, AG490, RT112 and J82 cells initially underwent G0/G1

arrest,with RT112 cells subsequently exhibiting S phase arrest. Phosphorylation

of STAT1Tyr701, STAT3Tyr705 and Ser727 and STAT5Tyr694 was inhibited by DIM, as

was adhesion of J82cells to collagen, an effect that was enhanced when STAT1 or 3

was reduced by siRNA. However, over-expression of STAT3C partially rescued the

DIM inhibitory effect on collagen-mediated adhesion. Migration of both lines was

inhibited by DIM, while transfection of constitutively active STAT3C enhanced

migration of RT112 cells. DIM induced cell cycle arrest and apoptosisin three

cell lines with different degrees of radioresistance. Taken together, these

results suggest that inhibition of STAT signalling and/or treatment with DIM may

decrease invasiveness of bladder cancer. DIM can induce apoptosis in cell lines

which are radioresistant, so in combination with radiotherapy may be useful in

overcomingsuch resistance.

 

PMID: 22920439 

 

J Recept Signal Transduct Res. 2012 Oct;32(5):271-8. Epub 2012 Jul 31.

 

Effect of diindolylmethane on Ca(2+) homeostasis and viability in PC3 human

prostate cancer cells.

 

Tsai JY, Chou CT, Liu SI, Liang WZ, Kuo CC, Liao WC, Lin KL, Hsu SS, Lu YC, Huang

JK, Jan CR.

 

Department of Surgery, Kaohsiung Veterans General Hospital , Kaohsiung , Taiwan.

 

The effect of the natural product diindolylmethane on cytosolic Ca(2+)

concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was

explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure

[Ca(2+)](i). Diindolylmethane at concentrations of 20-50 ÁM induced [Ca(2+)](i)

rise in a concentration-dependent manner. The response was reduced partly by

removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by

nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic

acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic

reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone

(BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise.

Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced

[Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced

diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 50-100 ÁM,

diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic

effect was not altered by chelating cytosolic Ca(2+) with

1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI

staining data implicate that diindolylmethane (50 and 100 ÁM) induced apoptosis

in a concentration-dependent manner. In conclusion, diindolylmethane induced a

[Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release

from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive

store-operated Ca(2+) channels. Diindolylmethane caused cell death in which

apoptosis may participate.

 

PMID: 22845469 

 

J Nutr Biochem. 2012 Jul 20.

 

3,3'-Diindolylmethane induces activating transcription factor 3 (ATF3) via ATF4

in human colorectal cancer cells.

 

Lee SH, Min KW, Zhang X, Baek SJ.

 

Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine,

University of Tennessee, Knoxville, TN 37996-4542, USA.

 

3,3'-Diindolylmethane (DIM) is a major in vivo condensation product of

indole-3-carbinol, which is present in cruciferous vegetables. Although these

compounds have been widely implicated in antitumorigenic and proapoptotic

properties in animal as well as in vitro models of cancer, the underlying

cellular mechanisms regulated by DIM are only partially understood. Activating

transcription factor 3 (ATF3) is a member of the ATF/c-AMP response

element-binding (CREB) subfamily of the basic-region leucine zipper family and

has been known to induce apoptosis in human colorectal cancer (CRC) cells. The

present study was performed to elucidate the molecular mechanism of ATF3

induction by DIM in human CRC cells. The DIM treatment induced apoptosis and

induced ATF3 gene expression at protein and messenger RNA levels. DIM increased

ATF3 promoter activity, and the region of -84 to +34 within ATF3 promoter was

responsible for promoter activation by DIM. This region contained an ATF binding

site. Deletion and point mutation of the ATF binding site (-23 to -16) abolished

ATF3 promoter activation by DIM, and overexpression of ATF4 enhanced ATF3

transactivation. Chromatin immunoprecipitation assay confirmed the binding of

ATF4 in the ATF3 promoter. Inhibition of ATF4 expression by small interference

RNA results in repression of DIM-induced ATF3 expression. The current study

demonstrates that DIM stimulates ATF3 expression through ATF4-mediated pathway

and subsequently induces apoptosis in human CRC cells.

 

PMID: 22819556 

 

Toxicol Appl Pharmacol. 2012 Sep 15;263(3):345-51. Epub 2012 Jul 16.

 

3,3'-Diindolylmethane, but not indole-3-carbinol, inhibits histone deacetylase

activity in prostate cancer cells.

 

Beaver LM, Yu TW, Sokolowski EI, Williams DE, Dashwood RH, Ho E.

 

Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science

Center, Corvallis, OR 97331, USA; School of Biological and Population Health

Sciences, Oregon State University, 103 Milam Hall, Corvallis, OR 97331, USA.

 

Increased consumption of cruciferous vegetables is associated with a reduced risk

of developing prostate cancer. Indole-3-carbinol (I3C) and 3,3'-diindolylmethane

(DIM) are phytochemicals derived from cruciferous vegetables that have shown

promise in inhibiting prostate cancer in experimental models. Histone deacetylase

(HDAC) inhibition is an emerging target for cancer prevention and therapy. We

sought to examine the effects of I3C and DIM on HDACs in human prostate cancer

cell lines: androgen insensitive PC-3 cells and androgen sensitive LNCaP cells.

I3C modestly inhibited HDAC activity in LNCaP cells by 25% but no inhibition of

HDAC activity was detected in PC-3 cells. In contrast, DIM significantly

inhibited HDAC activity in both cell lines by as much as 66%. Decreases in HDAC

activity correlated with increased expression of p21, a known target of HDAC

inhibitors. DIM treatment caused a significant decrease in the expression of

HDAC2 protein in both cancer cell lines but no significant change in the protein

levels of HDAC1, HDAC3, HDAC4, HDAC6 or HDAC8 was detected. Taken together, these

results show that inhibition of HDAC activity by DIM may contribute to the

phytochemicals' anti-proliferative effects in the prostate. The ability of DIM to

target aberrant epigenetic patterns, in addition to its effects on detoxification

of carcinogens, may make it an effective chemopreventive agent by targeting

multiple stages of prostate carcinogenesis.

 

PMID: 22800507 

 

J Biol Chem. 2012 Aug 17;287(34):28745-54. Epub 2012 Jul 6.

 

Diindolylmethane-mediated Gli1 Protein Suppression Induces Anoikis in Ovarian

Cancer Cells in Vitro and Blocks Tumor Formation Ability in Vivo.

 

Kandala PK, Srivastava SK.

 

From the Department of Biomedical Sciences and Cancer Biology Center, Texas Tech

University Health Sciences Center, Amarillo, Texas 79106.

 

Anoikis is a cell death that occurs due to detachment of a cell from the

extracellular matrix (ECM). Resistance to anoikis is a primary feature of a cell

that undergoes metastasis. In this study for the first time, we demonstrated the

potential role of Gli1 in anoikis resistance. Treatment of various ovarian cancer

cells by different concentrations of diindolylmethane (DIM), an active ingredient

of cruciferous vegetables, reduced the anoikis resistance in a

concentration-dependent manner. Reduction in anoikis resistance was associated

with a decrease in the expression of Gli1 and an increase in the cleavage of

poly(ADP-ribose) polymerase (PARP). Sonic hedgehog (Shh) treatment not only

increased the expression of Gli1, but also blocked anoikis induced by DIM and

abrogated the change in the expression of Gli1 and cleaved PARP by DIM. To

confirm the role of Gli1, hedgehog inhibitor cyclopamine, Gli1 siRNA and

Gli1(-/-) mouse embryonic fibroblasts (MEFs) were used. Cyclopamine treatment

alone significantly reduced anoikis resistance in A2780 and OVCAR-429 cells.

Cyclopamine-mediated reduction in anoikis resistance was associated with reduced

expression of Gli1 and induction of cleaved PARP. Shh treatment blocked

cyclopamine-induced anoikis. Silencing Gli1 expression induced anoikis and

cleavage of PARP in A2780 and OVCAR-429 cells. Furthermore, Gli1(-/-) MEFs were

more sensitive to anoikis compared with Gli1(+/+) MEFs. Our in vivo studies

established that DIM- or cyclopamine-treated ovarian cancer cells under

suspension culture conditions drastically lost their ability of tumor formation

in vivo in mice. Taken together, our results establish that Gli1 is a critical

player in anoikis resistance in ovarian cancer.

 

PMID: 22773833  [PubMed - in process]

 

Oncol Rep. 2012 Sep;28(3):1063-8. doi: 10.3892/or.2012.1877. Epub 2012 Jun 20.

 

Anti-proliferative and pro-apoptotic effects of 3,3'-diindolylmethane in human

cervical cancer cells.

 

Zhu J, Li Y, Guan C, Chen Z.

 

Department of Gynecology, Renmin Hospital of Wuhan University, Wuchang, Wuhan

430060, PR China.

 

The antitumor effects of Indo-3-carbinol (I3C) have been proven in many human

carcinoma cells. However, the roles of 3,3-diindolylmethane (DIM), an important

polymer converted from I3C under pH 5.0-7.0, on the growth and proliferation of

cervical cancer HeLa and SiHa cells still remain unrevealed. In the present

study, we investigated the potential anti-proliferative and pro-apoptotic effects

of DIM on HeLa and SiHa cells. Cell proliferation was detected by Cell Counting

kit-8 and apoptosis was analyzed by flow cytometry. In addition, morphological

changes accompanying cell apoptosis were observed using an inverted microscope

after Hoechst 33258 staining. In addition, expression changes of proteins

involved in the MAPK and PI3K pathways were determined by western blotting. DIM

treatment inhibited the proliferation and induced apoptosis of HeLa and SiHa

cells significantly in a time- and dose-dependent manner. Moreover, SiHa cells

were more sensitive to DIM treatment than HeLa cells (P<0.05). In addition, the

expression of ERK, p38 and p-p38, which are involved in the MAPK pathway, was

downregulated by DIM treatment. Another protein involved in the MAPK pathway,

JNK, was upregulated. Furthermore, DIM treatment significantly suppressed the

expression of Akt, p-Akt, PI3K p110α, PI3K p110β, PI3K class III, GSK3-β, p-PDK1

and p-c-Raf which are involved in the PI3K pathway. These results demonstrate

that DIM exerts antitumor effects on HeLa and SiHa cells through its

anti-proliferative and pro-apoptotic roles, especially for SiHa cells. The

molecular mechanism for these effects may be related to its regulatory effects on

MAPK and PI3K pathway and apoptosis proteins. DIM may be a preventive and

therapeutic agent against cervical cancer.

 

PMID: 22736073

 

Biochem Biophys Res Commun. 2012 Jul 20;424(1):45-51. Epub 2012 Jun 19.

 

Diindolilmethane (DIM) selectively inhibits cancer stem cells.

 

Semov A, Iourtchenco L, Lin Fang L, Shengmin L, Xu Y, Su X, Muyjnek E, Kiselev V,

Alakhov V.

 

Supratek Pharma Inc., Montreal, Quebec, Canada. alexandre.semov@supratek.com

 

Epidemiologic studies repeatedly have shown chemopreventive effects of

cruciferous vegetables. Indole-3-carbinol (I3C) and its metabolite

diindolylmethane (DIM) were identified in these plants as active ingredients and

theirs anti-tumor activities were confirmed in multiple in vitro and in vivo

experiments. Here, we demonstrate that DIM is a selective and potent inhibitor of

cancer stem cells (CSCs). In several cancer cell lines, DIM inhibited tumor

sphere formation at the concentrations 30-300 times lower than concentrations

required for growth inhibition of parental cells cultured as adherent culture. We

also found that treatment with DIM overcomes chemoresistance of CSCs to

cytotoxics, such as paclitaxel, doxorubicin, and SN-38. Pre-treatment of tumor

spheres with DIM before implantation to mice significantly retarded the growth of

primary tumors compared to tumors formed by untreated tumor spheres. The

concentrations of DIM required to suppress CSCs formation are in the close range

to those achievable in human plasma after oral dosing of the compound. Therefore,

DIM can potentially be used in cancer patients, either alone, or in combinations

with existing drugs.

 

PMID: 22727906 

 

Nutr Cancer. 2012 Aug;64(6):879-88. Epub 2012 Jun 20.

 

Modulation of CYP1A1, CYP1A2 and CYP1B1 expression by cabbage juices and indoles

in human breast cell lines.

 

Szaefer H, Licznerska B, Krajka-Kuźniak V, Bartoszek A, Baer-Dubowska W.

 

Department of Pharmaceutical Biochemistry, Poznań University of Medical Sciences,

Poznań, Poland.

 

Epidemiological studies have shown that consumption of cabbage and sauerkraut is

connected with significant reduction of breast cancer incidences. Estrogens are

considered a major breast cancer risk factor and their metabolism by P450 enzymes

substantially contributes to carcinogenic activity. The aim of this study was to

investigate the effect of cabbage and sauerkraut juices of different origin on

the expression profile of the estrogen metabolism key enzymes (CYP1A1, CYP1A2,

CYP1B1) in breast cell lines MCF7, MDA-MB-231, and MCF10A. The effects of cabbage

juices were compared with that exerted by indole-3-carbinol (I3C) and

3,3'-diindolylmethane (DIM). The treatment with cabbage juices or indoles for

72 h affected the expression of CYP1 family genes in cell-type dependent manner.

Their induction was found in all cell lines, but the ratio of CYP1A1 to CYP1B1

was 1.22- to 10.6-fold in favor to CYP1A1 in MCF7 and MCF10A cells. Increased

levels of CYP1A2 in comparison with CYP1B1 were also observed in MCF7 cells. In

contrast, in MDA-MB-231 cells CYP1B1 was preferentially induced. Since the cell

lines investigated differ in invasion capacity, these results support

epidemiological observations and partly explain the mechanism of the

chemopreventive activity of white cabbage products.

 

PMID: 22716309 

 

Drug Discov Ther. 2012 Apr;6(2):94-101.

 

Regulation of Janus-activated kinase-2 (JAK2) by diindolylmethane in ovarian

cancer in vitro and in vivo.

 

Kandala PK, Srivastava SK.

 

Department of Biomedical Sciences and Cancer Biology Center, Texas Tech

University Health Sciences Center, Amarillo, TX, USA.

 

Janus-activated kinase-2 (JAK2) plays an important role in the activation of

signal transducer and activation of transcription 3 (STAT3), which is over

expressed in majority of ovarian tumors. We have reported previously that

diindolylmethane (DIM) induces apoptosis in ovarian cancer cells by inhibiting

STAT3. However, the role of JAK2 in our model was not yet understood and hence

evaluated in this report. SKOV-3 human ovarian cancer cells were used to evaluate

concentration and time dependent effects of DIM. Interleukin 3 (IL-3) and

epidermal growth factor (EGF) were used to activate JAK2. Tumor xenograft studies

were used to determine modulation of JAK2 in vivo. DIM treatment blocked the

phosphorylation of JAK2 at Tyr-1007 in a concentration-dependent manner. In a

time-dependent study, inhibition of JAK2 by DIM was as early as 1 h, which was

followed by the inhibition of STAT3 and survivin. IL-3-induced phosphorylation of

JAK2 and STAT3 was significantly blocked by DIM. IL-3 treatment blocked

DIM-induced apoptosis. EGF treatment resulted in the activation of JAK2 and STAT3

but suppressed by DIM. These results indicate the involvement of cytokines and

growth factors in the activation of JAK2/STAT3 and that DIM suppress their

activation. Furthermore, DIM in combination with cisplatin drastically reduced

the phosphorylation of JAK2 when compared to cisplatin alone. Western blot

analysis of tumors from DIM treated mice showed significant inhibition of JAK2

activation as compared with controls. These findings provide a rationale for

further clinical investigation of DIM for its potential use alone or in

combination with chemotherapy of ovarian cancer.

 

PMID: 22622019 

 

Neuro Endocrinol Lett. 2012;33(2):133-7.

 

An active product of cruciferous vegetables, 3,3'-diindolylmethane, inhibits

invasive properties of extravillous cytotrophoblastic cells.

 

Ribaux P, Irion O, Cohen M.

 

Laboratoire d'Hormonologie, Department of Gynaecology and Obstetrics, HUG,

MaternitÚ, Geneva, Switzerland.

 

OBJECTIVES: During implantation, human trophoblastic cells have to proliferate,

migrate and invade pregnant uterus. A natural product of cruciferous vegetables,

3,3'-diindolylmethane (DIM), is known to induce some stress response genes (such

as glucose-regulated protein 78 kDa (GRP78)) and to have anti-invasive and

pro-apoptotic effects on tumor cells. Therefore, we have investigated the

potential effect of DIM on invasive extravillous cytotrophoblasts (evCTBs) cells.

MATERIALS AND METHODS: evCTBs were purified from first trimester trophoblasts and cultured in presence or not of DIM for 48h. In order to evaluate invasive

properties of cells, they were seeded on collagen-coated insert following boyden

chamber principle and matrix metalloproteinases (MMPs) and GRP78 expression was

evaluated by qPCR.

RESULTS: We showed that DIM decreases (p=0.013) invasive properties of evCTBs. In

parallel, we determined that MMP-2, -7 and -9 which are involved in evCTBs

invasion and known to be regulated by DIM, are not affected by DIM in evCTBs. In

contrast, MMP-1 mRNA is induced (p=0.03) and MMP-12 is decreased (p=0.01) in DIM

treated cells. Moreover, DIM treatment does not affect GRP78 mRNA expression in

evCTBs.

CONCLUSIONS: Collectively, the present results provide evidence that DIM does not

impact evenly on evCTBs and cancer cells.

 

PMID: 22592193

 

J Exp Clin Cancer Res. 2012 May 16;31:46.

 

A selective aryl hydrocarbon receptor modulator 3,3'-Diindolylmethane inhibits

gastric cancer cell growth.

 

Yin XF, Chen J, Mao W, Wang YH, Chen MH.

 

Department of Gastroenterology, the First Affiliated Hospital of Sun Yat-Sen

University, Guangzhou, People's Republic of China.

 

BACKGROUND: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription

factor associated with gastric carcinogenesis. 3,3'-Diindolylmethane (DIM) is a

relatively non-toxic selective AhR modulator. This study was to detect the

effects of DIM on gastric cancer cell growth.

METHODS: Gastric cancer cell SGC7901 was treated with DIM at different

concentrations (0,10,20,30,40,50 μmol/L) with or without an AhR antagonist,

resveratrol. The expression of AhR and Cytochrome P4501A1 (CYP1A1), a classic

target gene of AhR pathway, were detected by RT-PCR and Western blot; cell

viability was measured by MTT assay, and the changes in cell cycle and apoptosis

were analyzed by flow cytometry.

RESULTS: RT-PCR and western-blot showed that with the increase of the

concentration of DIM, AhR protein gradually decreased and CYP1A1 expression

increased, suggesting that DIM activated the AhR pathway and caused the

translocation of AhR from cytoplasm to nucleus. MTT assay indicated that the

viability of SGC7901 cells was significantly decreased in a concentration- and

time-dependent manner after DIM treatment and this could be partially reversed by

resveratrol. Flow cytometry analysis showed that DIM arrested cell cycle in G1

phase and induced cell apoptosis.

CONCLUSION: Selective aryl hydrocarbon receptor modulator 3,3'-Diindolylmethane

inhibits SGC7901 cell proliferation by inducing apoptosis and delaying cell cycle

progression. AhR may be a potential therapeutic target for gastric cancer

treatment.

 

PMID: 22592002

 

Oncotarget. 2012 Apr;3(4):435-49.

 

Regulation of macroautophagy in ovarian cancer cells in vitro and in vivo by

controlling glucose regulatory protein 78 and AMPK.

 

Kandala PK, Srivastava SK.

 

Department of Biomedical Sciences and Cancer Biology Center, Texas Tech

University Health Sciences Center, Amarillo, TX, USA.

 

In this study we show that diindolylmethane (DIM) induces autophagy in ovarian

cancer cells by regulating endoplasmic reticulum (ER) stress and AMPK. Treatment

of SKOV-3, OVCAR-3 and TOV-21G ovarian cancer cells with varying concentrations

of DIM for 24 hours resulted in a concentration dependent induction of autophagy

as measured by flowcytometry. Electron microscopy confirmed the presence of

autophagosomes in DIM treated cells. Western blot analysis showed that DIM

treatment increased the expression of LC3B, a hall mark of autophagy as well as

p62 and Atg 12 proteins that are accumulated during autophagy. Autophagy

inhibitors bafilomycin or chloroquine inhibited DIM induced autophagy.

Furthermore, DIM treatment significantly increased the expression of ER stress

regulators such as Grp78, IRE1 and GADD153. Cycloheximide or ER stress inhibitor

mithramycin not only blocked ER stress proteins that were activated by DIM but

also autophagy. Silencing Grp78 or GADD 153 significantly blocked the expression

of LC3B and p62 indicating that autophagy in our model is mediated by ER stress.

Knocking out LC3B inhibited DIM induced autophagy. DIM treatment increased the

cytosolic calcium levels which lead to the activation of AMPK in our model.

Chelating cytosolic calcium with BAPT-AM abrogated not only the phosphorylation

of AMPK but also prevented DIM induced autophagy. Inhibiting AMPK by a chemical

inhibitor or siRNA blocked the induction of LC3B or p62, indicating that DIM

mediated autophagy requires activation of AMPK. Oral administration of DIM

significantly suppressed SKOV-3 tumor xenografts in nude mice. Activation of ER

stress and autophagy were observed in the tumors of DIM treated mice. Taken

together, these results suggest that induction of autophagy by DIM in ovarian

cancer cells was associated with ER stress and AMPK activation.

 

PMID: 22564965 

 

J Transl Med. 2012 May 1;10:81.

 

Molecular target based combinational therapeutic approaches in thyroid cancer.

 

Rajoria S, Suriano R, George AL, Kamat A, Schantz SP, Geliebter J, Tiwari RK.

 

Department of Microbiology and Immunology, New York Medical College, Valhalla,

New York, 10595, USA. raj_tiwari@nymc.edu.

 

ABSTRACT:BACKGROUND: Thyroid cancer, as with other types of cancer, is dependent

on angiogenesis for its continued growth and development. Interestingly, estrogen

has been shown to contribute to thyroid cancer aggressiveness in vitro, which is

in full support of the observed increased incidence of thyroid cancer in women

over men. Provided that estrogen has been observed to contribute to increased

angiogenesis of estrogen responsive breast cancer, it is conceivable to speculate

that estrogen also contributes to angiogenesis of estrogen responsive thyroid

cancer.

METHODS: In this study, three human thyroid cancer cells (B-CPAP, CGTH-W-1, ML-1)

were treated with estrogen alone or estrogen and anti-estrogens (fulvestrant and

3,3'-diindolylmethane, a natural dietary compound) for 24 hours. The cell culture

media was then added to human umbilical vein endothelial cell (HUVECs) and

assayed for angiogenesis associated events. Vascular endothelial growth factor

(VEGF) levels were also quantified in the conditioned media so as to evaluate if

it is a key player involved in these observations.

RESULTS: Conditioned medium from estrogen treated thyroid cancer cells enhanced

phenotypical changes (proliferation, migration and tubulogenesis) of endothelial

cells typically observed during angiogenesis. These phenotypic changes observed

in HUVECs were determined to be modulated by estrogen induced secretion of VEGF

by the cancer cells. Lastly, we show that VEGF secretion was inhibited by the

anti-estrogens, fulvestrant and 3,3'-diindolylmethane, which resulted in

diminished angiogenesis associated events in HUVECs.

CONCLUSION: Our data establishes estrogen as being a key regulator of VEGF

secretion/expression in thyroid cells which enhances the process of angiogenesis

in thyroid cancer. These findings also suggest the clinical utility of

anti-estrogens as anti-angiogenic compounds to be used as a therapeutic means to

treat thyroid cancer. We also observed that 3,3'-diindolylmethane is a promising

naturally occurring anti-estrogen which can be used as a part of therapeutic

regimen to treat thyroid cancer.

 

PMID: 22548798 

 

PLoS One. 2012;7(4):e34975. Epub 2012 Apr 13.

 

3,3'-Diindolylmethane induces G1 arrest and apoptosis in human acute T-cell

lymphoblastic leukemia cells.

 

Shorey LE, Hagman AM, Williams DE, Ho E, Dashwood RH, Benninghoff AD.

 

Department of Environmental and Molecular Toxicology, Oregon State University,

Corvallis, Oregon, United States of America.

 

Certain bioactive food components, including indole-3-carbinol (I3C) and

3,3'-diindolylmethane (DIM) from cruciferous vegetables, have been shown to

target cellular pathways regulating carcinogenesis. Previously, our laboratory

showed that dietary I3C is an effective transplacental chemopreventive agent in a

dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic

lymphoma. The primary objective of the present study was to extend our

chemoprevention studies in mice to an analogous human neoplasm in cell culture.

Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in

human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL

cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly

reduced cell proliferation and viability at concentrations 8- to 25-fold lower

than the parent compound I3C. DIM (7.5 ÁM) arrested CEM and HSB2 cells at the

G(1) phase of the cell cycle and 15 ÁM DIM significantly increased the percentage

of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein

expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3

(CCND3); DIM also significantly altered expression of eight transcripts related

to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8,

TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure

to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts

in immunodeficient SCID mice, reduced final tumor size by 44% and increased the

apoptotic index compared to control-fed mice. Taken together, our results

demonstrate a potential for therapeutic application of DIM in T-ALL.

 

PMID: 22514694 

 

Mol Carcinog. 2012 Apr 11. doi: 10.1002/mc.21906.

 

Pleiotropic effects of the sirtuin inhibitor sirtinol involves

concentration-dependent modulation of multiple nuclear receptor-mediated pathways

in androgen-responsive prostate cancer cell LNCaP.

 

Wang TT, Schoene NW, Kim EK, Kim YS.

 

Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research

Center, Agricultural Research Service, U.S. Department of Agriculture,

Beltsville, Maryland.

 

Sirtinol is a purported specific inhibitor of the nicotinamide adenine

dinucleotide (NAD)-dependent type III histone deacetylase (also known as

sirtuin). Sirtinol has been used extensively to identify

chemopreventive/chemotherapeutic agents that modulate the sirtuins. However, the

molecular effect of sirtinol other than serving as sirtuin inhibitor in cells is

less clear. The present study addressed this deficiency in the literature. Based

on structural similarity with plant-derived cancer preventive/therapeutic

compounds such as 3', 3'-diindolylmethane, resveratrol, and genistein, we

hypothesized that sirtinol may act on pathways similar to that affected by these

compounds in the human prostate cancer cell LNCaP. We found that treatment of

LNCaP cells with sirtinol led to concentration-dependent effects on multiple

pathways. Sirtinol inhibited LNCaP cell cycle and growth that was correlated with

up-regulation of cyclin-dependent kinase inhibitor 1A mRNA and protein levels.

This effect of sirtinol may due in part to modulation of androgen, estrogen, and

insulin-like growth factor-1 mediated pathways as sirtinol treatment led to

inhibition of mRNA and protein expression of marker genes involved in these

pathways. We also found sirtinol activates aryl hydrocarbon-dependent pathways in

LNCaP cells. The effects of sirtinol were observed at 25 ÁM, a concentration

lower than Ki (38 ÁM) for sirtuin activity. Based on these results we reasoned

that sirtinol exerts pleiotropic effects in cells and that biological effects of

sirtinol may not be due solely to inhibition of sirtuin. ę 2012 Wiley

Periodicals, Inc.

 

PMID: 22495798 

 

PLoS One. 2012;7(3):e33729. Epub 2012 Mar 19.

 

Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the

acquisition of cancer stem cell signatures that are attenuated by Diindolylmethane (DIM).

 

Kong D, Heath E, Chen W, Cher ML, Powell I, Heilbrun L, Li Y, Ali S, Sethi S,

Hassan O, Hwang C, Gupta N, Chitale D, Sakr WA, Menon M, Sarkar FH.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, Michigan, United States of America.

 

The emergence of castrate-resistant prostate cancer (CRPC) contributes to the

high mortality of patients diagnosed with prostate cancer (PCa), which in part

could be attributed to the existence and the emergence of cancer stem cells

(CSCs). Recent studies have shown that deregulated expression of microRNAs

(miRNAs) contributes to the initiation and progression of PCa. Among several

known miRNAs, let-7 family appears to play a key role in the recurrence and

progression of PCa by regulating CSCs; however, the mechanism by which let-7

family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2

(EZH2), a putative target of let-7 family, was demonstrated to control stem cell

function. In this study, we found loss of let-7 family with corresponding

over-expression of EZH2 in human PCa tissue specimens, especially in higher

Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors

decreased EZH2 expression and repressed clonogenic ability and sphere-forming

capacity of PCa cells, which was consistent with inhibition of EZH2 3'UTR

luciferase activity. We also found that the treatment of PCa cells with DIM

up-regulated let-7 and down-regulated EZH2 expression, consistent with

inhibition of self-renewal and clonogenic capacity. Moreover, DIM intervention

in our on-going phase II clinical trial in patients prior to radical

prostatectomy showed upregulation of let-7 consistent with down-regulation of

EZH2 expression in PCa tissue specimens after DIM intervention. These results

suggest that the loss of let-7 mediated increased expression of EZH2 contributes

to PCa aggressiveness, which could be attenuated by DIM treatment, and thus

DIM is likely to have clinical impact.

 

PMID: 22442719 

 

 PLoS One. 2012;7(3):e33011. Epub 2012 Mar 7.

 

Targeting bone remodeling by isoflavone and 3,3'-diindolylmethane in the context

of prostate cancer bone metastasis.

 

Li Y, Kong D, Ahmad A, Bao B, Sarkar FH.

 

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State

University School of Medicine, Detroit, Michigan, United States of America.

 

Prostate cancer (PCa) bone metastases have long been believed to be osteoblastic

because of bone remodeling leading to the formation of new bone. However, recent

studies have shown increased osteolytic activity in the beginning stages of PCa

bone metastases, suggesting that targeting both osteolytic and osteoblastic

mediators would likely inhibit bone remodeling and PCa bone metastasis. In this

study, we found that PCa cells could stimulate differentiation of osteoclasts and

osteoblasts through the up-regulation of RANKL, RUNX2 and osteopontin, promoting

bone remodeling. Interestingly, we found that formulated isoflavone and

3,3'-diindolylmethane (DIM) were able to inhibit the differentiation of

osteoclasts and osteoblasts through the inhibition of cell signal transduction in

RANKL, osteoblastic, and PCa cell signaling. Moreover, we found that isoflavone

and DIM down-regulated the expression of miR-92a, which is known to be

associated with RANKL signaling, EMT and cancer progression. By pathway and

network analysis, we also observed the regulatory effects of isoflavone and

DIM on multiple signaling pathways such as AR/PSA, NKX3-1/Akt/p27, MITF, etc.

Therefore, isoflavone and DIM with their multi-targeted effects could be

useful for the prevention of PCa progression, especially by attenuating bone

metastasis mechanisms.

 

PMID: 22412975

 

PLoS One. 2012;7(2):e31783. Epub 2012 Feb 21.

 

3,3'-Diindolylmethane exhibits antileukemic activity in vitro and in vivo through

a Akt-dependent process.

 

Gao N, Cheng S, Budhraja A, Liu EH, Chen J, Chen D, Yang Z, Luo J, Shi X, Zhang

Z.

 

3,3'-Diindolylmethane (DIM), one of the active products derived from Brassica

plants, is a promising antitumor agent. The present study indicated that DIM

significantly induced apoptosis in U937 human leukemia cells in dose- and

time-dependent manners. These events were also noted in other human leukemia

cells (Jurkat and HL-60) and primary human leukemia cells (AML) but not in normal

bone marrow mononuclear cells. We also found that DIM-induced lethality is

associated with caspases activation, myeloid cell leukemia-1 (Mcl-1)

down-regulation, p21(cip1/waf1) up-regulation, and Akt inactivation accompanied

by c-jun NH2-terminal kinase (JNK) activation. Enforced activation of Akt by a

constitutively active Akt construct prevented DIM-mediated caspase activation,

Mcl-1 down-regulation, JNK activation, and apoptosis. Conversely, DIM lethality

was potentiated by the PI3K inhibitor LY294002. Interruption of the JNK pathway

by pharmacologic or genetic approaches attenuated DIM-induced caspases

activation, Mcl-1 down-regulation, and apoptosis. Lastly, DIM inhibits tumor

growth of mouse U937 xenograft, which was related to induction of apoptosis and

inactivation of Akt, as well as activation of JNK. Collectively, these findings

suggest that DIM induces apoptosis in human leukemia cell lines and primary human

leukemia cells, and exhibits antileukemic activity in vivo through Akt

inactivation and JNK activation.

 

PMID: 22363731

 

In Vivo. 2012 Mar-Apr;26(2):207-11.

 

3,3'-Diindolylmethane increases serum interferon-γ levels in the K14-HPV16

transgenic mouse model for cervical cancer.

 

Sepkovic DW, Raucci L, Stein J, Carlisle AD, Auborn K, Ksieski HB, Nyirenda T,

Bradlow HL.

 

The David and Alice Jurist Institute for Research, Hackensack University Medical

Center, Hackensack, NJ, USA. dsepkovic@humed.com

 

While cervical cancer incidence and mortality rates have declined in the United

States, this cancer represents a worldwide threat. Human papilloma viral

infection causes cervical neoplasia (CIN). 3,3'-Diindolylmethane (DIM) prevents

or inhibits the progression from cervical dysplasia to cancer. The aim of this

study is to determine the most effective dose of DIM given continuously in food,

that significantly increases serum interferon gamma levels (IFN-γ) in the

K14-HPV16 transgenic mouse model for cervical cancer.MATERIALS AND METHODS: Five

doses of DIM in food were administered to the mouse model for 20 weeks. Serum

Interferon gamma (IFN-γ) levels and estrogen metabolite levels were quantified.

RESULTS: At 1000 ppm DIM, serum IFN-γ concentrations were significantly increased

(p<0.0396). The estrogen metabolites were unchanged. IFN-γ concentrations in CIN

free mice and the percentage of CIN free transgenic mice were well correlated

(r=0.88).

DISCUSSION: Significant increases in IFN-γ serum concentrations that correlate

with the percentage of CIN free mice in each group indicate that 1000 ppm of DIM

in food may be the most effective dose for future studies. These results may

eventually lead to new and effective vaccination strategies in women already

infected with the human papilloma virus.

 

PMID: 22351660 

 

Oncol Rep. 2012 May;27(5):1669-73. doi: 10.3892/or.2012.1662. Epub 2012 Jan 27.

 

3,3'-Diindolylmethane suppresses growth of human esophageal squamous cancer cells

by G1 cell cycle arrest.

 

Kim SJ, Lee JS, Kim SM.

 

Department of Physiology, Institute for Medical Sciences, Chonbuk National

University Medical School, Jeonju 561-180, Republic of Korea.

 

3,3'-Diindolylmethane (DIM), an active metabolite of indole-3-carbinol, is

thought to have antitumor effects in experimental animals and induce apoptosis in

various cancer cells. However, the biological functions of DIM in human

esophageal cancer cells are unknown. Thus, the purpose of this study was to

investigate the cytotoxic effects of DIM in human esophageal squamous cell

carcinoma (ESCC) cells to elucidate the molecular mechanism of cell death. Three

human ESCC cell lines (TT, TE-8 and TE-12) were used to test the response to DIM.

MTT, cell cycle and western blot analyses were conducted. DIM significantly

inhibited the proliferation of ESCC cells in a dose- and time-dependent manner.

The percentage of G1 phase cells increased 48 h after being treated with DIM. DIM

also reduced cyclin D1, cyclin E2, cyclin-dependent kinase (CDK) 4 and CDK 6

activities, and increased p15 and p27 levels. Additionally, DIM diminished

pro-caspase-9 protein expression levels and induced increased cleaved poly

(ADP-ribose) polymerase levels. These results indicate that DIM leads to G1 phase

cell cycle arrest and induces apoptosis by activating caspase-9 in ESCC cells.

 

PMID: 22293900

 

Chem Biol Interact. 2012 Feb 5;195(3):224-30. Epub 2012 Jan 24.

 

The dietary phytochemical 3,3'-diindolylmethane induces G2/M arrest and apoptosis

in oral squamous cell carcinoma by modulating Akt-NF-κB, MAPK, and p53 signaling.

 

Weng JR, Bai LY, Chiu CF, Wang YC, Tsai MH.

 

Department of Biological Science and Technology, China Medical University, 91

Hsueh-Shih Road, Taichung 40402, Taiwan. columnster@gmail.com

 

In light of the growing incidence of oral cancer in Taiwan, this study is aimed

at investigating the antitumor activity of 3,3'-diindolylmethane (DIM), an active

metabolite of the phytochemical indole-3-carbinol (I3C), in oral squamous cell

carcinoma (OSCC). DIM exhibited substantially higher antiproliferative potency

than I3C in three OSCC cell lines with IC(50) values in SCC2095, SCC9, and SCC15

cells, respectively, of 22 versus 168μM, 25 versus 176μM, and 29versus 300μM.

Flow cytometric analysis and Comet assay indicated that DIM suppressed the

viability of SCC2095 cells by inducing apoptosis and G2/M arrest. Western blot

analysis of various signaling markers revealed the ability of DIM to target

pathways mediated by Akt, mitogen-activated protein (MAP) kinases, nuclear factor

(NF)-κB, and p53, of which the concerted action underlined its antitumor

efficacy. The concomitant inactivation of Akt and MAP kinases in response to DIM

facilitated the dephosphorylation of the proapoptotic protein Bad at Ser-136 and

Ser-112, respectively. Through endoplasmic reticulum (ER) stress, DIM stimulated

the activation of p53 via Ser-15 phosphorylation, leading to increased expression

of the BH3-only proapoptotic Bcl-2 members Puma and Noxa. Together, these changes

decreased the mitochondrial threshold for apoptosis. G2/M arrest might be

attributable to the suppressive effect of DIM on the expression of cyclin B1 and

cdc25c. As many downstream effectors of the Akt-NF-κB pathway, including glycogen

synthase kinase 3β, IκB kinase α, and cyclooxygenase-2, have been shown to

promote oral tumorigenesis, the ability of DIM to inhibit this signaling axis

underscores its chemopreventive potential in oral cancer.

 

PMID: 22290291

 

BMC Med. 2012 Jan 26;10:9.

 

Diindolylmethane suppresses ovarian cancer growth and potentiates the effect of

cisplatin in tumor mouse model by targeting signal transducer and activator of

transcription 3 (STAT3).

 

Kandala PK, Srivastava SK.

 

Department of Biomedical Sciences and Cancer Biology Center, Texas Tech

University Health Sciences Center, Amarillo, TX 79106, USA.

sanjay.srivastava@ttuhsc.edu.

 

ABSTRACT:BACKGROUND: Signal transducer and activator of transcription 3 (STAT3)

is activated in majority of ovarian tumors and confers resistance to cisplatin

treatment in patients with ovarian cancer. We have reported previously that

diindolylmethane (DIM) inhibits the growth of ovarian cancer cells. However, to

date the exact mechanism by which DIM induces growth suppressive effects has not

been clear. In this report the mode of action of DIM is investigated.

METHODS: Six human ovarian cancer cell lines and an ovarian tumor xenograft

animal model were used to study the effect of diindolylmethane alone or in

combination with cisplatin.

RESULTS: Diindolylmethane treatment induced apoptosis in all six ovarian cancer

cell lines. Phosphorylation of STAT3 at Tyr-705 and Ser-727 was reduced by DIM in

a concentration-dependent manner. In addition, diindolylmethane treatment

inhibited nuclear translocation, DNA binding, and transcriptional activity of

STAT3. Interleukin (IL)-6-induced phosphorylation of STAT3 at Tyr-705 was

significantly blocked by DIM. Overexpression of STAT3 by gene transfection

blocked DIM-induced apoptosis. In addition, DIM treatment reduced the levels of

IL-6 in ovarian cancer cells and in the tumors. DIM treatment also inhibited cell

invasion and angiogenesis by suppressing hypoxia-inducible factor 1α (HIF-1α) and

vascular epithelial growth factor (VEGF). Importantly, diindolylmethane treatment

potentiated the effects of cisplatin in SKOV-3 cells by targeting STAT3. Oral

administration of 3 mg diindolylmethane per day and subsequent administration of

cisplatin substantially inhibited in vivo tumor growth. Western blotting analysis

of tumor lysates indicated increased apoptosis and reduced STAT3 activation.

CONCLUSIONS: These findings provide a rationale for further clinical

investigation of DIM alone or in combination for chemoprevention and/or

chemotherapy of ovarian cancer.

 

PMID: 22280969

 

J Pharmacol Exp Ther. 2012 Apr;341(1):24-32. Epub 2011 Dec 28.

 

Blocking epidermal growth factor receptor activation by 3,3'-diindolylmethane

suppresses ovarian tumor growth in vitro and in vivo.

 

Kandala PK, Wright SE, Srivastava SK.

  

Genetic alterations, including the overexpression of epidermal growth factor

receptor (EGFR) (in approximately 70% of ovarian tumors), play a crucial role in

the signal transduction pathways that regulate key cellular functions, such as

cell survival and proliferation, and are responsible for compromising traditional

chemotherapy. 3,3'-Diindolylmethane (DIM) is an indole compound present in

Brassica vegetables. In our previous studies, we demonstrated that BR-DIM, a

formulated version of DIM, suppressed the growth of ovarian cancer cells by

causing cell cycle arrest and apoptosis. In the present study, we delineated the

mechanism by which DIM suppressed the growth of SKOV-3, OVCAR-3, and TOV-21G

human ovarian cancer cells. DIM treatment caused significant down-regulation of

the constitutive EGFR protein level as well as phosphorylation of EGFR at

Tyr1068, Tyr992, Tyr845, and Tyr1173 in various ovarian cancer cells. To

determine whether DIM suppressed the activation of EGFR by activating

phosphorylation, cells were treated with epidermal growth factor. Epidermal

growth factor treatment significantly blocked the DIM-mediated inhibition of EGFR

activation and apoptosis in both SKOV-3 and OVCAR-3 cells. In addition, DIM

treatment drastically reduced the phosphorylation of mitogen-activated protein

kinase kinase (MEK) and extracellular signal-regulated kinase (ERK), which are

downstream to EGFR, without affecting their protein levels. DIM treatment also

inhibited the kinase activity of ERK, as observed by the down-regulation of

phospho-E twenty-six like transcription factor 1 (p-ELK1) in all three ovarian

cancer cell lines. DIM significantly suppressed the growth of ovarian tumors in

vivo. Tumor growth suppressive effects of DIM in SKOV-3 tumor xenografts were

associated with reduced phosphorylation of EGFR, MEK, and ERK. These results

indicate that DIM induces apoptosis in ovarian cancer cells by inhibiting the

EGFR-ERK pathway in vitro and in vivo.

 

PMID: 22205686

 

Front Biosci (Elite Ed). 2012 Jan 1;4:410-25.

 

Novel targets for detection of cancer and their modulation by chemopreventive

natural compounds.

 

Ahmad A, Sakr WA, Rahman KM.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, MI 48201, USA.

 

Cancer affects the lives of millions of people. Several signaling pathways have

been proposed as therapeutic targets for cancer therapy, and many more continue

to be validated. With the identification and validation of therapeutic targets

comes the question of designing novel strategies to effectively counter such

targets. Natural compounds from dietary sources form the basis of many ancient

medicinal systems. They are pleiotropic i.e. they act on multiple targets, and,

therefore, are often the first agents to be tested against a novel therapeutic

target. This review article summarizes the knowledge so far on some actively

pursued targets - Notch, CXCR4, Wnt and sonic hedgehog (shh) pathways, the

process of epithelial-mesenchymal transition (EMT) as well as molecular markers

such as uPA-uPAR, survivin, FoxM1, and the microRNAs. We have performed an

extensive survey of literature to list modulation of these targets by natural

agents such as curcumin, indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM),

resveratrol, epigallocatechin-3-gallate (EGCG), genistein etc. We believe that

this review will stimulate further research for elucidating and appreciating the

value of these wonderful gifts from nature.

 

PMID: 22201883

 

J Biomed Biotechnol. 2012;2012:256178. Epub 2011 Nov 13.

 

The indolic diet-derivative, 3,3'-diindolylmethane, induced apoptosis in human

colon cancer cells through upregulation of NDRG1.

 

Lerner A, Grafi-Cohen M, Napso T, Azzam N, Fares F.

 

Pediatric Gastroenterology and Nutrition Unit, Carmel Medical Center, Haifa

34362, Israel.

 

N-myc downstream regulated gene-1 participates in carcinogenesis, angiogenesis,

metastases, and anticancer drug resistance. In the present study, we analyzed the

expression pattern of N-myc downstream regulated gene-1 following treatment of

human colonic cancer cell lines; HCT-116 (well differentiated with wild-type p53

gene) and Colo-320 (poorly differentiated with mutant p53 gene), with

3,3'-diindolylmethane, a well-established proapoptotic agent product derived from

indole-3-carbinol. Treatment of Colo-320 and HCT-116 with 3,3'-diindolylmethane

disclosed inhibition of cell viability in a dose-dependent manner, mediated

through apoptosis induction. The increased expression of N-myc downstream

regulated gene-1 was detected only in poorly differentiated colon cancer cells,

Colo-320 cell line. Our results suggest that N-myc downstream regulated gene-1

expression is enhanced by 3,3'-diindolylmethane in poorly differentiated cells

and followed by induction of apoptosis. 3,3'-diindolylmethane induced apoptosis

may represent a new regulator of N-myc downstream regulated gene-1 in poorly

differentiated colonic cancer cells.

 

PMID: 22187533

 

Cancer Lett. 2012 May 1;318(1):86-92. Epub 2011 Dec 9.

 

DIM impairs radiation-induced survival pathways independently of androgen

receptor expression and augments radiation efficacy in prostate cancer.

 

Singh-Gupta V, Banerjee S, Yunker CK, Rakowski JT, Joiner MC, Konski AA, Sarkar

FH, Hillman GG.

 

Department of Radiation Oncology, Barbara Ann Karmanos Cancer Institute, Wayne

State University School of Medicine, Detroit, MI 48201, USA.

 

Increased consumption of cruciferous vegetables is associated with decreased risk

in prostate cancer (PCa). The active compound in cruciferous vegetables appears

to be the self dimerized product [3,3'-diindolylmethane (DIM)] of

indole-3-carbinol (I3C). Nutritional grade DIM has proven

safe in a Phase I trial in PCa. We investigated the anti-cancer activity of DIM

as a new biological approach to improve the effects of radiotherapy for hormone

refractory prostate cancer cells, which were either positive or negative for

androgen receptor (AR) expression. DIM inhibited cell growth in a

dose-dependent manner in both PC-3 (AR-) and C4-2B (AR+) cell lines. DIM was

effective at increasing radiation-induced cell killing in both cell lines,

independently of AR expression. DIM inhibited NF-κB and HIF-1α DNA activities

and blocked radiation-induced activation of these transcription factors in both

PC-3 and C4-2B cells. In C4-2B (AR+) cells, AR expression and nuclear

localization were significantly increased by radiation. However, DIM abrogated

the radiation-induced AR increased expression and trafficking to the nucleus,

which was consistent with decreased PSA secretion. In vivo, treatment of PC-3

prostate tumors in nude mice with DIM and radiation resulted in significant

primary tumor growth inhibition and control of metastasis to para-aortic lymph

nodes. These studies demonstrate that DIM augments radiation-induced cell

killing and tumor growth inhibition. DIM impairs critical survival signaling

pathways activated by radiation, leading to enhanced cell killing. These novel

observations suggest that DIM could be used as a safe compound to enhance the

efficacy of radiotherapy for castrate-resistant PCa.

 

PMID: 22155105

  

Toxicol Lett. 2011 Oct 10;206(2):218-28. Epub 2011 Jul 27.

 

3,3'-Diindolylmethane induces immunotoxicity via splenocyte apoptosis in neonatal

mice.

 

Roh YS, Cho A, Islam MR, Cho SD, Kim J, Kim JH, Lee JW, Lim CW, Kim B.

 

Biosafety Research Institute and Department of Pathology, College of Veterinary

Medicine, Chonbuk National University, Jeonju, South Korea.

 

3,3'-Diindolylmethane (DIM), a major product of indole-3-carbinol derived from

vegetables of the genus Brassica, exhibits chemotherapeutic activity and various

immune modulatory effects in animal models and in vitro studies. Although

extensive studies have only focused on DIM's beneficial effects, the toxic

effects of DIM on the immune systems have not been clearly elucidated. The aim of

this study was to explore the immunotoxic effects of DIM in a neonatal mouse and

to further evaluate whether DIM administration affects rotavirus (RV)-induced

gastroenteritis. Interestingly, multiple immunotoxic effects were observed in the

DIM treated group, including decreases in various immune cells (F4/80(+),

CD11c(+), CD19(+), and CD3(+) cells) in the spleen, induction of splenic white

pulp atrophy, an increase in immune cell apoptosis, and decreased expression of

various toll-like receptors (TLRs) in the spleen and small intestine. Apoptosis

was notably promoted by up-regulating caspase-3 activity and by the change in the

ratio of Bcl-2/Bax activities. Finally, oral administration of DIM led to

deterioration of RV-induced intestinal disease and delayed viral clearance in the

intestine and MLNs. Our results indicate that oral administration of DIM in

neonatal mice induces immunotoxicity and hampers efficient RV clearance in the

intestine. This new information about the immunotoxic roles of DIM in a newborn

mouse model may provide valuable clues for the development of a safe supplement,

especially one designed for human infants.

 

PMID: 21820497

 

Nutr Cancer. 2011;63(5):790-4. Epub 2011 Jun 22.

 

Inhibition of fatty acid synthase and Sp1 expression by 3,3'-diindolylmethane in

human breast cancer cells.

 

Saati GE, Archer MC.

 

Department of Nutritional Sciences, Faculty of Medicine, University of Toronto,

Toronto, Ontario, Canada.

 

The putative cancer preventive agent 3,3'-diindolylmethane (DIM) is formed in the

acidic environment of the stomach following consumption of indole-3-carbinol

(I3C), which is present in vegetables of the Brassica genus. We have recently

shown that the transcription factor Sp1 is involved in the regulation of both

proliferation and de novo lipogenesis in cancer cells. Here we show that DIM

inhibits the proliferation of 3 human breast cancer cell lines, MCF-7,

MDA-MB-231, and SKBr-3, and concomitantly inhibits the expression of Sp1 and

fatty acid synthase (FAS). There were no DIM-related effects on the proliferation

or expression of Sp1 or FAS in the nontumorigenic human breast epithelial cell

line MCF-10A. These results suggest that inhibition of Sp1 and/or FAS expression

could contribute to the anticancer properties of the dietary indoles.

 

PMID: 21767081 

 

Mol Cell Biochem. 2011 Dec;358(1-2):345-54. Epub 2011 Jul 15.

 

3,3'-Diindolylmethane inhibits breast cancer cell growth via miR-21-mediated

Cdc25A degradation.

 

Jin Y.

 

Laboratory of Cell Cycle and Cancer, College of Life Sciences, Capital Normal

University, HaiDian District, Beijing 100048, China. lantianmeiyu1985@gmail.com

 

3,3'-Diindolylmethane (DIM) is a potential cancer preventive phytochemical

derived from Brassica vegetables. The effects of DIM on cell-cycle regulation in

both estrogen-dependent MCF-7 and estrogen receptor negative p53 mutant

MDA-MB-468 human breast cancer cells were assessed in this study. DIM inhibited

the breast cancer cell growth in vitro and in vivo, and caused cell-cycle arrest

by down-regulating protein levels of cell-cycle related kinases CDK1, CDK2, CDK4,

and CDK6, as well as Cyclin B1 and Cdc25A. Meanwhile, it was revealed that

Ser(124) phosphorylation of Cdc25A is primarily responsible for the DIM-induced

Cdc25A degradation. Furthermore, treatment of MCF-7 cells with DIM increased

miR-21 expression and down-regulated Cdc25A, resulting in an inhibition of breast

cancer cell proliferation. These observations collectively suggest that by

differentially modulating cellular signaling pathways DIM is able to arrest the

cell-cycle progression of human breast cancer cells.

 

PMID: 21761201 

 

Mutat Res. 2011 Jul-Oct;728(1-2):47-66. Epub 2011 Jun 15.

 

Attenuation of multi-targeted proliferation-linked signaling by

3,3'-diindolylmethane (DIM): from bench to clinic.

 

Banerjee S, Kong D, Wang Z, Bao B, Hillman GG, Sarkar FH.

  

Emerging evidence provide credible support in favor of the potential role of

bioactive products derived from ingesting cruciferous vegetables such as

broccoli, brussel sprouts, cauliflower and cabbage. Among many compounds,

3,3'-diindolylmethane (DIM) is generated in the acidic environment of the stomach

following dimerization of indole-3-carbinol (I3C) monomers present in these

classes of vegetables. Both I3C and DIM have been investigated for their use in

preventing, inhibiting, and reversing the progression of cancer - as a

chemopreventive agent. In this review, we summarize an updated, wide-ranging

pleiotropic anti-tumor and biological effects elicited by DIM against tumor

cells. It is unfeasible to point one single target as basis of cellular target of

action of DIM. We emphasize key cellular and molecular events that are

effectively modulated in the direction of inducing apoptosis and suppressing cell

proliferation. Collectively, DIM orchestrates signaling through Ah receptor,

NF-κB/Wnt/Akt/mTOR pathways impinging on cell cycle arrest, modulation of key

cytochrome P450 enzymes, altering angiogenesis, invasion, metastasis and

epigenetic behavior of cancer cells. The ability of DIM to selectively induce

tumor cells to undergo apoptosis has been observed in preclinical models, and

thus it has been speculated in improving the therapeutic efficacy of other

anticancer agents that have diverse molecular targets. Consequently, DIM has

moved through preclinical development into Phase I clinical trials, thereby

suggesting that DIM could be a promising and novel agent either alone or as an

adjunct to conventional therapeutics such as chemo-radio and targeted therapies.

An important development has been the availability of DIM formulation with

superior bioavailability for humans. Therefore, DIM appears to be a promising

chemopreventive agent or chemo-radio-sensitizer for the prevention of tumor

recurrence and/or for the treatment of human malignancies.

 

PMID: 21703360

 

Cancer Prev Res (Phila). 2011 Sep;4(9):1495-506. Epub 2011 Jun 16.

 

Inactivation of AR/TMPRSS2-ERG/Wnt signaling networks attenuates the aggressive

behavior of prostate cancer cells.

 

Li Y, Kong D, Wang Z, Ahmad A, Bao B, Padhye S, Sarkar FH.

 

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State

University School of Medicine, Detroit, MI 48201, USA.

 

The development of prostate cancer and its progression to castrate-resistant

prostate cancer (CRPC) after antiandrogen ablation therapy are driven by

persistent biological activity of androgen receptor (AR) signaling. Moreover,

studies have shown that more than 50% of human prostate cancers overexpress ERG

(v-ets avian erythroblastosis virus E26 oncogene related gene) due to

AR-regulated TMPRSS2-ERG fusion gene. However, the reported roles of TMPRSS2-ERG fusion in cancer progression are not clear. In this study, we investigated the

signal transduction in the AR/TMPRSS2-ERG/Wnt signaling network for studying the

aggressive behavior of prostate cancer cells and further assessed the effects of

DIM and CDF [natural agents-derived synthetic formulation and analogue of

3,3'-diindolylmethane (DIM) and curcumin, respectively, with improved

bioavailability] on the regulation of AR/TMPRSS2-ERG/Wnt signaling. We found that

activation of AR resulted in the induction of ERG expression through TMPRSS2-ERG

fusion. Moreover, we found that ERG overexpression and nuclear translocation

activated the activity of Wnt signaling. Furthermore, forced overexpression of

ERG promoted invasive capacity of prostate cancer cells. More important, we found

that DIM and CDF inhibited the signal transduction in the AR/TMPRSS2-ERG/Wnt

signaling network, leading to the inactivation of Wnt signaling consistent with

inhibition of prostate cancer cell invasion. In addition, DIM and CDF

inhibited proliferation of prostate cancer cells and induced apoptotic cell

death. On the basis of our findings, we conclude that because DIM and CDF

downregulate multiple signaling pathways including AR/TMPRSS2-ERG/Wnt signaling,

these agents could be useful for designing novel strategies for the prevention

and/or treatment of prostate cancer.

 

PMID: 21680704

 

Biopharm Drug Dispos. 2011 Jul;32(5):289-300. doi: 10.1002/bdd.759. Epub 2011 Jun 8.

 

Pharmacodynamics of dietary phytochemical indoles I3C and DIM: Induction of

Nrf2-mediated phase II drug metabolizing and antioxidant genes and synergism with

isothiocyanates.

 

Saw CL, Cintrˇn M, Wu TY, Guo Y, Huang Y, Jeong WS, Kong AN.

 

Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State

University of New Jersey, Piscataway, NJ 08854, USA.

 

The antioxidant response element (ARE) is a critical regulatory element for the

expression of many phase II drug metabolizing enzymes (DME), phase III

transporters and antioxidant enzymes, mediated by the transcription factor Nrf2.

The aim of this study was to examine the potential activation and synergism of

Nrf2-ARE-mediated transcriptional activity between four common phytochemicals

present in cruciferous vegetables; the indoles: indole-3-carbinol (I3C),

3,3'-diindolylmethane (DIM); and the isothiocyanates (ITCs): phenethyl

isothiocyanate (PEITC) and sulforaphane (SFN). The cytotoxicity of the compounds

was determined in a human liver hepatoma cell line (HepG2-C8). The combination

index was calculated to assess the synergistic effects on the induction of

ARE-mediated gene expressions. Quantitative real-time polymerase chain reaction

(qPCR) was employed to measure the mRNA expressions of Nrf2 and Nrf2-mediated

genes. I3C and DIM showed less cytotoxicity than SFN and PEITC. Compared with

I3C, DIM was found to be a stronger inducer of ARE. Synergism was observed after

combined treatments of 6.25 Ám I3C + 1 Ám SFN, 6.25 Ám I3C + 1 Ám PEITC and 6.25

Ám DIM + 1 Ám PEITC, while an additive effect was observed for 6.25 Ám DIM + 1 Ám

SFN. Induction of endogenous Nrf2, phase II genes (GSTm2, UGT1A1 and NQO1) and

antioxidant genes (HO-1 and SOD1) was also observed. In summary, the indole I3C

or DIM alone could induce or syngergistically induce in combination with the ITCs

SFN or PEITC, Nrf2-ARE-mediated gene expression, which could potentially enhance

cancer chemopreventive activity.

 

PMID: 21656528

 

J Phys Chem A. 2011 Jul 7;115(26):7700-8. Epub 2011 Jun 7.

 

Dimer radical cations of indole and indole-3-carbinol: localized and delocalized

radical cations of diindolylmethane.

 

Błoch-Mechkour A, Bally T, Marcinek A.

 

Department of Chemistry, University of Fribourg, CH-1700, Fribourg, Switzerland.

 

Extending our previous study on the title species (J. Phys. Chem. A2010, 114,

6787), we investigated the dimer cations that are formed on oxidation of the

glucobrassin derivatives indole-3-carbinol (I3C) and diindolylmethane (DIM) and

of parent indole (I). Radiolysis in ionic liquid and Ar matrices shows that, at

sufficiently high concentrations and/or on annealing the solid glasses, intense

intermolecular charge-resonance (CR) absorption bands in the NIR herald the

formation of sandwich-type dimer cations. The molecular and electronic structure

of these species is modeled by calculations with the double-hybrid B2-PLYP-D

density functional method which yields predictions in good accord with

experiment. The radical cation of DIM also shows a CR band, but unlike in the

case of I and I3C, its occurrence is not dependent on the concentration but

instead on the solvent: in ionic liquid the CR band is initially absent and

arises only on annealing, whereas in Ar matrices it is present from the outset

and undergoes blue shifting and sharpening on annealing. These puzzling findings

are rationalized on the basis of B2-PLYP-D calculations which predict that

neutral DIM exists in the form of two conformers, present in different relative

amounts in the two experiments, which on vertical ionization form distinct

radical cations, a nonsymmetric one where the odd electron is largely localized

on one of the two indole moieties and one with C(2) symmetry where charge and

spin are completely delocalized over both halves of the molecule, thus giving

rise to an intramolecular CR transition. On annealing, the nonsymmetric cation

relaxes to a similarly delocalized structure with C(s) symmetry, thus explaining

the observed increase and the shift of the CR band. We believe that DIM(Ľ+)

represents the first example of a radical cation which can exist under the same

conditions as a localized and a delocalized complex cation.

 

PMID: 21648387

 

Oncol Rep. 2011 Sep;26(3):731-6. doi: 10.3892/or.2011.1316. Epub 2011 May 23.

 

Effects of 5,6-benzoflavone, indole-3-carbinol (I3C) and diindolylmethane (DIM)

on chemically-induced mammary carcinogenesis: is DIM a substitute for I3C?

 

Lubet RA, Heckman BM, De Flora SL, Steele VE, Crowell JA, Juliana MM, Grubbs CJ.

 

The abilities of 5,6-benzoflavone (5,6-BF, a synthetic flavonoid),

indole-3-carbinol (I3C, a plant derived product) or diindolylmethane (DIM, a

condensation product of I3C) to alter the induction of mammary cancers induced by

the carcinogens 7,12-dimethylbenzanthracene (DMBA) or N-methyl-N-nitrosourea

(MNU) were evaluated. Interestingly, the first two agents act as aryl hydrocarbon

receptor (AhR) agonists, while DIM does not. The agents were initially examined

for their ability to inhibit DMBA-induced mammary carcinogenesis. Agents were

administered for 14 days starting 7 days prior to a single dose of the

carcinogen. Evaluated over an extensive range of doses (165, 550 and 1650 ppm in

the diet), 5,6-BF caused a dose-dependent decrease of mammary cancers. In

addition, 5,6-BF at doses of 1650 and 165 ppm in the diet blocked the induction

of DMBA-induced DNA adducts in the mammary gland by approximately 85% and 45%,

respectively. In contrast, DIM (180 or 20 mg/kg BW/day) failed to block induction

of DMBA tumors. The effect of these agents on the promotion/progression phase of

carcinogenesis using the MNU mammary cancer model was also determined. 5,6-BF

(1650 or 165 ppm in the diet), I3C (180 or 60 mg/kg BW/day administered by

gavage), or DIM (180 or 60 mg/kg BW/day by gavage) were initiated 5 days after

the administration of MNU, and continually thereafter. 5,6-BF decreased MNU-

induced mammary tumor multiplicity by 40-60%. I3C reduced tumor multiplicity at

the high dose, while DIM at either dose had minimal effects on tumor

multiplicity. Thus, 5,6-BF and I3C were highly effective against initiation of

DMBA-induced mammary carcinogenesis, and were also effective against MNU-induced

tumors during the promotion/progression phase of carcinogenesis. In contrast, DIM

had minimal effects in either model; arguing that administration of DIM is not

analogous to administration of I3C.

 

PMID: 21617870  [PubMed - indexed for MEDLINE]

 

Free Radic Res. 2011 Aug;45(8):941-9. Epub 2011 May 26.

 

3,3'-Diindolylmethane but not indole-3-carbinol activates Nrf2 and induces Nrf2

target gene expression in cultured murine fibroblasts.

 

Ernst IM, Schuemann C, Wagner AE, Rimbach G.

 

Institute of Human Nutrition and Food Science, Christian-Albrechts-University

Kiel , Hermann-Rodewald-Strasse 6, 24118 Kiel, Germany.

 

There is increasing interest in the gene-regulatory activity of Brassica

vegetable derived phytochemicals such as 3,3'-diindolylmethane (DIM) and

indole-3-carbinol (I3C). DIM is formed under acidic conditions by dimerization of

I3C. This study compared the Nrf2 activating potential of DIM and I3C in murine

fibroblasts (NIH3T3). In contrast to its precursor I3C, DIM induces the

transactivation of Nrf2. Furthermore, Nrf2 targets such as HO-1, γGCS and NQO1

were increased on the mRNA and protein levels following DIM treatment. DIM was

less potent than sulforaphane (used as positive control) in inducing

Nrf2-dependent gene expression. The present data suggest that the dimerization of

I3C to DIM increases its Nrf2 inducing activity.

 

PMID: 21615272

 

Eur J Cancer Prev. 2011 Sep;20(5):417-25.

 

Chemopreventive effects of synthetic C-substituted diindolylmethanes originating

from cruciferous vegetables in human oral cancer cells.

 

Shin JA, Shim JH, Choi ES, Leem DH, Kwon KH, Lee SO, Safe S, Cho NP, Cho SD.

 

Department of Oral Pathology, School of Dentistry, Chonbuk National University,

Republic of Korea.

 

Diindolylmethane (DIM), an isothiocyanate found in cruciferous vegetables, has

been shown to have cancer chemopreventive effects. A series of synthetic

C-substituted DIMs (C-DIMs) analogs was developed, including DIM-C-pPhtBu and

DIM-C-pPhC6H5, which exhibited better inhibitory activity in cancer cells than

DIM. This study examined the effects of C-DIMs on the growth of human oral cancer

cells. DIM-C-pPhtBu and DIM-C-pPhC6H5 decreased the number of viable KB cells and

induced caspase-dependent apoptosis. The apoptotic cell death was accompanied by

a change in Bax/Bcl-2 ratio and damage to mitochondrial membrane potential

through the induction of death receptor 5 and the cleavage of Bid and caspase 8.

Studies on the mechanism of action showed that the apoptotic cell death induced

by DIM-C-pPhtBu and DIM-C-pPhC6H5 was mediated by endoplasmic reticulum stress.

In addition, C-DIMs inhibited cell proliferation and induced PARP cleavage

through death receptor 5 and CHOP in HEp-2 and HN22 cells. This provides the

first evidence that synthetic C-DIMs originating from cruciferous vegetables

induce apoptosis in human oral cancer cells through the endoplasmic reticulum

stress pathway.

 

PMID: 21597379

 

Mol Carcinog. 2011 Apr 22.

Broccoli-derived phytochemicals indole-3-carbinol and 3,3'-diindolylmethane
exerts concentration-dependent pleiotropic effects on prostate cancer cells:
Comparison with other cancer preventive phytochemicals.

Wang TT, Schoene NW, Milner JA, Kim YS.

Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research
Center, Agricultural Research Service, U.S. Department of Agriculture,
Beltsville, Maryland.

In the present studies, we utilized prostate cancer cell culture models to
elucidate the mechanisms of action of broccoli-derived phytochemicals
3,3'-diindolylmethane (DIM) and indole-3-carbinol (I3C). We found DIM and I3C at
1-5 ÁM inhibited androgen and estrogen-mediated pathways and induced xenobiotic
metabolism pathway. By contrast, DIM and I3C induced cyclin inhibitors,
indicators of stress/DNA damage, only at ≥25 ÁM. We also demonstrated that an
inhibitory effect of DIM and I3C on cell growth involves inhibition of
insulin-like growth factor-1 receptor expression. More importantly, we showed
that differences in efficacies and mechanisms existed between DIM and I3C. These
included differences in effective concentrations, a differential effect on
androgen receptor binding, and a differential effect on xenobiotic metabolic
pathway through aryl hydrocarbon receptor-dependent and -independent mechanism.
Furthermore we determined that several other diet-derived cancer protective
compounds, similar to DIM and I3C, exhibited pleiotrophic effects on signaling
pathways that included proliferation, cell cycle, and nuclear receptors-mediated
pathways. However, the efficacies and mechanisms of these compounds vary. We also
showed that some cellular pathways are not likely to be affected by DIM or I3C
when circulating concentration of orally ingested DIM or I3C is considered. Based
on our results, a model for cancer protective effects of DIM and I3C was
proposed.

PMID: 21520295

Cancer Prev Res (Phila). 2011 Mar 7.

Results from a dose response study using 3, 3'-diindolylmethane in the K14-HPV16
transgenic mouse model: Cervical histology.

Sepkovic DW, Stein J, Carlisle AD, Ksieski HB, Auborn K, Raucci L, Nyirenda T,
Bradlow HL.

1Research, Hackensack University Medical Center.

The Human Papilloma Virus is the major cause of cervical cancer. Viral infection
initiates cervical intraepithelial neoplasia which progresses through several
stages to cervical cancer. The objective of this study is to identify the minimum
effective dose of diindolylmethane that prevents the progression from cervical
dysplasia to carcinoma in situ. We document cervical histology in K14-HPV16 mice
receiving different doses of diindolylmethane. Urinary diindolylmethane
concentrations are reported. Diindolylmethane could enhance the efficacy of human
papilloma virus vaccines, creating a new therapeutic use for these vaccines in
women already infected with the virus. Five doses (0 to 2500ppm) of
diindolylmethane were incorporated into each mouse diet. The reproductive tract
was serially sectioned and urine was obtained for analysis of urinary
diindolylmethane. The results indicate that 62% of mice receiving 1,000ppm
diindolylmethane, remained dysplasia free after 20 weeks compared to 16% of mice
receiving no diindolylmethane and 18% receiving 500ppm. 1000ppm of
3,3'-diindolylmethane in the diet completely suppressed the development of
cervical cancer. Urinary diindolylmethane levels increased significantly as
diindolylmethane in food increased. These findings imply usefulness for
diindolylmethane in the search to prevent cervical cancer when used in
combination with prophylactic or therapeutic vaccines.

PMID: 21383027

Carcinogenesis. 2011 Mar 1.

Activation of Nuclear TR3 (Nr4a1) by A Diindolylmethane Analog Induces Apoptosis
and Proapoptotic Genes in Pancreatic Cancer Cells and Tumors.

Yoon K, Lee SO, Cho SD, Kim K, Khan S, Safe S.

Institute of Bioscience and Technology, Texas A&M Health Science Center, 2121 W.
Holcombe Blvd., Houston, TX 77030, USA.

NR4A1 (Nur77, TR3) is overexpressed in pancreatic tumors and activation of TR3 by
1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (DIM-C-pPhOCH(3)) inhibits cell
and tumor growth and induces apoptosis. Microarray analysis demonstrates that in
L3.6pL pancreatic cancer cells DIM-C-pPhOCH(3) induces genes associated with
metabolism, homeostasis, signal transduction, transcription, stress, transport,
immune responses, growth inhibition, and apoptosis. Among the most highly induced
growth inhibitory and proapoptotic genes including activating transcription
factor 3 (ATF3), p21, cystathionase, dual specificity phosphatase 1, and growth
differentiation factor 15, RNA interference studies demonstrated that induction
of all but the later gene by DIM-C-pPhOCH(3) were TR3-dependent. We also observed
that DIM-C-pPhOCH(3) induced Fas ligand and tumor necrosis factor-related
apopotosis-inducing ligand (TRAIL) and induction of TRAIL was ATF3-dependent.
Results of this and previous studies demonstrate that TR3 is unique among nuclear
receptors since nuclear TR3 is activated or deactivated by diindolylmethane
derivatives to induce different apoptotic and growth inhibitory pathways that
inhibit pancreatic cancer cell and tumor growth.

PMID: 21362629

Prostate. 2011 Feb 14. doi: 10.1002/pros.21356.

Antiandrogenic and growth inhibitory effects of ring-substituted analogs of
3,3'-diindolylmethane (Ring-DIMs) in hormone-responsive LNCaP human prostate
cancer cells.

Abdelbaqi K, Lack N, Guns ET, Kotha L, Safe S, Sanderson JT.

INRS-Institut Armand-Frappier, UniversitÚ du QuÚbec, Laval, QC, Canada.

BACKGROUND: Cruciferous vegetables protect against prostate cancer.
Indole-3-carbinol (I3C) and its major metabolite 3,3'-diindolylmethane (DIM),
exhibit antitumor activities in vitro and in vivo. Several synthetic
ring-substituted dihaloDIMs (ring-DIMs) appear to have increased anticancer
activity. METHODS: Inhibition of LNCaP prostate cancer cell growth was measured
by a WST-1 cell viability assay. Cytoplasmic and nuclear proteins were analyzed
by immunoblotting and immunofluorescence. Androgen receptor (AR) activation was
assessed by measuring prostate-specific antigen (PSA) expression and using LNCaP
cells containing human AR and an AR-dependent probasin promoter-green fluorescent
protein (GFP) construct. RESULTS: Like DIM, several ring-substituted dihaloDIM
analogs, namely 4,4'-dibromo-, 4,4'-dichloro-, 7,7'-dibromo-, and
7,7'-dichloroDIM, significantly inhibited DHT-stimulated growth of LNCaP cells at
concentrations ≥1 ÁM. We observed structure-dependent differences for the effects
of the ring-DIMs on AR expression, nuclear AR accumulation and PSA levels in
LNCaP cells after 24 hr. Both 4,4'- and 7,7'-dibromoDIM decreased AR protein and
mRNA levels, whereas 4,4'- and 7,7'-dichloroDIM had minimal effect. All four
dihaloDIMs (10 and 30 ÁM) significantly decreased PSA protein and mRNA levels.
Immuofluorescence studies showed that only the dibromoDIMs increased nuclear
localization of AR. All ring-DIMs caused a concentration-dependent decrease in
fluorescence induced by the synthetic androgen R1881 in LNCaP cells transfected
with wild-type human AR and an androgen-responsive probasin promoter-GFP gene
construct, with potencies up to 10-fold greater than that of DIM. CONCLUSION: The
antiandrogenic effects of ring-DIMs suggest they may form the basis for the
development of novel agents against hormone-sensitive prostate cancer, alone or
in combination with other drugs.


PMID: 21321979

PLoS One. 2011 Jan 18;6(1):e15879.

Estrogen induced metastatic modulators MMP-2 and MMP-9 are targets of
3,3'-diindolylmethane in thyroid cancer.

Rajoria S, Suriano R, George A, Shanmugam A, Schantz SP, Geliebter J, Tiwari RK.

Department of Microbiology and Immunology, New York Medical College, Valhalla,
New York, United States of America.

BACKGROUND: Thyroid cancer is the most common endocrine related cancer with
increasing incidences during the past five years. Current treatments for thyroid
cancer, such as surgery or radioactive iodine therapy, often require patients to
be on lifelong thyroid hormone replacement therapy and given the significant
recurrence rates of thyroid cancer, new preventive modalities are needed. The
present study investigates the property of a natural dietary compound found in
cruciferous vegetables, 3,3'-diindolylmethane (DIM), to target the metastatic
phenotype of thyroid cancer cells through a functional estrogen receptor.
METHODOLOGY/PRINCIPAL FINDINGS: Thyroid cancer cell lines were treated with
estrogen and/or DIM and subjected to in vitro adhesion, migration and invasion
assays to investigate the anti-metastatic and anti-estrogenic effects of DIM. We
observed that DIM inhibits estrogen mediated increase in thyroid cell migration,
adhesion and invasion, which is also supported by ER-α downregulation (siRNA)
studies. Western blot and zymography analyses provided direct evidence for this
DIM mediated inhibition of E(2) enhanced metastasis associated events by virtue
of targeting essential proteolytic enzymes, namely MMP-2 and MMP-9.
CONCLUSION/SIGNIFICANCE: Our data reports for the first time that DIM displays
anti-estrogenic like activity by inhibiting estradiol enhanced thyroid cancer
cell proliferation and in vitro metastasis associated events, namely adhesion,
migration and invasion. Most significantly, MMP-2 and MMP-9, which are known to
promote and enhance metastasis, were determined to be targets of DIM. This
anti-estrogen like property of DIM may lead to the development of a novel
preventive and/or therapeutic dietary supplement for thyroid cancer patients by
targeting progression of the disease.

PMID: 21267453

Thyroid. 2011 Mar;21(3):299-304. Epub 2011 Jan 22.

3,3'-diindolylmethane modulates estrogen metabolism in patients with thyroid
proliferative disease: a pilot study.

Rajoria S, Suriano R, Parmar PS, Wilson YL, Megwalu U, Moscatello A, Bradlow HL,
Sepkovic DW, Geliebter J, Schantz SP, Tiwari RK.

Department of Microbiology and Immunology, New York Medical College, Valhalla,
New York 10595, USA.

BACKGROUND: The incidence of thyroid cancer is four to five times higher in women
than in men, suggesting a role for estrogen (E₂) in the pathogenesis of thyroid
proliferative disease (TPD) that comprises cancer and goiter. The objective of
this study was to investigate the antiestrogenic activity of
3,3'-diindolylmethane (DIM), a bioactive compound derived from cruciferous
vegetables, in patients with TPD.
METHODS: In this limited phase I clinical trial study, patients found to have TPD
were administered 300 mg of DIM per day for 14 days. Patients subsequently
underwent a total or partial thyroidectomy, and tissue, urine, and serum samples
were collected. Pre- and post-DIM serum and urine samples were analyzed for DIM
levels as well as estrogen metabolites. DIM levels were also determined in
thyroid tissue samples.
RESULTS: DIM was detectable in thyroid tissue, serum, and urine of patients after
14 days of supplementation. Urine analyses revealed that DIM modulated estrogen
metabolism in patients with TPD. There was an increase in the ratio of
2-hydroxyestrones (C-2) to 16α-hydroxyestrone (C-16), consistent with
antiestrogenic activity that results in more of C-2 product compared with C-16.
CONCLUSION: Our data suggest that DIM enhances estrogen metabolism in TPD
patients and can potentially serve as an antiestrogenic dietary supplement to
help reduce the risk of developing TPD. The fact that DIM is detected in thyroid
tissue implicates that it can manifest its antiestrogenic activity in situ to
modulate TPD.

PMID: 21254914

Mol Carcinog. 2011 Feb;50(2):100-12. doi: 10.1002/mc.20698. Epub 2010 Nov 8.

3,3'-Diindolylmethane inhibits prostate cancer development in the transgenic
adenocarcinoma mouse prostate model.

Cho HJ, Park SY, Kim EJ, Kim JK, Park JH.

Department of Food Science and Nutrition, Hallym University, Chuncheon, Korea.

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol,
which is present in cruciferous vegetables and has been reported to possess
anti-carcinogenic properties. In the present study, we examined whether DIM
inhibits the development of prostate cancer using the transgenic adenocarcinoma
mouse prostate (TRAMP) model. DIM feeding inhibited prostate carcinogenesis in
TRAMP mice, reduced the number of cells expressing the SV40 large tumor antigen
and proliferating cell nuclear antigen, and increased the number of terminal dUTP
nick-end labeling-positive cells in the dorsolateral lobes of the prostate.
Additionally, DIM feeding reduced the expression of cyclin A, cyclin-dependent
kinase (CDK)2, CDK4, and Bcl-xL, and increased p27 and Bax expression. To assess
the mechanisms by which DIM induces apoptosis, LNCaP and DU145 human prostate
cancer cells were cultured with various concentrations of DIM. DIM induced a
substantial reduction in the numbers of viable cells and induced apoptosis in
LNCaP and DU145 cells. DIM increased the cleavage of caspase-9, -7, -3, and poly
(ADP-ribose) polymerase (PARP). DIM increased mitochondrial membrane permeability
and the translocation of cytochrome c and Smac/Diablo from the mitochondria.
Additionally, DIM induced increases in the levels of cleaved caspase-8, truncated
Bid, Fas, and Fas ligand, and the caspase-8 inhibitor Z-IETD-FMK was shown to
mitigate DIM-induced apoptosis and the cleavage of caspase-3, PARP, and Bid.
These results indicate that DIM inhibits prostate carcinogenesis via induction of
apoptosis and inhibition of cell cycle progression. DIM induces apoptosis in
prostate cancer cells via the mitochondria- and death receptor-mediated pathways.

PMID: 21229607

Expert Opin Ther Targets. 2011 Feb;15(2):195-206. Epub 2011 Jan 5.

Targeting NR4A1 (TR3) in cancer cells and tumors.

Lee SO, Li X, Khan S, Safe S.

Institute of Bioscience and Technology, Texas A&M Health Science Center, 2121 W.
Holcombe Boulevard, Houston, TX 77030, USA.

INTRODUCTION: Nuclear receptor 4A1(NR4A1) (testicular receptor 3 (TR3), nuclear
hormone receptor (Nur)77) is a member of the nuclear receptor superfamily of
transcription factors and is highly expressed in multiple tumor types. RNA
interference studies indicate that NR4A1 exhibits growth-promoting, angiogenic
and prosurvival activity in most cancers. AREAS COVERED: Studies on several
apoptosis-inducing agents that activate nuclear export of NR4A1, which
subsequently forms a mitochondrial NR4A1-bcl-2 complex that induces the intrinsic
pathway for apoptosis are discussed. Cytosporone B and related compounds that
induce NR4A1-dependent apoptosis in cancer cells through both modulation of
nuclear NR4A1 and nuclear export are discussed. A relatively new class of
diindolylmethane analogs (C-DIMs) including
1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (DIM-C-pPhOCH(3)) (NR4A1
activator) and 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH)
(NR4A1 deactivator) are discussed in more detail. These anticancer drugs (C-DIMs)
act strictly through nuclear NR4A1 and induce apoptosis in cancer cells and
tumors. EXPERT OPINION: It is clear that NR4A1 plays an important pro-oncogenic
role in cancer cells and tumors, and there is increasing evidence that this
receptor can be targeted by anticancer drugs that induce cell death via
NR4A1-dependent and -independent pathways. Since many of these compounds exhibit
relatively low toxicity, they represent an important class of mechanism-based
anticancer drugs with excellent potential for clinical applications.

PMID: 21204731

Curr Drug Targets. 2010 Dec 15. [Epub ahead of print]

Cancer Chemoprevention by Targeting the Epigenome.

Huang J, Plass C, Gerhńuser C.

Division Epigenomics and Cancer Risk Factors, German Cancer Research Center,
Heidelberg, Germany. c.gerhauser@dkfz.de.

The term "epigenetics" refers to modifications in gene expression caused by
heritable, but potentially reversible, changes in DNA methylation and chromatin
structure. Given the fact that epigenetic modifications occur early in
carcinogenesis and represent potentially initiating events in cancer development,
they have been identified as promising new targets for prevention strategies. The
present review will give a comprehensive overview of the current literature on
chemopreventive agents and their influence on major epigenetic mechanisms, that
is DNA methylation, histone acetylation and methylation, and microRNAs, both in
vitro and in rodent and human studies, taking into consideration specific
mechanisms of action, target sites, concentrations, methods used for analysis,
and outcome. Chemopreventive agents with reported mechanisms targeting the
epigenome include micronutrients (folate, selenium, retinoic acid, Vit. E),
butyrate, polyphenols (from green tea, apples, coffee, and other dietary
sources), genistein and soy isoflavones, parthenolide, curcumin, ellagitannin,
indol-3-carbinol (I3C) and diindolylmethane (DIM), mahanine, nordihydroguaiaretic
acid (NDGA), lycopene, sulfur-containing compounds from Allium and cruciferous
vegetables (sulforaphane, phenylethyl isothiocyanate (PEITC), phenylhexyl
isothiocyanate (PHI), diallyldisulfide (DADS), allyl mercaptan (AM)), antibiotics
(mithramycin A, apicidin), pharmacological agents (celecoxib, DFMO,
5-aza-2'-deoxycytidine and zebularine), compounds affecting sirtuin activity
(resveratrol, dihydrocoumarin, cambinol), inhibitors of histone acetyl
transferases (anacardic acid, garcinol, ursodeoxycholic acid), and relatively
unexplored modulators of histone lysine methylation (chaetocin, polyamine
analogues, n-3 polyunsaturated fatty acids). Their effects on global DNA
methylation, tumor suppressor genes silenced by promoter methylation, histone
modifications, and miRNAs deregulated during carcinogenesis have potential impact
on multiple mechanisms relevant for chemoprevention, including signal
transduction mediated by nuclear receptors and transcription factors such as
NF-κB, cell cycle progression, cellular differentiation, apoptosis induction,
senescence and others. In vivo studies that demonstrate the functional relevance
of epigenetic mechanisms for chemopreventive efficacy are still limited. Future
research will need to identify best strategies for chemopreventive intervention,
taking into account the importance of epigenetic mechanisms for gene regulation.

PMID: 21158707

Int J Cancer. 2010 Dec 10. [Epub ahead of print]

3, 3'-diindolylmethane enhances taxotere-induced growth inhibition of breast
cancer cells through down-regulation of FoxM1.

Ahmad A, Ali S, Wang Z, Ali AS, Sethi S, Sakr WA, Raz A, Rahman KW.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI.

Emerging evidence suggests that the transcription factor Forkhead Box M1 (FoxM1)
is associated with aggressive human carcinomas, including breast cancer. Since
elevated expression of FoxM1 has been observed in human breast cancers, FoxM1 has
attracted much attention in recent years as a potential target for the prevention
and/or therapeutic intervention in breast cancer. However, no information is
currently available regarding how down-regulation of FoxM1 could be achieved for
breast cancer prevention and therapy. Here we report for the first time that 3,
3'-diindolylmethane (DIM), a non-toxic dietary chemopreventive agent could
effectively down-regulate FoxM1 in various breast cancer cell lines. Using gene
transfection, real-time RT-PCR, Western blotting, invasion and MTT assays, we
found that DIM could enhance Taxotere-induced growth inhibition of breast cancer
cells, and decreased invasive capacity of breast cancer cells was observed after
either treatment alone or the combination. These effects were associated with
down-regulation of FoxM1. We also found that knock-down of FoxM1 expression by
siRNA transfection increased DIM-induced cell growth inhibition, whereas
over-expression of FoxM1 by cDNA transfection attenuated DIM-induced cell growth
inhibition, suggesting the mechanistic role of FoxM1. Most importantly, the
combination treatment significantly inhibited tumor growth in SCID mice, and the
results were correlated with the down-regulation of FoxM1 in tumor remnants. We
conclude that inactivation of FoxM1 and its target genes by DIM could enhance the
therapeutic efficacy of Taxotere in breast cancer, which could be a useful
strategy for the prevention and/or treatment of breast cancer.

PMID: 21154750

Oncol Rep. 2011 Feb;25(2):491-7. doi: 10.3892/or.2010.1076. Epub 2010 Dec 7.

3,3'-diindolylmethane inhibits migration and invasion of human cancer cells
through combined suppression of ERK and AKT pathways.

Rajoria S, Suriano R, Wilson YL, Schantz SP, Moscatello A, Geliebter J, Tiwari
RK.

Department of Microbiology and Immunology, New York Medical College, Valhalla,
New York, NY 10595, USA.

Metastasis of cancer is a multifactorial disease and is the main cause of death
in patients with malignancy. This disease demands treatments which may target
multiple dysregulated cellular pathways in cancer cells. The anti-tumor and
anti-metastatic properties of natural products in cancer prevention have been
confirmed by several epidemiological studies, with cruciferous vegetables being
especially protective against many cancers. In this study, we evaluated the
anti-carcinogenic effects of 3,3'-diindolylmethane (DIM), which is a bioactive
compound present in cruciferous vegetables and a widely used dietary supplement,
on events commonly observed during metastasis using in vitro adhesion, migration
and invasion assays. Our results indicate that DIM inhibits human cancer cell in
vitro proliferation, adhesion, migration and invasion. Western blot analyses show
that this inhibition of cell proliferation by DIM is exerted by combined
suppression of AKT and ERK pathways. Furthermore, DIM also leads to
down-regulation of G1-S cell cycle markers: cyclin D1, cdk6 and cdk4. These
findings may lead to development of a novel preventive and/or therapeutic dietary
supplement for patients diagnosed with cancer or predisposed to developing
certain cancers.

PMID: 21152869

Mol Carcinog. 2010 Nov 8. [Epub ahead of print]

3,3'-diindolylmethane inhibits prostate cancer development in the transgenic
adenocarcinoma mouse prostate model.

Cho HJ, Park SY, Kim EJ, Kim JK, Park JH.

Department of Food Science and Nutrition, Hallym University, Chuncheon, Korea.

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol,
which is present in cruciferous vegetables and has been reported to possess
anti-carcinogenic properties. In the present study, we examined whether DIM
inhibits the development of prostate cancer using the transgenic adenocarcinoma
mouse prostate (TRAMP) model. DIM feeding inhibited prostate carcinogenesis in
TRAMP mice, reduced the number of cells expressing the SV40 large tumor antigen
and proliferating cell nuclear antigen, and increased the number of terminal dUTP
nick-end labeling-positive cells in the dorsolateral lobes of the prostate.
Additionally, DIM feeding reduced the expression of cyclin A, cyclin-dependent
kinase (CDK)2, CDK4, and Bcl-xL, and increased p27 and Bax expression. To assess
the mechanisms by which DIM induces apoptosis, LNCaP and DU145 human prostate
cancer cells were cultured with various concentrations of DIM. DIM induced a
substantial reduction in the numbers of viable cells and induced apoptosis in
LNCaP and DU145 cells. DIM increased the cleavage of caspase-9, -7, -3, and poly
(ADP-ribose) polymerase (PARP). DIM increased mitochondrial membrane permeability
and the translocation of cytochrome c and Smac/Diablo from the mitochondria.
Additionally, DIM induced increases in the levels of cleaved caspase-8, truncated
Bid, Fas, and Fas ligand, and the caspase-8 inhibitor Z-IETD-FMK was shown to
mitigate DIM-induced apoptosis and the cleavage of caspase-3, PARP, and Bid.
These results indicate that DIM inhibits prostate carcinogenesis via induction of
apoptosis and inhibition of cell cycle progression. DIM induces apoptosis in
prostate cancer cells via the mitochondria- and death receptor-mediated pathways.

PMID: 21061271

Free Radic Biol Med. 2011 Jan 15;50(2):228-36. Epub 2010 Oct 26.

3,3'-Diindolylmethane decreases VCAM-1 expression and alleviates experimental
colitis via a BRCA1-dependent antioxidant pathway.

Huang Z, Zuo L, Zhang Z, Liu J, Chen J, Dong L, Zhang J.

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing, People's Republic of China.

Reactive oxygen species (ROS) exhibit a key role in the pathogenesis of
inflammatory bowel disease (IBD). 3,3'-Diindolylmethane (DIM) can protect against
oxidative stress in a breast cancer susceptibility gene 1 (BRCA1)-dependent
manner. The aim of this study was to examine the therapeutic effects of DIM in
experimental colitis and investigate the possible mechanisms underlying its
effects on intestinal inflammation. The therapeutic effects of DIM were studied
in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Pathological
markers of colitis severity, antioxidant activity, and ROS generation in colonic
tissue were measured. The impact of DIM on ROS-induced endothelial vascular cell
adhesion molecule 1 (VCAM-1) expression and leukocyte-endothelial cell
interaction was further investigated in cultures of endothelial cells and in the
TNBS-induced colitis model. Administration of DIM was demonstrated to attenuate
experimental colitis, as judged by pathological indices. DIM could effectively
stimulate the expression of BRCA1 in vitro and in vivo and reduce ROS generation,
leading to the inhibition of VCAM-1 expression and leukocyte-endothelial cell
adhesion, and finally resulted in an alleviation of experimental colitis. DIM has
shown anti-IBD activity in animal models by inhibiting ROS-induced VCAM-1
expression and leukocyte recruitment via a BRCA1-dependent antioxidant pathway
and thus may offer potential treatments for IBD patients.

PMID: 21034812

Am J Transl Res. 2010 Jul 23;2(4):402-11.

A phase I dose-escalation study of oral DIM (3,3'-Diindolylmethane) in

castrate-resistant, non-metastatic prostate cancer.

Heath EI, Heilbrun LK, Li J, Vaishampayan U, Harper F, Pemberton P, Sarkar FH.

Karmanos Cancer Institute, Wayne State University Detroit, MI, USA.

3, 3'-diindolylmethane (DIM) modulates estrogen metabolism and acts as an
anti-androgen which down-regulates the androgen receptor and prostate specific
antigen (PSA). We conducted a dose-escalation, phase I study of DIM with

objectives to determine the maximum tolerated dose (MTD), toxicity
profile, and phar-macokinetics (PK) of DIM, and to assess its effects on serum
PSA and quality of life (QoL).PATIENTS AND METHODS: Cohorts of 3-6 patients
received escalating doses of twice daily oral DIM providing DIM at 75 mg, then
150 mg, 225 mg, and 300 mg. Toxicity was evaluated monthly. Serum PSA and QoL
were measured at baseline, monthly during treatment, and at end of study.
RESULTS: 12 patients with castrate-resistant, non-metastatic, PSA relapse
prostate cancer were treated over 4 dose cohorts; 2 patients (at 150 mg and 225
mg, respectively) underwent intra-patient dose escalation, by one dose level.
After oral administration of the first dose of DIM, the plasma exposure to DIM
appeared dose proportional at doses ranging from 75 to 300 mg, with the mean
C(max) and mean AUC(last) increasing from 41.6 to 236.4 ng/ml and from 192.0 to
899.0 ng/ml*h, respectively. Continued relatively stable systemic exposure to DIM
was achieved following twice daily oral administration of DIM. Minimal
toxicity was observed. Two of the four patients treated at 300 mg had grade 3
asymptomatic hyponatremia (AH) discovered on routine blood work. The other 2
patients at this dose had no AH. Therefore, the maximum tolerated dose (MTD) was
deemed to be 300 mgand the recommended phase II dose (RP2D) of DIM was 225 mg
twice daily. One patient without AH at 225 mg experienced a 50% PSA decline. One
patient with DIM dose of 225 mg had PSA stabilization. The other 10 patients
had an initial deceleration of their PSA rise (decrease in slope), but eventually
progressed based on continual PSA rise or evidence of metastatic disease. Ten
patients completed monthly QoL reports for a mean of 6 months (range: 1-13). QoL
measures emotional functioning may have held up somewhat better over time than
their physical functioning.


CONCLUSION: DIM was well tolerated. Increasing systemic exposure to DIM was
achieved with the increase of DIM dose. Modest efficacy was demonstrated.
Patients' QoL varied over time with length of treatment. Phase II studies are
recommended at the dose of 225 mg orally twice daily.

PMID: 20733950

Anticancer Drugs. 2010 Oct;21(9):814-22.

3,3'-Diindolylmethane negatively regulates Cdc25A and induces a G2/M arrest by
modulation of microRNA 21 in human breast cancer cells.

Jin Y, Zou X, Feng X.

Capital Normal University, Beijing, China. lantianmeiyu1985@gmail.com

3,3'-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived
from Brassica vegetables. In this study, we assessed the effects of DIM on cell
cycle regulation in both estrogen-dependent MCF-7 and estrogen receptor negative
p53 mutant MDA-MB-468 human breast cancer cells. In-vitro culture studies showed
that DIM dose dependently inhibited the proliferation of both cells. In addition,
in-vivo xenograft model showed that DIM strongly inhibited the development of
human breast tumors. Fluorescence activated cell sorter analysis showed a
DIM-mediated G2/M cell cycle arrest in MCF-7 and MDA-MB-468 cells. Western blot
analysis showed that DIM downregulated the expression of cyclin-dependent kinases
2 and 4 and Cdc25A, which plays an important role in G2/M phase. Furthermore,
treatment of MCF-7 cells with DIM, which increased microRNA 21 expression, caused
a downregulation of Cdc25A, resulting in an inhibition of breast cancer cell
proliferation. Taken together, our data show that DIM is able to stop the cell
cycle progression of human breast cancer cells regardless of their
estrogen-dependence and p53 status, by differentially modulating cell cycle
regulatory pathways. The modulation of microRNA 21 mediates the DIM cell cycle
regulator effect in MCF-7 cells.

PMID: 20724916

Cancer Metastasis Rev. 2010 Sep;29(3):383-94.

The role of nutraceuticals in the regulation of Wnt and Hedgehog signaling in
cancer.

Sarkar FH, Li Y, Wang Z, Kong D.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, 740 HWCRC, 4100 John R Street, Detroit, MI 48201,
USA.

Multiple cellular signaling pathways have been involved in the processes of
cancer cell invasion and metastasis. Among many signaling pathways, Wnt and
Hedgehog (Hh) signaling pathways are critically involved in embryonic
development, in the biology of cancer stem cells (CSCs) and in the acquisition of
epithelial to mesenchymal transition (EMT), and thus this article will remain
focused on Wnt and Hh signaling. Since CSCs and EMT are also known to be
responsible for cancer cell invasion and metastasis, the Wnt and Hedgehog
signaling pathways are also intimately associated with cancer invasion and
metastasis. Emerging evidence suggests the beneficial role of chemopreventive
agents commonly known as nutraceutical in cancer. Among many such agents, soy
isoflavones, curcumin, green tea polyphenols, 3,3'-diindolylmethane, resveratrol,
lycopene, vitamin D, etc. have been found to prevent, reverse, or delay the
carcinogenic process. Interestingly, these agents have also shown to prevent or
delay the progression of cancer, which could in part be due to their ability to
attack CSCs or EMT-type cells by attenuating the Wnt and Hedgehog signaling
pathways. In this review, we summarize the current state of our knowledge on the
role of Wnt and Hedgehog signaling pathways, and their targeted inactivation by
chemopreventive agents (nutraceuticals) for the prevention of tumor progression
and/or treatment of human malignancies.

PMID: 20711635

 

Mol Carcinog. 2010 Jul;49(7):672-83.
 
 3,3'-diindolylmethane suppresses 12-O-tetradecanoylphorbol-13-acetate-induced
 inflammation and tumor promotion in mouse skin via the downregulation of
 inflammatory mediators.
 
 Kim EJ, Park H, Kim J, Park JH.
 
 Center for Efficacy Assessment and Development of Functional Foods and Drugs,
 Hallym University, Chuncheon, Korea.
 
 3,3'-Diindolylmethane (DIM) is a major acid-condensation product of
 indole-3-carbinol and is present in cruciferous vegetables. In this study, we
 evaluated the effects of DIM on antiinflammatory and antitumor promotion activity
 in mouse skin and explored the relevant mechanisms. When
 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied topically to the mouse ear
 to induce inflammation, DIM pretreatment effectively inhibited TPA-induced ear
 edema formation. To evaluate the mechanisms underlying DIM's antiinflammatory
 effects, DIM was topically treated to the shaved backs of mice 30 min before TPA
 treatment. DIM inhibited the TPA-induced increases in the expression of
 cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), chemokine (C-X-C
 motif) ligand (CXCL) 5, and interleukin (IL)-6 in mouse skin. DIM also inhibited
 nuclear factor-kappa B (NF-kappaB)'s DNA binding activity, the nuclear
 translocation of p65, and the degradation of inhibitor of kappaB (IkappaB) alpha
 in TPA-stimulated mouse skin. Furthermore, DIM reduced TPA-induced increases in
 the activity of extracellular signal regulated protein kinase (ERK)-1/2 and
 IkappaB kinase (IKK). When mouse skin papillomas were initiated via the topical
 application of 7,12-dimethylbenz[alpha]anthracene (DMBA) and promoted with
 repeated topical applications of TPA, repeated topical applications of DIM prior
 to each TPA treatment significantly suppressed the incidence and multiplicity of
 the papillomas. DIM also reduced the expression of COX-2 and iNOS, ERK
 phosphorylation, and the nuclear translocation of p65 in papillomas.
 Collectively, these results show that DIM exerts antiinflammatory and
 chemopreventive effects in mouse skin via the downregulation of COX-2, iNOS,
 CXCL5, and IL-6 expression, which may be mediated by reductions in NF-kappaB
 activation.
 
 PMID: 20564344

 

Pharm Res. 2010 Jun;27(6):1027-41.

Regulation of microRNAs by natural agents: an emerging field in chemoprevention
and chemotherapy research.

Li Y, Kong D, Wang Z, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, Michigan 48201, USA.

In recent years, microRNAs have received greater attention in cancer research.
These small, non-coding RNAs could inhibit target gene expression by binding to
the 3' untranslated region of target mRNA, resulting in either mRNA degradation
or inhibition of translation. miRNAs play important roles in many normal
biological processes; however, studies have also shown that aberrant miRNA
expression is correlated with the development and progression of cancers. The
miRNAs could have oncogenic or tumor suppressor activities. Moreover, some miRNAs
could regulate formation of cancer stem cells and epithelial-mesenchymal
transition phenotype of cancer cells which are typically drug resistant.
Furthermore, miRNAs could be used as biomarkers for diagnosis and prognosis, and
thus miRNAs are becoming emerging targets for cancer therapy. Recent studies have
shown that natural agents including curcumin, isoflavone, indole-3-carbinol,
3,3'-diindolylmethane, (-)-epigallocatechin-3-gallate, resveratrol, etc. could
alter miRNA expression profiles, leading to the inhibition of cancer cell growth,
induction of apoptosis, reversal of epithelial-mesenchymal transition, or
enhancement of efficacy of conventional cancer therapeutics. These emerging
results clearly suggest that specific targeting of miRNAs by natural agents could
open newer avenues for complete eradication of tumors by killing the
drug-resistant cells to improve survival outcome in patients diagnosed with
malignancies.

PMID: 20306121

 

Drug Resist Update. 2010 Jun;13(3):57-66.

Implication of microRNAs in drug resistance for designing novel cancer therapy.

Sarkar FH, Li Y, Wang Z, Kong D, Ali S.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, MI 48201, USA.

Recently, microRNAs (miRNAs) have received increasing attention in the field of
cancer research. miRNAs play important roles in many normal biological processes;
however, the aberrant miRNA expression and its correlation with the development
and progression of cancers is an emerging field. Therefore, miRNAs could be used
as biomarkers for diagnosis of cancer and prediction of prognosis. Importantly,
some miRNAs could regulate the formation of cancer stem cells and the acquisition
of epithelial-mesenchymal transition, which are critically associated with drug
resistance. Moreover, some miRNAs could target genes related to drug sensitivity,
resulting in the altered sensitivity of cancer cells to anti-cancer drugs.
Emerging evidences have also shown that knock-down or re-expression of specific
miRNAs by synthetic anti-sense oligonucleotides or pre-miRNAs could induce drug
sensitivity, leading to increased inhibition of cancer cell growth, invasion, and
metastasis. More importantly, recent studies have shown that natural agents
including isoflavone, 3,3'-diindolylmethane, and (-)-epigallocatechin-3-gallate
altered miRNA expression profiles, leading to an increased sensitivity of cancer
cells to conventional therapeutics. These emerging results suggest that specific
targeting of miRNAs by different approaches could open new avenues for cancer
treatment through overcoming drug resistance and thereby improve the outcome of
cancer therapy.

PMID: 20236855

 

Curr Drug Targets. 2010 Jun 1;11(6):652-66.

Anticancer properties of indole compounds: mechanism of apoptosis induction and
role in chemotherapy.

Ahmad A, Sakr WA, Rahman KM.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI 48201, USA.

Indole compounds, obtained from cruciferous vegetables, have been investigated
for their putative anti-cancer properties. Studies with indole-3-carbinol (I3C)
and its dimeric product, 3, 3' diindolylmethane (DIM), have indicated efficacy of
these compounds against a number of human cancers. Available as well as emerging
data suggests that these compounds act on a number of cellular signaling pathways
leading to their observed biological effects. Such pleiotropic effects of these
compounds are also considered crucial for their chemosensitization activity
wherein they help reduce the toxicity and resistance against conventional
chemotherapeutic drugs. These observations have major clinical implications
especially in chemotherapy. Through this review, we have attempted to update
current understanding on the state of anti-cancer research involving indole
compounds. We have also summarized the available literature on modulatory effects
of indoles on molecular targets such as survivin, uPA/uPAR and signaling pathways
such as the NF-kappaB pathway, which are important for the apoptosis-inducing and
chemosensitizing properties of these compounds.

PMID: 20298156

 

J Cell Biochem. 2010 May;110(1):171-81.

Concurrent inhibition of NF-kappaB, cyclooxygenase-2, and epidermal growth factor
receptor leads to greater anti-tumor activity in pancreatic cancer.

Ali S, Banerjee S, Schaffert JM, El-Rayes BF, Philip PA, Sarkar FH.

Division of Hematology/Oncology, Karmanos Cancer Institute, Wayne State
University, Detroit, Michigan 48201, USA.

Inactivation of survival pathways such as NF-kappaB, cyclooxygenase (COX-2), or
epidermal growth factor receptor (EGFR) signaling individually may not be
sufficient for the treatment of advanced pancreatic cancer (PC) as suggested by
recent clinical trials. 3,3'-Diindolylmethane (DIM) is an inhibitor of
NF-kappaB and COX-2 and is a well-known chemopreventive agent. We hypothesized
that the inhibition of NF-kappaB and COX-2 by DIM concurrently with the
inhibition of EGFR by erlotinib will potentiate the anti-tumor effects of
cytotoxic drug gemcitabine, which has been tested both in vitro and in vivo.
Inhibition of viable cells in seven PC cell lines treated with DIM, erlotinib,
or gemcitabine alone or their combinations was evaluated using
3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Significant inhibition in cell viability was observed in PC cells expressing high
levels of COX-2, EGFR, and NF-kappaB proteins. The observed inhibition was
associated with an increase in apoptosis as assessed by ELISA. A significant
down-regulation in the expression of COX-2, NF-kappaB, and EGFR in BxPC-3,
COLO-357, and HPAC cells was observed, suggesting that simultaneous targeting of
EGFR, NF-kappaB, and COX-2 is more effective than targeting either signaling
pathway separately. Our in vitro results were further supported by in vivo
studies showing that DIM in combination with erlotinib and gemcitabine was
significantly more effective than individual agents. Based on our preclinical in
vitro and in vivo results, we conclude that this multi-targeted combination could
be developed for the treatment of PC patients whose tumors express high levels of
COX-2, EGFR, and NF-kappaB.

PMID: 20213764

Mol Pharmacol. 2010 May 5.

Activation of Chk2 by 3,3' Diinolylmethane is required for causing G2/M cell
cycle arrest in human ovarian cancer cells.

Kandala PK, Srivastava SK.

Texas Tech University Health Sciences Center.

We evaluated the effect of 3,3'-diindolylmethane (DIM) in ovarian cancer cells.
DIM treatment inhibited the growth of SKOV-3, TOV-21G and OVCAR-3 ovarian cancer
cells in both dose and time-dependent manner with effective concentrations
ranging from 40muM to 100muM. Growth inhibitory effects of DIM were mediated by
cell cycle arrest in G2/M phase in all the three cell lines. G2/M arrest was
associated with DNA damage as indicated by phosphorylation of H(2)A.X at Ser 139
and activation of Chk2 in all the three cell lines. Other G2/M regulatory
molecules such as Cdc25C, Cdk1, Cyclin B1 were downregulated by DIM.
Cycloheximide or Chk2 inhibitor pretreatment abrogated not only activation of
Chk2 but also G2/M arrest and apoptosis mediated by DIM. To further establish the
involvement of Chk2 in DIM-mediated G2/M arrest, cells were transfected with
dominant negative Chk2 (DN-Chk2). Blocking Chk2 activation by DN-Chk2 completely
protected cells from DIM- mediated G2/M arrest. These results were further
confirmed in Chk2 knock out DT40 lymphoma cells where DIM failed to cause cell
cycle arrest. These results clearly indicate the requirement of Chk2 activation
to cause G2/M arrest by DIM in ovarian cancer cells. Moreover, blocking Chk2
activation also abrogates the apoptosis-inducing effects of DIM. Further our
results show that DIM treatment cause ROS generation. Blocking ROS generation by
NAC protect the cells from DIM- mediated G2/M arrest and apoptosis. Our results
establish Chk2 as a potent molecular target of DIM in ovarian cancer cells and
provide the rationale for further clinical investigation of DIM.

PMID: 20444961

Cancer Lett. 2010 Mar 16.

3,3'-Diindolylmethane (DIM) inhibits the growth and invasion of drug-resistant
human cancer cells expressing EGFR mutants.

Rahimi M, Huang KL, Tang CK.

Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown
University Medical Center, Washington, DC 20057, United States.

Epidermal Growth Factor Receptor (EGFR) mutants are associated with resistance to
chemotherapy, radiation, and targeted therapies. Here we found that the
phytochemical 3,3'-Diindolylmethane (DIM) can inhibit the growth and also the
invasion of breast cancer, glioma, and non-small cell lung cancer cells
regardless of which EGFR mutant is expressed and the drug-resistant phenotype.
DIM reduced an array of growth factor signaling pathways and altered cell cycle
regulators and apoptotic proteins favoring cell cycle arrest and apoptosis.
Therefore, DIM may be used in treatment regimens to inhibit cancer cell growth
and invasion, and potentially overcome EGFR mutant-associated drug resistance.

PMID: 20299148
 

Endocrinology. 2010 Apr;151(4):1662-7.

Selective activation of estrogen receptor-beta target genes by
3,3'-diindolylmethane.

Vivar OI, Saunier EF, Leitman DC, Firestone GL, Bjeldanes LF.

Department of Nutritional Science and Toxicology, University of California,
Berkeley, Berkeley, California 94720-3104, USA.

3,3'-Diindolylmethane (DIM) is a natural compound found in cruciferous vegetables
that has antiproliferative and estrogenic activity. However, it is not clear
whether the estrogenic effects are mediated through estrogen receptor (ER)alpha,
ERbeta, or both ER subtypes. We investigated whether DIM has ER subtype
selectivity on gene transcription. DIM stimulated ERbeta but not ERalpha
activation of an estrogen response element upstream of the luciferase reporter
gene. DIM also selectively activated multiple endogenous genes through ERbeta.
DIM did not bind to ERbeta, indicating that it activates genes by a
ligand-independent mechanism. DIM causes ERbeta to bind regulatory elements and
recruit the steroid receptor coactivator (SRC)-2 coactivator, which leads to the
activation of ER target genes. Silencing of SRC-2 inhibited the activation of ER
target genes, demonstrating that SRC-2 is required for transcriptional activation
by DIM. Our results demonstrate that DIM is a new class of ERbeta-selective
compounds, because it does not bind to ERbeta, but instead it selectively
recruits ERbeta and coactivators to target genes.

PMID: 20160136

Cancer Res. 2010 Feb 15;70(4):1486-95.

MiR-146a suppresses invasion of pancreatic cancer cells.

Li Y, Vandenboom TG 2nd, Wang Z, Kong D, Ali S, Philip PA, Sarkar FH.

Departments of Pathology and Internal Medicine, Barbara Ann Karmanos Cancer
Institute, Wayne State University School of Medicine, Detroit, Michigan 48201,
USA.

The aggressive course of pancreatic cancer is believed to reflect its unusually
invasive and metastatic nature, which is associated with epidermal growth factor
receptor (EGFR) overexpression and NF-kappaB activation. MicroRNAs (miRNA) have
been implicated in the regulation of various pathobiological processes in cancer,
including metastasis in pancreatic cancer and in other human malignancies. In
this study, we report lower expression of miR-146a in pancreatic cancer cells
compared with normal human pancreatic duct epithelial cells. Reexpression of
miR-146a inhibited the invasive capacity of pancreatic cancer cells with
concomitant downregulation of EGFR and the NF-kappaB regulatory kinase
interleukin 1 receptor-associated kinase 1 (IRAK-1). Cellular mechanism studies
revealed crosstalk between EGFR, IRAK-1, IkappaBalpha, NF-kappaB, and MTA-2, a
transcription factor that regulates metastasis. Treatment of pancreatic cancer
cells with the natural products 3,3'-diinodolylmethane (DIM) or isoflavone, which
increased miR-146a expression, caused a downregulation of EGFR, MTA-2, IRAK-1,
and NF-kappaB, resulting in an inhibition of pancreatic cancer cell invasion. Our
findings reveal DIM and isoflavone as nontoxic activators of a miRNA that can
block pancreatic cancer cell invasion and metastasis, offering starting points to
design novel anticancer agents.

PMID: 20124483

 

Cancer Res. 2010 Jan 12.

Chemopreventive Agent 3,3'-Diindolylmethane Selectively Induces Proteasomal
Degradation of Class I Histone Deacetylases.

Li Y, Li X, Guo B.

Authors' Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy,
North Dakota State University, Fargo, North Dakota.

3,3'-Diindolylmethane (DIM) is an anticancer agent that induces cell cycle arrest

and apoptosis. Here, we report that DIM can selectively induce proteasome-mediated

degradation of class I histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC8)

without affecting the class II HDAC proteins. DIM induced downregulation of class I

HDACs in human colon cancer cells in vitro and in vivo in tumor xenografts. HDAC

depletion relieved HDAC-mediated transcriptional inhibition of the cyclin-dependent

kinase inhibitors p21WAF1 and p27KIP2, significantly increasing their expression and

triggering cell cycle arrest in the G(2) phase of the cell cycle. Additionally, HDAC

depletion was associated with an induction of DNA damage that triggered apoptosis. Our

findings indicate that DIM acts to selectively target the degradation of class I HDACs.
Cancer Res; 70(2); 646-54.


PMID: 20068155

Eur J Cancer Prev.

The potential efficacy of 3,3'-diindolylmethane in prevention of prostate cancer
development.

Fares F, Azzam N, Appel B, Fares B, Stein A.

 

Department of Biology, Faculty of Science and Science Education, University of
Haifa bDepartment of Molecular Genetics, Carmel Medical Center cDepartment of
Urology, Carmel Medical Center, Faculty of Medicine, Technion-Israel Institute of
Technology, Haifa, Israel.

The objective of this study was to examine the efficacy of 3,3'-diindolylmethane
(DIM) in prevention of prostate cancer tumor development in an animal model.
Mouse prostate cancer cells (TRAMP-C2, 2x10) were injected subcutaneously into
three groups of C57BL/6 mice (10 mice in each group). Two groups were treated
earlier with DIM; 2 or 10 mg/kg each, and an additional control group was
injected with medium. Animals were treated for five more weeks until sacrificed.
Tumor sizes were measured biweekly. At the end of the experiment, mice were
sacrificed, and tumors were excised, weighed, measured and tested using
immunohistochemical studies. In addition blood samples were collected for
biochemical analysis. The results indicated that DIM significantly reduced tumor
development in treated animals when compared with controls. Tumors developed in
80% of controls and 40% and 60% of animals treated with 10 or 2 mg/kg of DIM,
respectively. Moreover, tumors that developed in treated animals were
significantly (P<0.001) smaller than in controls. Additionally, our results
indicated that DIM has no effect on animal weight or liver and kidney functions.
These results indicated that the DIM agent is not toxic and has an in-vivo
preventive effect against the development of prostate cancer in a mouse model.

PMID: 20010430

Gynecol Oncol. 2009 Nov 24.

Oral diindolylmethane (DIM): Pilot evaluation of a nonsurgical treatment for
cervical dysplasia.

Del Priore G, Gudipudi DK, Montemarano N, Restivo AM, Malanowska-Stega J, Arslan
AA.

Indiana University School of Medicine, Dept of Ob-Gyn, Div Gyn Oncology,
Indianapolis, IN, USA; Research Department, New York Downtown Hospital-member
NY-Presbyterian Healthcare, New York, NY, USA.

OBJECTIVE: Standard surgical treatment for CIN may impair fertility generating a
need for alternative treatment options. We tested the efficacy and toxicity of
oral DIM in the treatment of CIN 2 or 3 lesions. METHODS: Patients with
biopsy-proven cervical intraepithelial neoplasia (CIN) 2 or 3 scheduled for loop
electrosurgical excision procedure (LEEP) were randomized 2:1 to receive
diindolylmethane (DIM) orally at approximately 2 mg/kg/day for 12 weeks or placebo.

Subjects were evaluated every 3-4 months for 1 year. Analysis of
data up to 1 year was assessed including Pap smear, HPV, colposcopy, biopsy and
physical examination were performed at follow-up. Central pathology review
confirmed all histology diagnoses. RESULTS: To date, 64 subjects (mean age 28
years, range 18-61) have been enrolled (45 in the DIM arm, 19 in the placebo
arm), with 60 available for analysis. Average follow-up was 6 months. At
enrollment, 58% were diagnosed with CIN 2 and 42% with CIN 3, 57% of subjects
were Caucasian, 15% African American, 12% Hispanic and 17% Asian. During
treatment 2 subjects (3%) complained of nausea (grade 2) at the 3- to 4-month
visit. No systemic toxicities were observed (normal CBC, LFTs, comprehensive
metabolic). Forty-six subjects had biopsies at first follow-up (77%). Twenty-one
subjects (47%) in the DIM group had improved CIN with a decrease by 1-2 grades or
a normal result. Median time to improvement was 5 months. Improved Pap smear was
seen in 49% (22/45) with either a less severe abnormality or normal result.
Colposcopy improved in twenty-five subjects in the DIM group (56%). Of these 25
subjects, 21 (84%) had improved colposcopic impression, 13 (52%) had a decrease
in involved quadrants and 18 (72%) had a decrease in lesion number. Complete
colposcopic response was observed in 4 subjects (9%). Stratifying by level of
dysplasia, age, race, HPV status, tobacco use, contraceptive used did not alter
the results. At median follow-up of 6 months, 85% of subjects have not required
LEEP based on routine clinical triage of improving global assessment.

CONCLUSION: Oral DIM at 2 mg/kg/day is well tolerated with no
significant toxicity. We observed a high rate of clinically significant
improvement in confirmed CIN 2 or 3 lesions among both treatment groups in this
randomized clinical trial.

PMID: 19939441

J Nutr. 2010 Jan;140(1):1-6. Epub 2009 Nov 4.

Antiangiogenic effects of indole-3-carbinol and 3,3'-diindolylmethane are
associated with their differential regulation of ERK1/2 and Akt in tube-forming
HUVEC.

Kunimasa K, Kobayashi T, Kaji K, Ohta T.

Department of Food and Nutritional Sciences, Graduate School of Nutritional and
Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.

We previously reported that indole-3-carbinol (I3C), found in cruciferous
vegetables, suppresses angiogenesis in vivo and in vitro. However, the underlying
molecular mechanisms still remain unclear. Antiangiogenic effects of its major
metabolite, 3,3'-diindolylmethane (DIM), also have not been fully elucidated. In
this study, we investigated the effects of these indoles on angiogenesis and
tested a hypothesis that I3C and DIM inhibit angiogenesis and induce apoptosis by
affecting angiogenic signal transduction in human umbilical vein endothelial
cells (HUVEC). We found that I3C and DIM at 25 micromol/L significantly inhibited
tube formation and only DIM induced a significant increase in apoptosis in
tube-forming HUVEC. DIM showed a stronger antiangiogenic activity than I3C. At
the molecular level, I3C and DIM markedly inactivated extracellular
signal-regulated kinase 1/2 (ERK1/2) and the inhibitory effect of DIM was
significantly greater than that of I3C. DIM treatment also resulted in activation
of the caspase pathway and inactivation of Akt, whereas I3C did not affect them.
These results indicate that I3C and DIM had a differential potential in the
regulation of the 2 principal survival signals, ERK1/2 and Akt, in endothelial
cells. We also demonstrated that pharmacological inhibition of ERK1/2 and/or Akt
was enough to inhibit tube formation and induce caspase-dependent apoptosis in
tube-forming HUVEC. We conclude that both I3C and DIM inhibit angiogenesis at
least in part via inactivation of ERK1/2 and that inactivation of Akt by DIM is
responsible for its stronger antiangiogenic effects than those of I3C.

PMID: 19889811

J Nutr. 2009 Dec;139(12):2373-9. Epub 2009 Oct 28.

Oral administration of 3,3'-diindolylmethane inhibits lung metastasis of 4T1
murine mammary carcinoma cells in BALB/c mice.

Kim EJ, Shin M, Park H, Hong JE, Shin HK, Kim J, Kwon DY, Park JH.

Center for Efficacy Assessment and Development of Functional Foods and Drugs,
Hallym University, Chuncheon, Korea.

3,3'-diindolylmethane (DIM) is the major in vivo product of the acid-catalyzed
oligomerization of indole-3-carbinol present in cruciferous vegetables, and it
has been shown to exhibit anticancer properties. In this study, we assessed the
effects of DIM on the metastasis of 4T1 mouse mammary carcinoma cells. In vitro
culture studies showed that DIM dose-dependently inhibited the migration,
invasion, and adhesion of 4T1 cells at concentrations of 0-10 micromol/L without
attendant changes in cell viability. In an in vivo lung metastasis model, 4T1
cells (2 x 10(5) cells/mouse) were injected into the tail veins of syngeneic
female BALB/c mice. Beginning on the second day, the mice were subjected to
gavage with 0-10 mg DIM/(kg body weight x d) for 13 d. Oral DIM administration
resulted in a marked reduction in the number of pulmonary tumor nodules. DIM
treatment significantly reduced the levels of matrix metalloproteinase (MMP)-2,
MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and vascular cell adhesion
molecule (VCAM)-1 and increased TIMP-2 levels in the sera and lungs of mice
injected with 4T1 cells. Additionally, DIM treatment reduced the serum
concentrations of interleukin (IL)-1beta, IL-6 and tumor necrosis factor
(TNF)alpha. We have demonstrated that DIM profoundly inhibits the lung metastasis
of 4T1 cells, which was accompanied by reduced levels of MMP, adhesion molecules,
and proinflammatory cytokines. These results indicate that DIM has potential as
an antimetastatic agent for the treatment of breast cancer.

PMID: 19864400

Cancer Epidemiol Biomarkers Prev. 2009 Nov;18(11):2957-64. Epub 2009 Oct 27.

Diindolylmethane inhibits cervical dysplasia, alters estrogen metabolism, and
enhances immune response in the K14-HPV16 transgenic mouse model.

Sepkovic DW, Stein J, Carlisle AD, Ksieski HB, Auborn K, Bradlow HL.

The David and Alice Jurist Institute for Research, Hackensack University Medical
Center, Hackensack, New Jersey 07601, USA. dsepkovic@humed.com

This study was designed to establish whether 3,3'-diindolylmethane (DIM) can
inhibit cervical lesions, alter estrogen metabolism in favor of C-2
hydroxylation, and enhance immune function in the K14-HPV16 transgenic mouse
model. Mice were bred, genotyped, implanted with E(2) pellets (0.25 mg/90-day
release) under anesthesia, and divided into groups. Wild-type and transgenic mice
were given either AIN76A diet alone or with 2,000 ppm DIM for 12 weeks. Blood and
reproductive tracts were obtained. Blood was analyzed for estrogen metabolites
and IFN-gamma. The cervical transformation zone was sectioned and stained for
histology. Estradiol C-2 hydroxylation and serum IFN-gamma levels were
significantly increased over controls in wild-type and transgenic mice receiving
DIM. In wild-type mice without DIM, hyperplasia of the squamous epithelium was
observed. Wild-type mice fed DIM displayed a normal thin epithelium. In
transgenic mice without DIM, epithelial cell projections into the stroma
(papillae) were present. An additional degree of nuclear anaplasia in the stratum
espinosum was observed. Dysplastic cells were present. Transgenic mice fed DIM
displayed some mild hyperplasia of the squamous epithelium. DIM increases
estrogen C-2 hydroxylation in this model. Serum INF-gamma was increased,
indicating increased immune response in the DIM-fed animals. Histopathology
showed a marked decrease in cervical dsyplasia in both wild-type and transgenic
mice, indicating that DIM delays or inhibits the progression from cervical
dysplasia to cervical cancer. Using the K14-HPV16 transgenic mouse model, we have
shown that DIM inhibits the development of E6/E7 oncogene-induced cervical
lesions.

PMID: 19861518

Biochem Pharmacol. 2009 Oct 23. [Epub ahead of print]

3,3'-Diindolylmethane attenuates experimental arthritis and osteoclastogenesis.

Dong L, Xia S, Gao F, Zhang D, Chen J, Zhang J.

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing 210093, PR China.

3,3'-Diindolylmethane (DIM) is a natural compound formed during the autolysis of
glucobrassicin present in Brassica food plants. This study aimed to investigate
the therapeutic efficacies of DIM on experimental arthritis. The effects of DIM
on experimental arthritis were examined on a rat model of adjuvant-induced
arthritis (AIA), with daily AIA paw swelling observation and
histological/radiographic analysis. To elucidate the possible mechanisms of its
action, serum cytokine levels as well as the expression of receptor activator for
nuclear factor kappa B ligand (RANKL) in infected tissues were subsequently
analyzed. The impact of DIM on osteoclastogenesis was further investigated on a
mouse model of endotoxin-induced bone resorption (EIBR) and in vitro cultures of
fibroblast-like cells and osteoblasts, with RANKL expression being evaluated with
great interest. The administration of DIM was demonstrated to attenuate AIA in
animal models, as judged by clinical and histologic indices of inflammation and
tissue damage. On the one hand, DIM could reduce the expression of several
inflammatory cytokines, which was, however, not adequate to prevent the
development of the arthritis. On the other hand, DIM was shown to effectively
inhibit the expression of RANKL, leading to the blockade of osteoclastogenesis
and consequently an alleviation of experimental arthritis. Further in vitro and
in vivo studies confirmed the inhibition of RANKL by DIM. DIM has shown
anti-arthritis activity in animal models via inhibiting the expression of RANKL,
and thus may offer potential treatments for arthritis and associated disorders.

PMID: 19854159

Mol Endocrinol. 2009 Dec;23(12):1940-7. Epub 2009 Oct 16.

Minireview: modulation of hormone receptor signaling by dietary anticancer
indoles.

Firestone GL, Sundar SN.

Department of Molecular and Cell Biology, The University of California at
Berkeley, 94720-3200, USA.

Indole-3-carbinol and its diindole condensation product 3-3'-diindolylmethane are
dietary phytochemicals that have striking anticarcinogenic properties in human
cancer cells. Molecular, cellular, physiological, and clinical studies have
documented that both indole-3-carbinol and 3-3'-diindolylmethane have potent
endocrine modulating activities through a myriad of mechanisms. The focus of this
review is to discuss the evidence that directly links the anticancer actions of
these two indole compounds to the control of steroid receptor and growth factor
receptor signaling.

PMID: 19837944

Int J Oncol. 2009 Nov;35(5):1191-9.

1,1-Bis(3'-indolyl)-1-(p-bromophenyl)methane and related compounds repress
survivin and decrease gamma-radiation-induced survivin in colon and pancreatic
cancer cells.

Sreevalsan S, Jutooru I, Chadalapaka G, Walker M, Safe S.

Department of Veterinary Physiology and Pharmacology, Texas A&M University,
College Station, TX 77843-4466, USA.

1,1-Bis(3'-indolyl)-1-(p-bromophenyl)methane (DIM-C-pPhBr) and the 2,2'-dimethyl
analog (2,2'-diMeDIM-C-pPhBr) inhibit proliferation and induce apoptosis in SW480
colon and Panc28 pancreatic cancer cells. In this study, treatment with 10-20
microM concentrations of these compounds for 24 h induced cleaved PARP and
decreased survivin protein and mRNA expression in both cell lines. However,
results of time course studies show that DIM-C-pPhBr and 2,2'-diMeDIM-C-pPhBr
decrease survivin protein within 2 h after treatment, whereas survivin mRNA
levels were decreased only at later time-points indicating activation of
transcription-independent and -dependent pathways for downregulation of survivin.
In addition, we also observed that gamma-radiation inhibited pancreatic and colon
cancer cell growth and this was associated with enhanced expression of survivin
after 24 (SW480) or 24 and 48 h (Panc28) and correlated with previous studies on
the role of survivin in radiation-resistance. However, in cells co-treated with
gamma-radiation plus DIM-C-pPhBr or 2,2'-diMeDIM-C-pPhBr, induction of survivin
by gamma-radiation was inhibited after co-treatment with both compounds,
suggesting applications for these drugs in combination cancer chemotherapy with
gamma-radiation.

PMID: 19787275

J Cell Biochem. 2009 Nov 1;108(4):916-25.

Down-regulation of uPA and uPAR by 3,3'-diindolylmethane contributes to the
inhibition of cell growth and migration of breast cancer cells.

Ahmad A, Kong D, Wang Z, Sarkar SH, Banerjee S, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Center, Wayne State
University School of Medicine, Detroit, Michigan 48201, USA.

3,3'-Diindolylmethane (DIM) is a known anti-tumor agent against breast and other
cancers; however, its exact mechanism of action remains unclear. The urokinase
plasminogen activator (uPA) and its receptor (uPAR) system are involved in the
degradation of basement membrane and extracellular matrix, leading to tumor cell
invasion and metastasis. Since uPA-uPAR system is highly activated in aggressive
breast cancer, we hypothesized that the biological activity of DIM could be
mediated via inactivation of uPA-uPAR system. We found that DIM treatment as
well as silencing of uPA-uPAR led to the inhibition of cell growth and motility
of MDA-MB-231 cells, which was in part due to inhibition of VEGF and MMP-9.
Moreover, silencing of uPA-uPAR led to decreased sensitivity of these cells to
DIM indicating an important role of uPA-uPAR in DIM-mediated inhibition of
cell growth and migration. We also found similar effects of DIM on MCF-7, cells
expressing low levels of uPA-uPAR, which was due to direct down-regulation of
MMP-9 and VEGF, independent of uPA-uPAR system. Interestingly, over-expression of
uPA-uPAR in MCF-7 cells attenuated the inhibitory effects of DIM. Our results,
therefore, suggest that DIM down-regulates uPA-uPAR in aggressive breast
cancers but in the absence of uPA-uPAR, DIM can directly inhibit VEGF and MMP-9
leading to the inhibition of cell growth and migration of breast cancer cells.
(c) 2009 Wiley-Liss, Inc.

PMID: 19693769

 

Biochem Pharmacol. 2009 Sep 1;78(5):469-76. Epub 2009 May 9.

3,3'-Diindolylmethane induces a G(1) arrest in human prostate cancer cells
irrespective of androgen receptor and p53 status.

Vivar OI, Lin CL, Firestone GL, Bjeldanes LF.

Department of Nutritional Sciences and Toxicology, 119 Morgan Hall, University of
California, Berkeley, CA 94720, USA.

3,3'-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived
from Brassica vegetables. In this study we characterized the effect of DIM on
cell cycle regulation in both androgen-dependent LNCaP and androgen receptor
negative p53 mutant DU145 human prostate cancer cells. DIM had an
anti-proliferative effect on both LNCaP and DU145 cells, as it significantly
inhibited [3H]-thymidine incorporation. FACS analysis revealed a DIM-mediated
G(1) cell cycle arrest. DIM strongly inhibited the expression of cdk2 and cdk4
protein and increased the expression of the cell cycle inhibitor p27(Kip1)
protein in LNCaP and DU145 cells. Promoter deletion studies with p27(Kip1)
reporter gene constructs showed that this DIM-mediated increase in p27(Kip1) was
dependent on the Sp1 transcription factor. Moreover, using a dominant negative
inhibitor of p38 MAPK, we showed that the induction of p27(Kip1) and subsequent
G(1) arrest by DIM involve activation of the p38 MAPK pathway in the DU145 cells.
Taken together, our results indicate that DIM is able to stop the cell cycle
progression of human prostate cancer cells regardless of their
androgen-dependence and p53 status, by differentially modulating cell cycle
regulatory pathways. The Sp1 and p38 MAPK pathways mediate the DIM cell cycle
regulatory effect in DU145 cells.

PMID: 19433067

Cancer Res. 2009 Aug 15;69(16):6704-12. Epub 2009 Aug 4.

Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of
epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer
cells.

Li Y, VandenBoom TG 2nd, Kong D, Wang Z, Ali S, Philip PA, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, Michigan 48201, USA.

Pancreatic cancer is the fourth most common cause of cancer death in the United
States, and the aggressiveness of pancreatic cancer is in part due to its
intrinsic and extrinsic drug resistance characteristics, which are also
associated with the acquisition of epithelial-to-mesenchymal transition (EMT).
Emerging evidence also suggests that the processes of EMT are regulated by the
expression status of many microRNAs (miRNA), which are believed to function as
key regulators of various biological and pathologic processes during tumor
development and progression. In the present study, we compared the expression of
miRNAs between gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer
cells and investigated whether the treatment of cells with "natural agents"
[3,3'-diindolylmethane (DIM) or isoflavone] could affect the expression of
miRNAs. We found that the expression of miR-200b, miR-200c, let-7b, let-7c,
let-7d, and let-7e was significantly down-regulated in gemcitabine-resistant
cells, which showed EMT characteristics such as elongated fibroblastoid
morphology, lower expression of epithelial marker E-cadherin, and higher
expression of mesenchymal markers such as vimentin and ZEB1. Moreover, we found
that reexpression of miR-200 by transfection studies or treatment of
gemcitabine-resistant cells with either DIM or isoflavone resulted in the
down-regulation of ZEB1, slug, and vimentin, which was consistent with
morphologic reversal of EMT phenotype leading to epithelial morphology. These
results provide experimental evidence, for the first time, that DIM and
isoflavone could function as miRNA regulators leading to the reversal of EMT
phenotype, which is likely to be important for designing novel therapies for
pancreatic cancer.

PMID: 19654291

Cancer Res. 2009 Aug;69(15):6083-91. Epub 2009 Jul 21.

Low concentrations of diindolylmethane, a metabolite of indole-3-carbinol,
protect against oxidative stress in a BRCA1-dependent manner.

Fan S, Meng Q, Saha T, Sarkar FH, Rosen EM.

Department of Oncology, Georgetown University, Washington, District of
Columbia200 57, USA.

The indole-3-carbinol (I3C) metabolite 3,3'-diindolylmethane (DIM) is a proposed
cancer prevention agent for various tumor types, including breast cancer. Here,
we show that DIM up-regulates expression of the tumor suppressor protein BRCA1 in
carcinoma and normal cell types. Up-regulation of BRCA1 was dose and time
dependent, and it was observed at physiologically relevant micromolar and
submicromolar DIM concentrations when cells were exposed for 72 hours. Treatment
with the parent compound (I3C) or DIM (1 micromol/L) protected against cell
killing due to H(2)O(2) and other oxidants, and the protection was abrogated by
knockdown of BRCA1. DIM stimulated signaling by the antioxidant transcription
factor NFE2L2 (NRF2) through the antioxidant response element in a
BRCA1-dependent manner. We further showed that DIM rapidly stimulated
phosphorylation of BRCA1 on Ser (1387) and Ser (1524) and that these
phosphorylations are required for protection against oxidative stress.
DIM-induced phosphorylation of BRCA1 on Ser (1387) was dependent on
ataxia-telangiectasia mutated. Finally, in our assay systems, H(2)O(2)-induced
cell death was not due to apoptosis. However, a significant component of cell
death was attributable to autophagy, and both DIM and BRCA1 inhibited
H(2)O(2)-induced autophagy. Our findings suggest that low concentrations of DIM
protect cells against oxidative stress via the tumor suppressor BRCA1 by several
distinct mechanisms.

PMID: 19622773

Cancer Res. 2009 Aug 4.

Up-regulation of miR-200 and let-7 by Natural Agents Leads to the Reversal of
Epithelial-to-Mesenchymal Transition in Gemcitabine-Resistant Pancreatic Cancer
Cells.

Li Y, Vandenboom TG 2nd, Kong D, Wang Z, Ali S, Philip PA, Sarkar FH.

Departments of Pathology and Internal Medicine, Barbara Ann Karmanos Cancer
Institute, Wayne State University School of Medicine, Detroit, Michigan.

Pancreatic cancer is the fourth most common cause of cancer death in the United
States, and the aggressiveness of pancreatic cancer is in part due to its
intrinsic and extrinsic drug resistance characteristics, which are also
associated with the acquisition of epithelial-to-mesenchymal transition (EMT).
Emerging evidence also suggests that the processes of EMT are regulated by the
expression status of many microRNAs (miRNA), which are believed to function as
key regulators of various biological and pathologic processes during tumor
development and progression. In the present study, we compared the expression of
miRNAs between gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer
cells and investigated whether the treatment of cells with "natural agents"
[3,3'-diindolylmethane (DIM) or isoflavone] could affect the expression of
miRNAs. We found that the expression of miR-200b, miR-200c, let-7b, let-7c,
let-7d, and let-7e was significantly down-regulated in gemcitabine-resistant
cells, which showed EMT characteristics such as elongated fibroblastoid
morphology, lower expression of epithelial marker E-cadherin, and higher
expression of mesenchymal markers such as vimentin and ZEB1. Moreover, we found
that reexpression of miR-200 by transfection studies or treatment of
gemcitabine-resistant cells with either DIM or isoflavone resulted in the
down-regulation of ZEB1, slug, and vimentin, which was consistent with
morphologic reversal of EMT phenotype leading to epithelial morphology. These
results provide experimental evidence, for the first time, that DIM and
isoflavone could function as miRNA regulators leading to the reversal of EMT
phenotype, which is likely to be important for designing novel therapies for
pancreatic cancer. [Cancer Res 2009;69(16):6704-12].

PMID: 19654291

Cancer Treat Rev. 2009 Aug 4.

Harnessing the fruits of nature for the development of multi-targeted cancer
therapeutics.

Sarkar FH, Li Y.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, MI, USA.

Cancer cells exhibit deregulation in multiple cellular signaling pathways.
Therefore, treatments using specific agents that target only one pathway usually
fail in cancer therapy. The combination treatments using chemotherapeutic agents
with distinct molecular mechanisms are considered more promising for higher
efficacy; however, using multiple agents contributes to added toxicity. Emerging
evidence has shown that some "natural products" such as isoflavones,
indole-3-carbinol (I3C) and its in vivo dimeric product 3,3'-diindolylmethane
(DIM), and curcumin among many others, have growth inhibitory and apoptosis
inducing effects on human and animal cancer cells mediated by targeting multiple
cellular signaling pathways in vitro without causing unwanted toxicity in normal
cells. Therefore, these non-toxic "natural products" from natural resources could
be useful in combination with conventional chemotherapeutic agents for the
treatment of human malignancies with lower toxicity and higher efficacy. In fact,
recently increasing evidence from pre-clinical in vivo studies and clinical
trials have shown some success in support of the use of rational design of
multi-targeted therapies for the treatment of cancers using conventional
chemotherapeutic agents in combination with "natural products". These studies
have provided promising results and further opened-up newer avenues for cancer
therapy. In this review article, we have succinctly summarized the known effects
of "natural products" especially by focusing on isoflavones, indole-3-carbinol
(I3C) and its in vivo dimeric product 3,3'-diindolylmethane (DIM), and curcumin,
and provided a comprehensive view on the molecular mechanisms underlying the
principle of cancer therapy using combination of "natural products" with
conventional therapeutics.

PMID: 19660870

Cancer Res. 2009 Aug 1;69(15):6083-91. Epub 2009 Jul 21.

Low concentrations of diindolylmethane, a metabolite of indole-3-carbinol,
protect against oxidative stress in a BRCA1-dependent manner.

Fan S, Meng Q, Saha T, Sarkar FH, Rosen EM.

Department of Oncology, Georgetown University, Washington, District of
Columbia200 57, USA.

The indole-3-carbinol (I3C) metabolite 3,3'-diindolylmethane (DIM) is a proposed
cancer prevention agent for various tumor types, including breast cancer. Here,
we show that DIM up-regulates expression of the tumor suppressor protein BRCA1 in
carcinoma and normal cell types. Up-regulation of BRCA1 was dose and time
dependent, and it was observed at physiologically relevant micromolar and
submicromolar DIM concentrations when cells were exposed for 72 hours. Treatment
with the parent compound (I3C) or DIM (1 micromol/L) protected against cell
killing due to H(2)O(2) and other oxidants, and the protection was abrogated by
knockdown of BRCA1. DIM stimulated signaling by the antioxidant transcription
factor NFE2L2 (NRF2) through the antioxidant response element in a
BRCA1-dependent manner. We further showed that DIM rapidly stimulated
phosphorylation of BRCA1 on Ser (1387) and Ser (1524) and that these
phosphorylations are required for protection against oxidative stress.
DIM-induced phosphorylation of BRCA1 on Ser (1387) was dependent on
ataxia-telangiectasia mutated. Finally, in our assay systems, H(2)O(2)-induced
cell death was not due to apoptosis. However, a significant component of cell
death was attributable to autophagy, and both DIM and BRCA1 inhibited
H(2)O(2)-induced autophagy. Our findings suggest that low concentrations of DIM
protect cells against oxidative stress via the tumor suppressor BRCA1 by several
distinct mechanisms.

PMID: 19622773

Inflamm Bowel Dis. 2009 Aug;15(8):1164-73.

3,3'-diindolylmethane attenuates colonic inflammation and tumorigenesis in mice.

Kim YH, Kwon HS, Kim DH, Shin EK, Kang YH, Park JH, Shin HK, Kim JK.

Center for Efficacy Assessment and Development of Functional Foods and Drugs,
Hallym University, Chuncheon, South Korea.

BACKGROUND: 3,3-Diindolylmethane (DIM) is a major in vivo product of
acid-catalyzed oligomerization of indole-3-carbinol (I3C) derived from Brassica
food plants. Although DIM is known as a chemopreventive and chemotherapeutic
phytochemical, the effects of DIM on inflammation in vivo are still unknown. In
the present study we investigated the antiinflammatory effects of DIM on
experimental colitis and colitis-associated colorectal carcinogenesis. METHODS:
To determine if DIM has an antiinflammatory effect in vivo, we examined the
therapeutic effects of DIM in dextran sodium sulfate (DSS)-induced experimental
colitis and colitis-associated colon carcinogenesis induced by azoxymethane
(AOM)/DSS in BALB/c mice. RESULTS: Treatment with DIM significantly attenuated
loss of body weight, shortening of the colon, and severe clinical signs in a
colitis model. This was associated with a remarkable amelioration of the
disruption of the colonic architecture and a significant reduction in colonic
myeloperoxidase activity and production of prostaglandin E(2), nitric oxide, and
proinflammatory cytokines. Further, DIM administration dramatically decreased the
number of colon tumors in AOM/DSS mice. CONCLUSIONS: These results suggest that
DIM-mediated antiinflammatory action at colorectal sites may be therapeutic in
the setting of inflammatory bowel disease and colitis-associated colon cancer.

PMID: 19334074


Cancer Res. 2009 Jul 1;69(13):5592-600. Epub 2009 Jun 16.

3,3'-Diindolylmethane enhances chemosensitivity of multiple chemotherapeutic
agents in pancreatic cancer.

Banerjee S, Wang Z, Kong D, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, Michigan 48201, USA.

Clinical management of pancreatic cancer is a major problem, which is in part due
to both de novo and acquired resistance to conventional therapeutics. Here, we
present in vitro and in vivo preclinical evidence in support of
chemosensitization of pancreatic cancer cells by 3,3-diindolylmethane (DIM), a
natural compound that can be easily obtained by consuming cruciferous vegetables.
DIM pretreatment of pancreatic cancer cells led to a significantly increased
apoptosis (P < 0.01) with suboptimal concentrations of chemotherapeutic agents
(cisplatin, gemcitabine, and oxaliplatin) compared with monotherapy. It is known
that resistance to chemotherapy in pancreatic cancer is associated with
constitutively activated nuclear factor-kappaB (NF-kappaB), which becomes further
activated by chemotherapeutic drugs. Our data provide mechanistic evidence for
the first time showing that DIM potentiates the killing of pancreatic cancer
cells by down-regulation of constitutive as well as drug-induced activation of
NF-kappaB and its downstream genes (Bcl-xL, XIAP, cIAP, and survivin). Most
importantly, using an orthotopic animal model, we found reduction in tumor size
(P < 0.001) when DIM was given in combination with oxaliplatin compared with
monotherapy. This was accompanied by loss of phospho-p65 and down-regulation of
NF-kappaB activity and its downstream genes (Bcl-xL, survivin, and XIAP), which
correlated with reduced cell proliferation (as assessed by Ki-67 immunostaining
of tumor specimens) and evidence of apoptosis [as assessed by poly(ADP-ribose)
polymerase cleavage and terminal deoxynucleotidyl transferase-mediated dUTP nick
end labeling staining]. These results provide strong in vivo evidence in support
of our hypothesis that DIM could abrogate chemotherapeutic drug (cisplatin,
gemcitabine, and/or oxaliplatin)-induced activation of NF-kappaB, resulting in
the chemosensitization of pancreatic tumors to conventional therapeutics.

PMID: 19531648

Cancer Lett. 2009 Jun 18;278(2):201-9. Epub 2009 Feb 23.

Sensitization of squamous cell carcinoma to cisplatin induced killing by natural
agents.

Ali S, Varghese L, Pereira L, Tulunay-Ugur OE, Kucuk O, Carey TE, Wolf GT, Sarkar
FH.

Division of Hematology/Oncology, Karmanos Cancer Center, Wayne State University,
Detroit, MI, USA.

Cisplatin resistance is a major problem in the successful treatment of squamous
cell carcinoma (SCC). In the present study we showed, for the first time, that
the constitutive activation of NF-kappaB partly contributes to cisplatin
resistance and that the inactivation of NF-kappaB by natural agents [G2535
(isoflavone mixture containing genistein and diadzein), 3,3'-diindolylmethane
(DIM)] could overcome this resistance, resulting in the inhibition of cell growth

and induction of apoptosis, which might be an useful strategy for achieving better

treatment outcome in patients diagnosed with cisplatin-resistant tumors of SCC.

PMID: 19231069

Laryngoscope. 2009 Jun 18.

A controlled safety study of diindolylmethane in the immature rat model.

Elackattu AP, Feng L, Wang Z.

Department of Otolaryngology-Head and Neck Surgery, Boston University School of
Medicine, Boston, Massachusetts, U.S.A.

OBJECTIVES/HYPOTHESIS:: Diindolylmethane (DIM), a natural product from
cruciferous vegetables, has been shown to be a dietary component that has
inhibitory effects on some tumors (e.g., laryngeal papilloma). However, current
evidence to support its safety is based on adult humans or mature animals. There
is little to show on its safety in children. This study is designed to assess
safety in the young rat model. STUDY DESIGN:: Prospective controlled animal
study. METHODS:: Forty rats were separated into four treatment groups of 10 rats
each, based on the amount of study drug they received in their daily food: 1)
immature rats fed a low dose of DIM, (0.6 mg/kg/day); 2) immature rats fed a high
dose of DIM (6.0 mg/kg/day); 3) immature rats fed no DIM (control); and 4) adult
rats fed a high dose of DIM (6.0 mg/kg/day). At the conclusion of the study we
collected blood to compare serum chemistries and vitamin D levels, and harvested
organs to observe for any gross or histological changes among the groups.
Statistical methods involved one-way analysis of variance and pairwise
comparisons with Tukey multiple comparison adjustment. RESULTS:: Although our
numbers do not allow for statistical significance, there was no appreciable
difference in rat weights among the immature groups, nor was there appreciable
difference in serum chemistries, or gross or histological examination of liver,
kidney, and bone. CONCLUSIONS:: Diindolylmethane seems to have no adverse affects
on the rat. This adds evidence to the safety of this drug in the pediatric population as a
treatment option for recurrent respiratory papilloma.

PMID: 19544380

Cancer Prev Res (Phila Pa). 2009 Jun;2(6):581-9. Epub 2009 May 26.

3,3'-diindolylmethane enhances the efficacy of butyrate in colon cancer
prevention through down-regulation of survivin.

Bhatnagar N, Li X, Chen Y, Zhou X, Garrett SH, Guo B.

Department of Pharmaceutical Sciences, College of Pharmacy, North Dakota State
University, Fargo, North Dakota 58105, USA.

Butyrate is an inhibitor of histone deacetylase (HDAC) and has been extensively
evaluated as a chemoprevention agent for colon cancer. We recently showed that
mutations in the adenomatous polyposis coli (APC) gene confer resistance to HDAC
inhibitor-induced apoptosis in colon cancers. Here, we show that APC mutation
rendered colon cancer cells resistant to butyrate-induced apoptosis due to the
failure of butyrate to down-regulate survivin in these cells. Another
cancer-preventive agent, 3,3'-diindolylmethane (DIM), was identified to be able
to down-regulate survivin in colon cancers expressing mutant APC. DIM inhibited
survivin mRNA expression and promoted survivin protein degradation through
inhibition of p34(cdc2)-cyclin B1-mediated survivin Thr(34) phosphorylation.
Pretreatment with DIM enhanced butyrate-induced apoptosis in colon cancer cells
expressing mutant APC. DIM/butyrate combination treatment induced the expression
of proapoptotic Bax and Bak proteins, triggered Bax dimerization/activation, and
caused release of cytochrome c and Smac proteins from mitochondria. Whereas
overexpression of survivin blocked DIM/butyrate-induced apoptosis, knocking down
of survivin by small interfering RNA increased butyrate-induced apoptosis in
colon cancer cells. We further showed that DIM was able to down-regulate survivin
and enhance the effects of butyrate in apoptosis induction and prevention of
familial adenomatous polyposis in APC(min/+) mice. Thus, the combination of DIM
and butyrate is potentially an effective strategy for the prevention of colon
cancer.

PMID: 19470789

Cancer Prev Res (Phila Pa). 2009 Jun;2(6):566-71. Epub 2009 May 26.

3,3'-diindolylmethane induction of p75NTR-dependent cell death via the p38
mitogen-activated protein kinase pathway in prostate cancer cells.

Khwaja FS, Wynne S, Posey I, Djakiew D.

Department of Biochemistry, the Vincent T Lombardi Comprehensive Cancer Center,
Georgetown University Medical, University of the District of Columbia,
Washington, DC 20057-1436, USA.

The p75(NTR) functions as a tumor suppressor in prostate epithelial cells, where
its expression declines with progression to malignant cancer. Previously, we
showed that treatment with the nonsteroidal anti-inflammatory drug, indomethacin,
induced p75(NTR) expression in the T24 cancer cell line leading to
p75(NTR)-mediated decreased survival. Utilizing the indole moiety of indomethacin
as a pharmacophore, we identified in rank-order with least efficacy, ketorolac,
etodolac, indomethacin, 5-methylindole-3-acetic acid, indole-3-carbinol, and
3,3'-diindolylmethane (DIM) exhibiting greatest activity for induction of
p75(NTR) levels and inhibition of cell survival. Prostate (PC-3, DU-145) and
bladder (T24) cancer cells were more sensitive to DIM induction of
p75(NTR)-associated loss of survival than breast (MCF7) and fibroblast (3T3)
cells. Transfection of the PC-3 prostate cell line with a dominant-negative form
of p75(NTR) before DIM treatment significantly rescued cell survival
demonstrating a cause and effect relationship between DIM induction of p75(NTR)
levels and inhibition of survival. Furthermore, siRNA knockdown of the p38
mitogen-activated protein kinase (MAPK) protein prevented induction of p75(NTR)
by DIM in the PC-3 prostate cell line. DIM treatment induced phosphorylation of
p38 MAPK as early as within 1 minute. Collectively, we identify DIM as an indole
capable of inducing p75(NTR)-dependent apoptosis via the p38 MAPK pathway in
prostate cancer cells.

PMID: 19470787

J Cell Biochem. 2009 Jun 1;107(3):516-27.

Inactivation of uPA and its receptor uPAR by 3,3'-diindolylmethane (DIM) leads to
the inhibition of prostate cancer cell growth and migration.

Ahmad A, Kong D, Sarkar SH, Wang Z, Banerjee S, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Center, Wayne State
University School of Medicine, Detroit, Michigan 48201, USA.

3,3'-Diindolylmethane (DIM) has been studied for its putative anti-cancer
properties, especially against prostate cancer; however, its exact mechanism of
action remains unclear. We recently provided preliminary data suggesting
down-regulation of uPA during DIM (a clinically active DIM)-induced inhibition
of invasion and angiogenesis in prostate cancer cells. Since the expression and
activation of uPA plays important role in tumorigenicity, and high endogenous
levels of uPA and uPAR are found in advanced metastatic cancers, we investigated
their role in DIM-mediated inhibition of prostate cancer cell growth and
motility. Using PC3 cells, we found that DIM treatment as well as the silencing
of uPA and uPAR by siRNAs led to the inhibition of cell growth and motility.
Conversely, over-expression of uPA/uPAR in LNCaP and C4-2B cells resulted in
increased cell growth and motility, which was effectively inhibited by DIM.
Moreover, we found that uPA as well as uPAR induced the production of VEGF and
MMP-9, and that the down-regulation of uPA/uPAR by siRNAs or DIM treatment
resulted in the inhibition of VEGF and MMP-9 secretion which could be responsible
for the observed inhibition of cell migration. Interestingly, silencing of
uPA/uPAR led to decreased sensitivity to DIM indicating important role of
uPA/uPAR in DIM-mediated regulation of prostate cancer cell growth and
migration. Our data suggest that chemopreventive and/or therapeutic activity of
DIM is in part due to down-regulation of uPA-uPAR leading to reduced production
of VEGF/MMP-9 which ultimately leads to the inhibition of cell growth and
migration of aggressive prostate cancer cells.

PMID: 19330806

Mol Cancer Res. 2009 Jun;7(6):977-86. Epub 2009 May 26.

Estrogen receptor subtype- and promoter-specific modulation of aryl hydrocarbon
receptor-dependent transcription.

WihlÚn B, Ahmed S, Inzunza J, Matthews J.

Department of Biosciences and Nutrition at Novum, Karolinska Institutet,
Huddinge, Sweden.

In this study, we examined the role of estrogen receptors (ER) in aryl
hydrocarbon receptor (AHR)-dependent transactivation. Chromatin
immunoprecipitation assays showed that AHR agonists differentially induced
recruitment of ERalpha to the AHR target genes CYP1A1 and CYP1B1. Cotreatment
with 17beta-estradiol significantly increased beta-naphthoflavone (BNF)- and
2,3,7,8-tetrachlorodibenzo-p-dioxin-induced recruitment of ERalpha to CYP1A1,
whereas 3,3'-diindolylmethane induced promoter occupancy of ERalpha at CYP1A1
that was unaffected by cotreatment with 17beta-estradiol. Cyclical recruitment of
AHR and ERalpha to CYP1A1 was only observed in cells treated with BNF. Stable and
subtype-specific knockdown of ERalpha or ERbeta using shRNA showed that
suppression of ERalpha significantly reduced, whereas knockdown of ERbeta
significantly enhanced, AHR agonist-induced Cyp1a1 expression in HC11 mouse
mammary epithelial cells. AHR agonist-induced Cyp1b1 expression was reduced by
ERbeta knockdown but unaffected by ERalpha knockdown. The siRNA-mediated
knockdown of ERalpha in MCF-7 human breast cancer cells did not affect
2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent regulation of CYP1A1 and CYP1B1
mRNA expression. In agreement with our in vitro findings in the HC11 cells,
ERalpha knockout mice exhibit reduced BNF-dependent induction of Cyp1a1 mRNA.
These results establish ligand- and promoter-specific influences on the cyclical
recruitment patterns for AHR and show ER species-, subtype-, and
promoter-specific modulation of AHR-dependent transcription.

PMID: 19470599

BMC Gastroenterol. 2009 May 29;9:39.

Induction of G1 and G2/M cell cycle arrests by the dietary compound
3,3'-diindolylmethane in HT-29 human colon cancer cells.

Choi HJ, Lim do Y, Park JH.

Department of Food Science and Nutrition, Hallym University, Chuncheon, Republic
of Korea.

BACKGROUND: 3,3'-Diindolylmethane (DIM), an indole derivative produced in the
stomach after the consumption of broccoli and other cruciferous vegetables, has
been demonstrated to exert anti-cancer effects in both in vivo and in vitro
models. We have previously determined that DIM (0 - 30 micromol/L) inhibited the
growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In
this study, we evaluated the effects of DIM on cell cycle progression in HT-29
cells. METHODS: HT-29 cells were cultured with various concentrations of DIM (0 -
30 micromol/L) and the DNA was stained with propidium iodide, followed by flow
cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses,
immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK)
and cell division cycle (CDC)2 were conducted. RESULTS: The percentages of cells
in the G1 and G2/M phases were dose-dependently increased and the percentages of
cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced
DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and
the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also
increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of
cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors,
p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the
levels of CDC25C phosphatase and cyclin B1. CONCLUSION: Here, we have
demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells,
and this effect may be mediated by reduced CDK activity.

PMID: 19480695

Cancer Res. 2009 May 15;69(10):4468-75. Epub 2009 May 12.

3,3'-Diindolylmethane enhances taxotere-induced apoptosis in hormone-refractory
prostate cancer cells through survivin down-regulation.

Rahman KM, Banerjee S, Ali S, Ahmad A, Wang Z, Kong D, Sakr WA.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI 48201, USA. kmrahman@med.wayne.edu

Survivin, a member of inhibitor of apoptosis family, is associated with both
prostate cancer progression and drug resistance. Therefore, we hypothesized that
survivin may play a potentially important role in hormone-refractory prostate
cancer (HRPC) and bone metastatic disease; thus, targeting of survivin signaling
could enhance therapeutic efficacy in prostate cancer. 3,3'-Diindolylmethane
(DIM) has been known to have cancer chemoprevention activity. However, no
information is available regarding the down-regulation of survivin by DIM, which
could result in the chemosensitization of HRPC cells to Taxotere-induced killing.
We investigated the effect of DIM alone or in combination with Taxotere using
LNCaP and C4-2B prostate cancer cells. We observed that DIM enhanced
Taxotere-induced apoptotic death in both cell lines. These enhancing effects were
related to a decrease in survivin expression as well as androgen receptor and
nuclear factor-kappaB (NF-kappaB) DNA-binding activity. We also found that
knockdown of survivin expression by small interfering RNA transfection increased
DIM-induced cell growth inhibition and apoptosis, whereas overexpression of
survivin by cDNA transfection abrogated DIM-induced cell growth inhibition and
apoptosis in both prostate cancer cells. Importantly, luciferase assays showed a
significant reduction of survivin-Luc and NF-kappaB-Luc activity in prostate
cancer cells exposed to DIM and Taxotere. Furthermore, combination treatment
significantly inhibited C4-2B bone tumor growth, and the results were correlated
with the down-regulation of survivin. From these results, we conclude that
inactivation of survivin by DIM enhanced the therapeutic efficacy of Taxotere in
prostate cancer in general, which could be useful for the treatment of HRPC and
metastatic prostate cancer.

PMID: 19435906

Chem Pharm Bull (Tokyo). 2009 May;57(5):536-40.

Enhancement of chemically-induced HL-60 cell differentiation by
3,3'-diindolylmethane derivatives.

Noguchi-Yachide T, Tetsuhashi M, Aoyama H, Hashimoto Y.

Institute of Molecular & Cellular Biosciences, The University of Tokyo, Tokyo,
Japan.

3,3'-Diindolylmethane (DIM, 1) and its derivatives have been prepared, and their
enhancing effects on chemically-induced HL-60 cell differentiation were analyzed.
Among the prepared compounds, IndDIM (12) showed the most potent enhancing effect
on HL-60 cell differentiation induced by chemicals, including retinoids,
1,25-dihydroxyvitamin D(3), 12-O-tetradecanoyl phorbol-13-acetate and dimethyl
sulfoxide.

PMID: 19420792

J Biol Chem. 2009 May 1;284(18):12328-38. Epub 2009 Mar 13.

Lipid G protein-coupled receptor ligand identification using beta-arrestin
PathHunter assay.

Yin H, Chu A, Li W, Wang B, Shelton F, Otero F, Nguyen DG, Caldwell JS, Chen YA.

GPCR Platform, Genomics Institute of the Novartis Research Foundation, San Diego,
California 92121, USA.

A growing number of orphan G-protein-coupled receptors (GPCRs) have been reported
to be activated by lipid ligands, such as lysophosphatidic acid, sphingosine
1-phosphate (S1P), and cannabinoids, for which there are already well established
receptors. These new ligand claims are controversial due to either lack of
independent confirmations or conflicting reports. We used the beta-arrestin
PathHunter assay system, a newly developed, generic GPCR assay format that
measures beta-arrestin binding to GPCRs, to evaluate lipid receptor and ligand
pairing. This assay eliminates interference from endogenous receptors on the
parental cells because it measures a signal that is specifically generated by the
tagged receptor and is immediately downstream of receptor activation. We screened
a large number of newly "deorphaned" receptors (GPR23, GPR92, GPR55, G2A, GPR18,
GPR3, GPR6, GPR12, and GPR63) and control receptors against a collection of
approximately 400 lipid molecules to try to identify the receptor ligand in an
unbiased fashion. GPR92 was confirmed to be a lysophosphatidic acid receptor with
weaker responses to farnesyl pyrophosphate and geranylgeranyl diphosphate. The
putative cannabinoid receptor GPR55 responded strongly to AM251, rimonabant, and
lysophosphatidylinositol but only very weakly to endocannabinoids. G2A receptor
was confirmed to be an oxidized free fatty acid receptor. In addition, we
discovered that 3,3'-diindolylmethane, a dietary molecule from cruciferous
vegetables, which has known anti-cancer properties, to be a CB(2) receptor
partial agonist, with binding affinity around 1 microm. The anti-inflammatory
effect of 3,3'-diindolylmethane in RAW264.7 cells was shown to be partially
mediated by CB(2).

PMID: 19286662

J Cell Physiol. 2009 Apr;219(1):94-9.

Cell cycle-dependent effects of 3,3'-diindolylmethane on proliferation and
apoptosis of prostate cancer cells.

Chinnakannu K, Chen D, Li Y, Wang Z, Dou QP, Reddy GP, Sarkar FH.

Vattikuti Urology Institute, Henry Ford Health System, Detroit, Michigan, USA.

Epidemiological studies have shown that a diet rich in fruits and cruciferous
vegetables is associated with a lower risk of prostate cancer. Indole-3-carbinol
(I3C) and its dimeric product 3,3'-diindolylmethane (DIM) have been shown to
exhibit anti-tumor activity both in vitro and in vivo. Recently, we have reported
that DIM induced apoptosis and inhibited growth, angiogenesis, and invasion of

prostate cancer cells by regulating Akt, NF-kappaB, VEGF and the androgen receptor

(AR) signaling pathway. However, the precise molecular mechanism(s) by which DIM

inhibits prostate cancer cell growth and induces apoptosis have not been fully elucidated.

Most importantly, it is not known how DIM affects cell cycle regulators and proteasome

activity, which are  critically involved in cell growth and apoptosis. In this study, we

investigated the effects of DIM on proteasome activity and AR transactivation with

respect to DIM-mediated cell cycle regulation and induction of apoptosis in both
androgen-sensitive LNCaP and androgen-insensitive C4-2B prostate cancer cells. We
believe that our results show for the first time the cell cycle-dependent effects
of DIM on proliferation and apoptosis of synchronized prostate cancer cells
progressing from G(1) to S phase. DIM inhibited this progression by induction
of p27(Kip1) and down-regulation of AR. We also show for the first time that
DIM inhibits proteasome activity in S phase, leading to the inactivation of
NF-kappaB signaling and induction of apoptosis in LNCaP and C4-2B cells. These
results suggest that DIM could be a potent agent for the prevention and/or
treatment of both hormone sensitive as well as hormone-refractory prostate
cancer.

PMID: 19062173

Cell Signal. 2009 Mar 16. [Epub ahead of print]

Cellular signaling perturbation by natural products.

Sarkar FH, Li Y, Wang Z, Kong D.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, 740 Hudson Webber Cancer Research Center, 4100
John R, Detroit, MI 48201, USA.

Cancer cells are known to have alterations in multiple cellular signaling
pathways and because of the complexities in the communication between multiple
signaling networks, the treatment and the cure for most human malignancies is
still an open question. Perhaps, this is the reason why specific inhibitors that
target only one pathway have been typically failed in cancer treatment. However,
the in vitro and in vivo studies have demonstrated that some natural products
such as isoflavones, indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM),
curcumin, (-)-epigallocatechin-3-gallate (EGCG), resveratrol, lycopene, etc, have
inhibitory effects on human and animal cancers through targeting multiple
cellular signaling pathways and thus these "natural agents" could be classified
as multi-targeted agents. This is also consistent with the epidemiological
studies showing that the consumption of fruits, soybean and vegetables is
associated with reduced risk of several types of cancers. By regulating multiple
important cellular signaling pathways including NF-kappaB, Akt, MAPK, Wnt, Notch,
p53, AR, ER, etc, these natural products are known to activate cell death signals
and induce apoptosis in pre-cancerous or cancer cells without affecting normal
cells. Therefore, non-toxic "natural agents" harvested from the bounties of
nature could be useful either alone or in combination with conventional
therapeutics for the prevention of tumor progression and/or treatment of human
malignancies.

PMID: 19298854

Cancer Prev Res (Phila Pa). 2009 Mar;2(3):251-6. Epub 2009 Feb 17.

Toxic and chemopreventive ligands preferentially activate distinct aryl
hydrocarbon receptor pathways: implications for cancer prevention.

Okino ST, Pookot D, Basak S, Dahiya R.

Department of Urology, San Francisco Veterans Affairs Medical Center and the
University of California at San Francisco, San Francisco, California 94121, USA.

The aryl hydrocarbon receptor (AhR) is a ligand-activated regulatory protein that
controls estrogen action through two distinct pathways. In one pathway, AhR acts
as a transcription factor that induces the expression of the CYP1 family of
estrogen-metabolizing genes; in the other pathway, AhR initiates the degradation
of the estrogen receptor and suppresses estrogen signaling. The AhR ligand
3,3'-diindolylmethane (DIM) is a beneficial dietary constituent that prevents
breast tumors in rodents and is associated with decreased breast cancer risk in
humans. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic AhR ligand that is
implicated in birth defects, infertility, and cancer. We analyzed MCF-7 cells to
gain insight into how two AhR ligands can exert such fundamentally different
health effects. We find that DIM and TCDD have differing abilities to activate
the distinct AhR-controlled pathways. TCDD strongly induces AhR-dependent CYP1
gene expression, whereas DIM is a relatively weak CYP1 inducer. DIM strongly
inhibits estrogen receptor-alpha expression and estrogen signaling, whereas TCDD
has a notably weaker effect on these processes. Small interfering RNA knockdown
of AhR confirms that the effects of DIM and TCDD are indeed AhR dependent. Our
findings reveal that DIM and TCDD each elicit a unique pattern of change in
pathways that control estrogen action; such patterns may determine if an AhR
ligand has beneficial or adverse health effects.

PMID: 19223575

Clin Cancer Res. 2009 Jan 15;15(2):543-52.

Enhancement of docetaxel anticancer activity by a novel diindolylmethane compound
in human non-small cell lung cancer.

Ichite N, Chougule MB, Jackson T, Fulzele SV, Safe S, Singh M.

College of Pharmacy and Pharmaceutical Sciences. Florida A&M University,
Tallahassee, Florida 32307, USA.

PURPOSE: This study was conducted to examine the cytotoxic effects of a
peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, 1,1-bis
(3'-indolyl)-1-(p-biphenyl) methane (DIM-C-pPhC(6)H(5)), alone and in combination
with docetaxel in vitro in A549 lung cancer cells and in vivo in nude mice
bearing A549 orthotopic lung tumors. EXPERIMENTAL DESIGN: Isobolographic method
was used to calculate combination index values from cell viability data.
Apoptosis was evaluated in A549 cells by terminal deoxynucleotidyl
transferase-mediated nick end labeling assay and measurement of cleaved
poly(ADP-ribose) polymerase level. Expression of proteins was studied by Western
blotting. A549 cells were implanted to induce orthotopic lung tumors in nude mice
and the efficacy of docetaxel, DIM-C-pPhC(6)H(5), or combination was determined.
Apoptosis and cleaved caspase-3 expression in the harvested tissues were studied
by terminal deoxynucleotidyl transferase-mediated nick end labeling and
immunohistochemistry, respectively. RESULTS: The combination index values
(0.36-0.9) suggested synergistic to additive effects of docetaxel +
DIM-C-pPhC(6)H(5) and resulted in the highest increase in percentage of apoptotic
cells and expression of cleaved poly(ADP-ribose) polymerase, Bax, and N-cadherin
compared with treatment with either agent. The combination also enhanced
procaspase-3 and -9 cleavage. In vivo, docetaxel + DIM-C-pPhC(6)H(5) reduced lung
weights by 57% compared with 39% by docetaxel or 22% by DIM-C-pPhC(6)H(5) alone,
induced apoptosis in 43% of the tumor cells compared with 29% and 22% in tumors
treated with docetaxel and DIM-C-pPhC(6)H(5), respectively, and increased
procaspase-3 cleavage compared with either agent alone. CONCLUSIONS: These
findings suggest potential benefit for use of docetaxel and DIM-C-pPhC(6)H(5)
combination in lung cancer treatment.

PMID: 19147759

J Nutr. 2009 Jan;139(1):26-32. Epub 2008 Dec 3.

Targeting of aryl hydrocarbon receptor-mediated activation of cyclooxygenase-2
expression by the indole-3-carbinol metabolite 3,3'-diindolylmethane in breast
cancer cells.

Degner SC, Papoutsis AJ, Selmin O, Romagnolo DF.

Department of Nutritional Sciences, University of Arizona, Tucson, AZ 85721, USA.

Ligands of the aryl hydrocarbon receptor (AhR) include the environmental
xenobiotic 2,3,7,8 tetrachlorodibenzo(p)dioxin (TCDD), polycyclic aryl
hydrocarbons, and the dietary compounds 3, 3'-diindolylmethane (DIM), a
condensation product of indol-3-carbinol found in Brassica vegetables, and the
phytoalexin resveratrol (RES). The AhR and its cofactors regulate the expression
of target genes at pentameric (GCGTG) xenobiotic responsive elements (XRE).
Because the activation of cyclooxygenase-2 (COX-2) expression by AhR ligands may
contribute to inflammation and tumorigenesis, we investigated the epigenetic
regulation of the COX-2 gene by TCDD and the reversal effects of DIM in MCF-7
breast cancer cells. Results of DNA binding and chromatin immunoprecipitation
(ChIP) studies documented that the treatment with TCDD induced the association of
the AhR to XRE harbored in the COX-2 promoter and control CYP1A1 promoter
oligonucleotides. The TCDD-induced binding of the AhR was reduced by
small-interfering RNA for the AhR or the cotreatment with synthetic
(3-methoxy-4-naphthoflavone) or dietary AhR antagonists (DIM, RES). In time
course ChIP studies, TCDD induced the rapid (15 min) occupancy by the AhR, the
histone acetyl transferase p300, and acetylated histone H4 (AcH4) at the COX-2
promoter. Conversely, the cotreatment of MCF-7 cells with DIM (10 micromol/L)
abrogated the TCDD-induced recruitment of the AhR and AcH4 to the COX-2 promoter
and the induction of COX-2 mRNA and protein levels. Taken together, these data
suggest that naturally occurring modulators of the AhR such as DIM may be
effective agents for dietary strategies against epigenetic activation of COX-2
expression by AhR agonists.

PMID: 19056653

J Oncol. 2009;2009:491985. Epub 2009 Mar 24.

Modulation of CXCR4, CXCL12, and Tumor Cell Invasion Potential In Vitro by
Phytochemicals.

Hsu EL, Chen N, Westbrook A, Wang F, Zhang R, Taylor RT, Hankinson O.

Molecular Toxicology Interdepartmental Doctoral Program, University of California
Los Angeles, Los Angeles, CA 90095, USA.

CXCR4 is a chemokine receptor frequently overexpressed on primary tumor cells.
Organs to which these cancers metastasize secrete CXCL12, the unique ligand for
CXCR4, which stimulates invasion and metastasis to these sites. Similar to our
previous work with the chemoprotective phytochemical, 3,3'-diindolylmethane
(DIM), we show here that genistein also downregulates CXCR4 and CXCL12 and
subsequently lowers the migratory and invasive potentials of breast and ovarian
cancer cells. Moreover, genistein and DIM elicit a significantly greater
cumulative effect in lowering CXCR4 and CXCL12 levels than either compound alone.
Our data suggest a novel mechanism for the protective effects of phytochemicals
against cancer progression and indicate that in combination, these compounds may
prove even more efficacious.

PMID: 19325924

Mol Pharmacol. 2009 Jan;75(1):35-43. Epub 2008 Oct 7.

Suppression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nitric-oxide
synthase 2 expression in astrocytes by a novel diindolylmethane analog protects
striatal neurons against apoptosis.

Carbone DL, Popichak KA, Moreno JA, Safe S, Tjalkens RB.

Department of Environmental and Radiological Health Sciences, Colorado State
University, Collins, CO 80523, USA.

The progressive debilitation of motor functions in Parkinson's disease (PD)
results from degeneration of dopaminergic neurons within the substantia nigra
pars compacta of the midbrain. Long-term inflammatory activation of microglia and
astrocytes plays a central role in the progression of PD and is characterized by
activation of the nuclear factor-kappaB (NF-kappaB) signaling cascade and
subsequent overproduction of inflammatory cytokines and nitric oxide (NO).
Suppression of this neuroinflammatory phenotype has received considerable
attention as a potential target for chemotherapy, but there are no currently
approved drugs that sufficiently address this problem. The data presented here
demonstrate the efficacy of a novel anti-inflammatory diindolylmethane class
compound, 1,1-bis(3'-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu), in
suppressing NF-kappaB-dependent expression of inducible nitric-oxide synthase
(NOS2) and NO production in astrocytes exposed to the parkinsonian neurotoxicant
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) through a mechanism distinct
from that described for the thiazolidinedione-class compound, rosiglitazone.
Chromatin immunoprecipitations revealed that micromolar concentrations of
DIM-C-pPhtBu prevented association of the p65 subunit of NF-kappaB with enhancer
elements in the Nos2 promoter but had little effect on DNA binding of either
peroxisome proliferator-activated receptor-gamma (PPAR-gamma) or the nuclear
corepressor NCoR2. Treatment with DIM-C-pPhtBu concomitantly suppressed NO
production and protein nitration in MPTP-activated astrocytes and completely
protected cocultured primary striatal neurons from astrocyte-dependent apoptosis.
These data demonstrate the efficacy of DIM-C-pPhtBu in preventing the activation
of NF-kappaB-dependent inflammatory genes in primary astrocytes and suggest that
this class of compounds may be effective neuroprotective anti-inflammatory agents
in vivo.

PMID: 18840677


J Nutr. 2008 Dec;138(12):2379-85.

3,3'-Diindolylmethane and genistein decrease the adverse effects of estrogen in
LNCaP and PC-3 prostate cancer cells.

Smith S, Sepkovic D, Bradlow HL, Auborn KJ.

Department of Microbiology and Immunology, Albert Einstein College of Medicine,
Bronx, NY 10461, USA.

Evidence suggests that 17beta-estradiol (E2) contributes to the risk of prostate
cancer (PCa), whereas the phytochemicals genistein from soy and
3,3'-diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous
vegetables, decrease the risk of PCa. This study examined the potential of these
phytochemicals to reduce the adverse effects of E2 on PCa. In LNCaP PCa cells (E2
sensitive), DIM decreased E2-induced proliferation. Genistein increased
proliferation at low concentrations and decreased proliferation at higher
concentrations; DIM abolished the increased proliferation by genistein. The E2
stimulation in LNCaP cells was consistent with dependence on the androgen
receptor, as evidenced by the inhibition of E2-induced proliferation with the
antiandrogen casodex, E2 stimulation of an androgen response element luciferase
reporter, and E2 stimulation of prostate-specific antigen (PSA) protein
expression. Both genistein and DIM abrogated the E2 stimulation of PSA. Genistein
and DIM altered major E2 metabolism pathways in LNCaP and PC-3 (E2 insensitive)
PCa cells by increasing the expression of the 2-hydoxylation enzyme cytochrome
P450 1A1 (CYP1A1) and the O-methylating enzyme catechol-o-methyltransferase
(COMT) as determined by real-time RT-PCR. The increase in COMT mRNA occurred only
when the combination of DIM and genistein (15 micromol/L) was used. Quantitation
by MS indicated increased 2-hydroxyestrogen and decreased 16alpha-hydroxyestrone,
a result that should result in less estrogenicity and increased amounts of the
anticancer metabolite 2-methoxyestrone. We conclude that DIM and genistein
decrease the effects of E2 that have the potential to promote PCa.

PMID: 19022961

Pharm Res. 2008 Sep;25(9):2117-24. Epub 2008 Apr 22.

Chemoprevention of pancreatic cancer: characterization of Par-4 and its
modulation by 3,3' diindolylmethane (DIM).

Azmi AS, Ahmad A, Banerjee S, Rangnekar VM, Mohammad RM, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, 9374 Scott Hall, 540 E Canfield, Detroit, Michigan 48201, USA.

PURPOSE: Cancer chemoprevention is defined as the use of natural, synthetic, or
biological agents to suppress, reverse or prevent the carcinogenic process from
turning into aggressive cancer. Prostate apoptosis response-4 (Par-4) is a unique
pro-apoptotic protein that selectively induces apoptosis in prostate cancer
cells. However, its role in other malignancies has not been fully explored. This
study tries to identify the functional significance of Par-4 in pancreatic
cancer. METHODS: Multiple molecular techniques such as Western blot analysis,
trypan blue assay for cell viability, MTT assay for cell growth inhibition and
Histone/DNA ELISA for apoptosis were used. RESULTS: Western blot analysis
revealed that 3,3'-diindolylmethane (DIM) a chemopreventive agent, specifically
its more bioavailable formulation, DIM, at low doses (20 micromol/L) induces
Par-4, in L3.6pl and Colo-357 pancreatic cancer cells. At similar doses, DIM
reduced cell viability and caused cell growth inhibition and apoptosis. Moreover,
DIM pre-treatment sensitized the cells to cytotoxic action of chemotherapeutic
drug gemcitabine through up-regulation of Par-4. CONCLUSION: The induction of
Par-4 is indirectly related to increased sensitivity and cell death through
apoptosis. To our knowledge the results reported here showed, for the first time,
the induction of Par-4 by chemopreventive agents, in general, and DIM, in
particular, in pancreatic cancer cells in vitro.

PMID: 18427961

 

Food Chem Toxicol. 2008 Jul;46(7):2451-8. Epub 2008 Apr 6.

Inhibitory effects of a dietary phytochemical 3,3'-diindolylmethane (DIM) on the
phenobarbital-induced hepatic CYP mRNA expression and CYP-catalyzed reactions in
female rats.

Parkin DR, Lu Y, Bliss RL, Malejka-Giganti D.

Veterans Affairs Medical Center, Minneapolis, MN 55417, USA.

3,3'-diindolylmethane (DIM), derived from indole-3-carbinol (I3C), is used as a
dietary supplement for its putative anticancer effects that include suppression
of mammary tumor growth in female rats. The mechanism of action DIM may involve
its interaction(s) with hepatic cytochromes P450 (CYPs) catalyzing oxidations of
17beta-estradiol (E2). Our study showed that DIM added to hepatic microsomes of
female Sprague-Dawley rats was primarily a competitive inhibitor of
beta-naphthoflavone (beta-NF)- or I3C-induced CYP1A1 probe activity, and a potent
mixed or uncompetitive inhibitor of phenobarbital (PB)-induced CYP2B1 or CYP2B2
probe activity, respectively. Microsomal metabolites of DIM were tentatively
identified as two mono-hydroxy isomers of DIM, each formed preferentially by
CYP1A1- or CYP2B1/2-catalyzed reaction. Evaluation of the effects of co-treatment
of rats with PB and DIM by a full factorial ANOVA showed that DIM decreased the
PB-induced CYP2B1 and CYP2B2 mRNA expression levels, and the rates of 2- and
4-hydroxylation of E2, and total E2 metabolite formation. The results suggest
that interactions of DIM, and/or its mono-hydroxy metabolites, with CYP2B1 and
CYP2B2 found to occur in hepatic microsomes upon addition of DIM or co-treatment
of rats with DIM affect the rates of relevant oxidations of E2, and potentially
protect against estrogen-dependent tumorigenesis.

PMID: 18486294

 

Carcinogenesis. 2008 Jun;29(6):1139-47. Epub 2008 May 5.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell
and tumor growth through activation of c-jun N-terminal kinase.

Lei P, Abdelrahim M, Cho SD, Liu S, Chintharlapalli S, Safe S.

Institute of Biosciences and Technology, Texas A&M University Health Science
Center, Houston, TX 77030-3303, USA.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) activate the orphan
receptors peroxisome proliferator-activated receptor gamma (PPARgamma) and Nur77
and induce receptor-dependent and -independent apoptotic pathways in colon and
other cancer cells. Structure-activity studies show that the p-bromo
(DIM-C-pPhBr) and p-fluoro (DIM-C-pPhF) analogs, which exhibit minimal activation
of Nur77 and PPARgamma, induce expression of CCAAT/enhancer-binding protein
homologous protein (CHOP/GADD153) in colon cancer cells. Moreover, among a series
of bromo and fluoro C-DIM analogs, their induction of CHOP was dependent on the
position of the phenyl substituents (para >/= meta >/= ortho) and required a free
indole group. DIM-C-pPhBr and DIM-C-pPhF not only induced CHOP but also activated
death receptor 5 (CHOP dependent), cleavage of caspase 8 and poly (ADP ribose)
polymerase (PARP) that is consistent with activation of the extrinsic pathway of
apoptosis. These responses were associated with the activation of c-jun
N-terminal kinase (JNK) pathway since inhibition of JNK inhibited induction of
the extrinsic apoptotic pathway by these C-DIMs. However, in contrast to
classical inducers of endoplasmic reticulum (ER) stress such as tunicamycin and
thapsigargin, the C-DIM compounds did not induce glucose-related protein 78 that
is a marker of ER stress. Proapoptotic and anticarcinogenic effects were also
observed in athymic nude mice bearing RKO cell xenografts and treated with 30
mg/kg/day DIM-C-pPhBr and this was accompanied by increased JNK phosphorylation
in the tumors. Thus, the anticarcinogenic activity of DIM-C-pPhBr in colon cancer
cells and tumors is related to a novel ER stress-independent activation of JNK.

PMID: 18460448

 

Cancer Let. 2008 Jun 28;265(1):113-23. Epub 2008 Apr 2.

CXCR4 and CXCL12 down-regulation: a novel mechanism for the chemoprotection of
3,3'-diindolylmethane (DIM) for breast and ovarian cancers.

Hsu EL, Chen N, Westbrook A, Wang F, Zhang R, Taylor RT, Hankinson O.

Molecular Toxicology Interdepartmental Doctoral Program, University of
California-Los Angeles, Los Angeles, CA 90095-1732, USA.

Cruciferous vegetables are thought to protect against numerous types of cancer.
3,3'-Diindolylmethane (DIM) is an acid-catalyzed product generated during the
consumption of cruciferous vegetables and appears to be chemoprotective for
breast cancer. The interaction between the chemokine receptor, CXCR4, and its
unique ligand, CXCL12, is known to mediate the progression and metastasis of
breast and other cancers. Organs to which these cancers metastasize secrete
CXCL12, which binds to CXCR4 expressed on the surface of primary cancer cells.
This process subsequently stimulates the invasive properties of the cancer cells
and attracts them to the preferred organ sites of metastases. We have found that
DIM down-regulates both CXCR4 and CXCL12 in MCF-7 and MDA-MB-231 breast cancer
cells as well as in BG-1 ovarian cancer cells at the transcriptional level and in
an estrogen-independent manner. We demonstrate that the potential of MDA-MB-231
and BG-1 cells for chemotaxis and invasion towards CXCL12, but not towards IL-6
or fetal bovine serum, respectively, is inhibited by DIM. Furthermore, we show
that DIM down-regulates CXCR4 under hypoxia and CXCL12 under estradiol-inducing
conditions. Our data suggest that one mechanism whereby DIM protects against
breast, ovarian, and possibly other cancers is through the repression of CXCR4
and/or CXCL12, thereby lowering the invasive and metastatic potential of these
cells.

PMID: 18378071

 

Mol Cancer Ther. 2008 Jun;7(6):1708-19.

Apoptosis-inducing effect of erlotinib is potentiated by 3,3'-diindolylmethane (DIM) in
vitro and in vivo using an orthotopic model of pancreatic cancer.

Ali S, Banerjee S, Ahmad A, El-Rayes BF, Philip PA, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI, USA.

Blockade of epidermal growth factor receptor (EGFR) by EGFR tyrosine kinase
inhibitors is insufficient for effective antitumor activity because of
independently activated survival pathways. A multitargeted approach may therefore
improve the outcome of anti-EGFR therapies. In the present study, we determined
the effects of 3,3'-diindolylmethane on cell viability and apoptosis with
erlotinib in vitro and in vivo using an orthotopic animal tumor model. BxPC-3 and
MIAPaCa cells with varying levels of EGFR and nuclear factor-kappaB (NF-kappaB)
DNA-binding activity were treated with DIM (20 micromol/L), erlotinib (2
micromol/L), and the combination. Cell survival and apoptosis was assessed by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and histone-DNA
ELISA. Electrophoretic mobility shift assay was used to evaluate NF-kappaB
DNA-binding activity. We found significant reduction in cell viability by both
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic
assays, induction of apoptosis, down-regulation of EGFR phosphorylation,
NF-kappaB DNA-binding activity, and expression of antiapoptotic genes in BxPC-3
cells when treated with the combination of erlotinib and DIM compared with
either agent alone. In contrast, no such effect was observed in MIAPaCa cells by
similar treatment. Most importantly, these in vitro results were recapitulated in
animal model showing that DIM in combination with erlotinib was much more
effective as an antitumor agent compared with either agent alone. These results
suggest that the utilization of DIM could be a useful strategy for achieving
better treatment outcome in patients with activated status of EGFR and NF-kappaB
in their tumors.

PMID: 18566242

Biochem Pharmacol. 2008 May 1;75(9):1858-67.

3,3'-diindolylmethane (DIM) reduces levels of HIF-1alpha and HIF-1 activity in hypoxic
cultured human cancer cells.

Riby JE, Firestone GL, Bjeldanes LF.

Department of Nutritional Sciences and Toxicology, 217 Morgan Hall, University of
California, Berkeley, CA 94720, USA.

3,3'-diindolylmethane (DIM) is a chemopreventive and chemotherapeutic
phytochemical derived from the metabolism of indoles found at high concentrations
in cruciferous vegetables. We have previously shown that DIM exhibits
anti-angiogenic properties in cultured vascular endothelial cells and in Matrigel
plug assays in rodents. In the present study, we demonstrate that DIM reduces the
level of hypoxia-inducible factor (HIF)-1alpha in hypoxic tumor cell lines, as
well as HIF-1 transcriptional activity as measured by a reporter assay. Moreover,
DIM inhibited the expression of HIF-1-responsive endogenous genes, resulting in
the reduced expression of key hypoxia responsive factors, VEGF, furin, enolase-1,
glucose transporter-1 and phosphofructokinase. DIM reduced the level of
HIF-1alpha in hypoxic cells by increasing the rate of the prolylhydroxylase- and
proteasome-mediated degradation of HIF-1alpha, and by decreasing the rate of
HIF-1alpha transcription. Using enzyme kinetics studies, we established that DIM
interacts with the oligomycin-binding site on the F0 transmembrane component of
mitochondrial F1F0-ATPase. The contributions of the resulting increases in levels
of ROS and O2 in hypoxic cells to the inhibitory effects of DIM on HIF-1alpha
expression are discussed. These studies are the first to show that DIM can
decrease the accumulation and activity of the key angiogenesis regulatory factor,
HIF-1alpha, in hypoxic tumor cells.

PMID: 18329003

Pharm Res. 2008 Apr 22

Chemoprevention of Pancreatic Cancer: Characterization of Par-4 and its
Modulation by 3,3' Diindolylmethane (DIM).

Azmi AS, Ahmad A, Banerjee S, Rangnekar VM, Mohammad RM, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, 9374 Scott Hall, 540 E Canfield, Detroit, Michigan, 48201, USA.

PURPOSE: Cancer chemoprevention is defined as the use of natural, synthetic, or
biological agents to suppress, reverse or prevent the carcinogenic process from
turning into aggressive cancer. Prostate apoptosis response-4 (Par-4) is a unique
pro-apoptotic protein that selectively induces apoptosis in prostate cancer
cells. However, its role in other malignancies has not been fully explored. This
study tries to identify the functional significance of Par-4 in pancreatic
cancer. METHODS: Multiple molecular techniques such as Western blot analysis,
trypan blue assay for cell viability, MTT assay for cell growth inhibition and
Histone/DNA ELISA for apoptosis were used. RESULTS: Western blot analysis
revealed that 3,3'-diindolylmethane (DIM) a chemopreventive agent, specifically
its more bioavailable formulation, DIM, at low doses (20 mumol/L) induces
Par-4, in L3.6pl and Colo-357 pancreatic cancer cells. At similar doses, DIM
reduced cell viability and caused cell growth inhibition and apoptosis. Moreover,
DIM pre-treatment sensitized the cells to cytotoxic action of chemotherapeutic
drug gemcitabine through up-regulation of Par-4. CONCLUSION: The induction of
Par-4 is indirectly related to increased sensitivity and cell death through
apoptosis. To our knowledge the results reported here showed, for the first time,
the induction of Par-4 by chemopreventive agents, in general, and DIM, in
particular, in pancreatic cancer cells in vitro.

PMID: 18427961

Cancer Let. 2008 Mar 28

CXCR4 and CXCL12 down-regulation: A novel mechanism for the chemoprotection of
3,3'-diindolylmethane (DIM) for breast and ovarian cancers.

Hsu EL, Chen N, Westbrook A, Wang F, Zhang R, Taylor RT, Hankinson O.

Molecular Toxicology Interdepartmental Doctoral Program, University of
California-Los Angeles, Los Angeles, CA 90095-1732, USA; Department of Pathology
and Laboratory Medicine, University of California-Los Angeles, Los Angeles, CA
90095-1732, USA; Jonsson Comprehensive Cancer Center, University of
California-Los Angeles, Los Angeles, CA 90095-1732, USA.

Cruciferous vegetables are thought to protect against numerous types of cancer.
3,3'-Diindolylmethane (DIM) is an acid-catalyzed product generated during the
consumption of cruciferous vegetables and appears to be chemoprotective for
breast cancer. The interaction between the chemokine receptor, CXCR4, and its
unique ligand, CXCL12, is known to mediate the progression and metastasis of
breast and other cancers. Organs to which these cancers metastasize secrete
CXCL12, which binds to CXCR4 expressed on the surface of primary cancer cells.
This process subsequently stimulates the invasive properties of the cancer cells
and attracts them to the preferred organ sites of metastases. We have found that
DIM down-regulates both CXCR4 and CXCL12 in MCF-7 and MDA-MB-231 breast cancer
cells as well as in BG-1 ovarian cancer cells at the transcriptional level and in
an estrogen-independent manner. We demonstrate that the potential of MDA-MB-231
and BG-1 cells for chemotaxis and invasion towards CXCL12, but not towards IL-6
or fetal bovine serum, respectively, is inhibited by DIM. Furthermore, we show
that DIM down-regulates CXCR4 under hypoxia and CXCL12 under estradiol-inducing
conditions. Our data suggest that one mechanism whereby DIM protects against
breast, ovarian, and possibly other cancers is through the repression of CXCR4
and/or CXCL12, thereby lowering the invasive and metastatic potential of these
cells.

PMID: 18378071

Cancer Res. 2008 Mar 15;68(6):1927-34.

Mammalian target of rapamycin repression by 3,3'-diindolylmethane (DIM) inhibits
invasion and angiogenesis in platelet-derived growth factor-D-overexpressing PC3
cells.

Kong D, Banerjee S, Huang W, Li Y, Wang Z, Kim HR, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI 48201, USA.

Platelet-derived growth factor-D (PDGF-D) is a newly recognized growth factor
known to regulate many cellular processes, including cell proliferation,
transformation, invasion, and angiogenesis. Recent studies have shown that PDGF-D
and its cognate receptor PDGFR-beta are expressed in prostate tumor tissues,
suggesting that PDGF-D might play an important role in the development and
progression of prostate cancer. However, the biological role of PDGF-D in
tumorigenesis remains elusive. In this study, we found that PDGF-D-overexpressing
PC3 cells (PC3 cells stably transfected with PDGF-D cDNA and referred to as PC3
PDGF-D) exhibited a rapid growth rate and enhanced cell invasion that was
associated with the activation of mammalian target of rapamycin (mTOR) and
reduced Akt activity. Rapamycin repressed mTOR activity and concomitantly
resulted in the activation of Akt, which could attenuate the therapeutic effects
of mTOR inhibitors. In contrast, DIM significantly inhibited both mTOR and Akt in

PC3 PDGF-D cells, which were correlated with decreased cell proliferation and invasion.
Moreover, conditioned medium from PC3 PDGF-D cells significantly increased the
tube formation of human umbilical vein endothelial cells, which was inhibited by
DIM treatment concomitant with reduced full-length and active form of PDGF-D.
Our results suggest that DIM could serve as a novel and efficient
chemopreventive and/or therapeutic agent by inactivation of both mTOR and Akt
activity in PDGF-D-overexpressing prostate cancer.

PMID: 18339874

Zhonghua Yi Xue Za Zhi. 2008 Mar 11;88(10):661-4.

3,3-diindolylmethane (DIM) enhances the inhibitory effect of idarubicin on the growth
of human prostate cancer cells (Article in Chinese)

Zhao YY, Zhou L, Pan YZ, Zhao LJ, Liu YN, Yu H, Li Y, Zhao XJ.

Prostate Diseases Prevention and Treatment Research Center, Jilin University,
Changchun 130021, China.

OBJECTIVE: To study the effects of idarubicin (IDA) combined with 3,
3-diindolylmethane (DIM) on the growth inhibition of human prostate cancer cells.
METHODS: Human prostate cancer cells of the line PC-3M were cultured and then
divided into the following groups: control group with solvent added into the
culture fluid; IDA groups, with IDA of the terminal concentrations of 0.5, 1 or 5
mg/L added into the culture fluid; DIM groups, with DIM of the terminal
concentrations of 30, 60 or 100 micromol/L added into the culture fluid; and DIM
+ IDA groups, with 0. 5 mg/L IDA and DIM 30, 60 or 100 micromol/L added into the
culture fluid. 48 h later the cell growth inhibition rate was detected by MTT
assay. Flow cytometry and acridine orange staining were used to detect the cell
cycle and apoptosis. RT-PCR and Western blotting were used to detect the mRNA and
protein expression of caspase 9, an apoptosis gene. RESULTS: Both IDA and DIM
dose-dependently inhibited the growth of the PC-3M cells. The growth inhibition
rate of the 60 micromol/L DIM + 0.5 mg/L IDA group was 69.9%, almost 10 times as
that of the 0.5 mg/L IDA group. The apoptosis rate of the 60 micromol/L DIM + 0.
5 mg/L IDA group was 47.0%, significantly higher than that of the 0.5 mg/L IDA
group (3.2%, P < 0.05). RT-PCR and Western blotting showed that the combination
of DIM and IDA significantly enhanced the mRNA and protein expression of caspase
9. CONCLUSION: DIM enhances the growth inhibition effect of IDA on human prostate
cancer cells by the mechanism of induction of apoptosis.

PMID: 18642764

Mol Cancer Ther. 2008 Feb;7(2):341-9.

Induction of growth arrest and apoptosis in human breast cancer cells by
3,3-diindolylmethane (DIM) is associated with induction and nuclear localization of
p27kip.

Wang Z, Yu BW, Rahman KM, Ahmad F, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI 48201, USA.

3,3'-Diindolylmethane (DIM) is a stable condensation product of
indole-3-carbanol, a potential breast cancer chemoprevention agent. Human breast
cancer cell lines were studied to better understand its mechanisms. In vitro
experiments were done in MCF-7, T47D, BT-20 and BT-474 cells using MTT, ELISA,
immunoblotting assays, reverse transcription-PCR, protein half-life, confocal
microscopy, cell fractionation, and immunoprecipitation assays. We found that DIM
inhibited the growth of all four breast cancer cell lines (IC(50)s, 25-56
micromol/L). Because BT-20 and BT-474 overexpressed Her-2 and activated Akt, and
BT-20 lacks estrogen receptor, these were studied further. In both cell lines,
DIM appeared to induce expression of p27(kip) protein before the loss of cell
viability and apoptosis. In BT-20 cells, DIM also inhibited expression of
activated Akt, but this appeared after p27(kip) induction. In both cell lines,
DIM induced p27(kip) transcript expression within 6 h. DIM prolonged the p27(kip)
protein half-life in BT-20 but not BT-474 cells. We also showed, for the first
time, that DIM induced nuclear localization of p27(kip) in both cell lines.
Moreover, in BT-20 cells, DIM induced a decrease in p27(kip) phosphorylation at
Thr(187), and its association with the 14-3-3 protein, which helped to explain
the protein half-life increase and nuclear localization, respectively. DIM
modulates p27(kip) through transcription, prolongation of protein half-life, and
nuclear localization. These effects appear to be independent of Her-2, Akt, or
estrogen receptor status and should support further study for its chemoprevention
potential in breast cancer.

PMID: 18281517

J Nutr. 2008 Jan;138(1):17-23.

 

3,3'-Diindolylmethane (DIM) suppresses the inflammatory response to lipopolysaccharide

in murine macrophages.

 

Cho HJ, Seon MR, Lee YM, Kim J, Kim JK, Kim SG, Park JH.

 

Center for Efficacy Assessment and Development of Functional Foods and Drugs,

Hallym University, Chuncheon 200-702, South Korea.

 

3,3'-Diindolylmethane (DIM), a major acid-condensation product of

indole-3-carbinol, has been shown to have multiple anticancer effects in

experimental models. Because recurrent or chronic inflammation has been

implicated in the development of a variety of human cancers, this study examined

the antiinflammatory effects of DIM and the underlying mechanisms using

lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages. DIM

significantly decreased the release of nitric oxide (NO), prostaglandin (PG)E2,

tumor necrosis factor alpha, interleukin (IL)-6, and IL-1beta by RAW264.7 cells

treated with LPS. DIM inhibited LPS-induced increases in protein levels of

inducible NO synthase (iNOS), which were accompanied by decreased iNOS mRNA

levels and transcriptional activity. The mRNA levels of phospholipase A2

decreased, whereas neither cyclooxygenases-2 protein nor transcript was altered

by DIM. In addition, DIM suppressed LPS-induced nuclear factor-kappaB (NF-kappaB)

transcriptional activity, NF-kappaB DNA-binding activity, translocation of p65

(RelA) to the nucleus, and degradation of inhibitor of kappaB alpha. Furthermore,

DIM decreased LPS-induced transcriptional activity of activator protein (AP)-1,

AP-1 DNA-binding activity, and phosphorylation of stress-activated protein

kinase/Jun-N-terminal kinase and c-Jun. We demonstrate that DIM inhibits

LPS-induced release of proinflammatory mediators in murine macrophages.

Downregulation of NF-kappaB and AP-1 signaling may be one of the mechanisms by

which DIM inhibits inflammatory responses.

 

PMID: 18156398

 

Mol Cancer Ther. 2007 Nov;6(11):3071-9.

 

Extended treatment with physiologic concentrations of dietary phytochemicals

results in altered gene expression, reduced growth, and apoptosis of cancer

cells.

 

Moiseeva EP, Almeida GM, Jones GD, Manson MM.

 

Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester,

University Road, Leicester LE1 7RH, United Kingdom.

 

Dietary phytochemicals exhibit chemopreventive potential in vivo through

persistent low-dose exposures, whereas mechanistic in vitro studies with these

agents generally use a high-dose single treatment. Because the latter approach is

not representative of an in vivo steady state, we investigated antitumor activity

of curcumin, 3,3'-diindolylmethane (DIM), epigallocatechin gallate (EGCG),

genistein, or indole-3-carbinol (I3C) in breast cancer MDA-MB-231 cells, exposed

in long-term culture to low concentrations, achievable in vivo. Curcumin and EGCG

increased cell doubling time. Curcumin, EGCG, and I3C inhibited clonogenic growth

by 55% to 60% and induced 1.5- to 2-fold higher levels of the basal caspase-3/7

activity. No changes in expression of cell cycle-related proteins or survivin

were found; however, I3C reduced epidermal growth factor receptor expression,

contributing to apoptosis. Because some phytochemicals are shown to inhibit DNA

and histone modification, modulation of expression by the agents in a set of

genes (cadherin-11, p21Cip1, urokinase-type plasminogen activator, and

interleukin-6) was compared with changes induced by inhibitors of DNA methylation

or histone deacetylation. The phytochemicals modified protein and/or RNA

expression of these genes, with EGCG eliciting the least and DIM the most changes

in gene expression. DIM and curcumin decreased cadherin-11 and increased

urokinase-type plasminogen activator levels correlated with increased cell

motility. Curcumin, DIM, EGCG, and genistein reduced cell sensitivity to

radiation-induced DNA damage without affecting DNA repair. This model has

revealed that apoptosis and not arrest is likely to be responsible for growth

inhibition. It also implicated new molecular targets and activities of the agents

under conditions relevant to human exposure.

 

PMID: 18025290 [PubMed - in process]

  

Mol Cancer Ther. 2007 Oct;6(10):2757-65. Epub 2007 Oct 3.

 

Inactivation of NF-kappaB by 3,3'-diindolylmethane (DIM) contributes to increased

apoptosis induced by chemotherapeutic agent in breast cancer cells.

 

Rahman KM, Ali S, Aboukameel A, Sarkar SH, Wang Z, Philip PA, Sakr WA, Raz A.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, 715 HWCRC, 4100 John R, Detroit, MI 48201, USA.

 

Constitutive activation of Akt or nuclear factor-kappaB (NF-kappaB) has been

reported to play a role in de novo resistance of cancer cells to chemotherapeutic

agents, which is a major cause of treatment failure in cancer chemotherapy.

Previous studies have shown that 3,3'-diindolylmethane (DIM), a major in vivo

acid-catalyzed condensation product of indole-3-carbinol, is a potent inducer of

apoptosis, inhibitor of tumor angiogenesis, and inactivator of Akt/NF-kappaB

signaling in breast cancer cells. However, little is known regarding the

inactivation of Akt/NF-kappaB that leads to chemosensitization of breast cancer

cells to chemotherapeutic agents, such as Taxotere. Therefore, we examined

whether the inactivation Akt/NF-kappaB signaling caused by DIM could sensitize

breast cancer cells to chemotherapeutic agents both in vitro and in vivo.

MDA-MB-231 cells were simultaneously treated with 15 to 45 micromol/L DIM and

0.5 to 1.0 nmol/L Taxotere for 24 to 72 h. Cell growth inhibition assay,

apoptosis assay, electrophoretic mobility shift assay, and Western blotting were

done. The combination treatment of 30 micromol/L DIM with 1.0 nmol/L Taxotere

elicited significantly greater inhibition of cell growth compared with either

agent alone. The combination treatment induced greater apoptosis in MDA-MB-231

cells compared with single agents. Moreover, we found that NF-kappaB activity was

significantly decreased in cells treated with DIM and Taxotere. We also have

tested our hypothesis using transfection studies, followed by combination

treatment with DIM/Taxotere, and found that combination treatment significantly

inhibited cell growth and induced apoptosis in MDA-MB-231 breast cancer cells

mediated by the inactivation of NF-kappaB, a specific target in vitro and in

vivo. These results were also supported by animal experiments, which clearly

showed that DIM sensitized the breast tumors to Taxotere, which resulted in

greater antitumor activity mediated by the inhibition of Akt and NF-kappaB.

Collectively, our results clearly suggest that inhibition of Akt/NF-kappaB

signaling by DIM leads to chemosensitization of breast cancer cells to

Taxotere, which may contribute to increased growth inhibition and apoptosis in

breast cancer cells. The data obtained from our studies could be a novel

breakthrough in cancer therapeutics by using nontoxic agents, such as DIM, in

combination with other conventional therapeutic agents, such as Taxotere.

  

PMID: 17913854

 

J Nutr Biochem. 2007 Aug 16 [Epub ahead of print]

 

3,3'-Diindolylmethane (DIM) stimulates murine immune function in vitro and in vivo.

 

Xue L, Pestka JJ, Li M, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720-3104, USA.

 

3,3'-Diindolylmethane (DIM), a major condensation product of indole-3-carbinol,

exhibits chemopreventive properties in animal models of cancer. Recent studies

have shown that DIM stimulates interferon-gamma (IFN-gamma) production and

potentiates the IFN-gamma signaling pathway in human breast cancer cells via a

mechanism that includes increased expression of the IFN-gamma receptor. The goal

of this study was to test the hypothesis that DIM modulates the murine immune

function. Specifically, the effects of DIM were evaluated in a panel of murine

immune function tests that included splenocyte proliferation, reactive oxygen

species (ROS) generation, cytokine production and resistance to viral infection.

DIM was found to induce proliferation of splenocytes as well as augment mitogen-

and interleukin (IL)-2-induced splenocyte proliferation. DIM also stimulated the

production of ROS by murine peritoneal macrophage cultures. Oral administration

of DIM, but not intraperitoneal injection, induced elevation of serum cytokines

in mice, including IL-6, granulocyte colony-stimulating factor (G-CSF), IL-12 and

IFN-gamma. Finally, in a model of enteric virus infection, oral DIM

administration to mice enhanced both clearance of reovirus from the GI tract and

the subsequent mucosal IgA response. Thus, DIM is a potent stimulator of immune

function. This property might contribute to the cancer inhibitory effects of this

indole.

 

PMID: 17707631 [PubMed - as supplied by publisher]

  

J Biol Chem. 2007 Jul 20;282(29):21542-50. Epub 2007 May 23.

 

Regulation of FOXO3a/beta-catenin/GSK-3beta signaling by 3,3'-diindolylmethane (DIM)

contributes to inhibition of cell proliferation and induction of apoptosis in

prostate cancer cells.

 

Li Y, Wang Z, Kong D, Murthy S, Dou QP, Sheng S, Reddy GP, Sarkar FH.

 

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State

University School of Medicine, Detroit, Michigan 48201, USA.

 

Previous studies from our laboratory have shown anti-proliferative and

pro-apoptotic effects of 3,3'-diindolylmethane (DIM) through regulation of Akt

and androgen receptor (AR) in prostate cancer cells. However, the mechanism by

which DIM regulates Akt and AR signaling pathways has not been fully

investigated. It has been known that FOXO3a and glycogen synthase kinase-3beta

(GSK-3beta), two targets of activated Akt, interact with beta-catenin, regulating

cell proliferation and apoptotic cell death. More importantly, FOXO3a, GSK-3beta,

and beta-catenin are all AR coregulators and regulate the activity of AR,

mediating the development and progression of prostate cancers. Here, we

investigated the molecular effects of DIM, a formulated DIM with higher

bioavailability, on Akt/FOXO3a/GSK-3beta/beta-catenin/AR signaling in

hormone-sensitive LNCaP and hormone-insensitive C4-2B prostate cancer cells. We

found that DIM significantly inhibited the phosphorylation of Akt and FOXO3a

and increased the phosphorylation of beta-catenin, leading to the inhibition of

cell growth and induction of apoptosis. We also found that DIM significantly

inhibited beta-catenin nuclear translocation. By electrophoretic mobility shift

and chromatin immunoprecipitation assays, we found that DIM inhibited FOXO3a

binding to the promoter of AR and promoted FOXO3a binding to the p27(KIP1)

promoter, resulting in the alteration of AR and p27(KIP1) expression, the

inhibition of cell proliferation, and the induction of apoptosis in both

androgen-sensitive and -insensitive prostate cancer cells. These results suggest

that DIM-induced cell growth inhibition and apoptosis induction are partly

mediated through the regulation of Akt/FOXO3a/GSK-3beta/beta-catenin/AR

signaling. Therefore, DIM could be a promising non-toxic agent for possible

treatment of hormone-sensitive but most importantly hormone-refractory prostate

cancers.

 

PMID: 17522055

 

Mol Med. 2007 Jan-Feb;13(1-2):69-78.

 

Interplay of genes regulated by estrogen and diindolylmethane (DIM) in breast cancer

cell lines.

 

Mulvey L, Chandrasekaran A, Liu K, Lombardi S, Wang XP, Auborn KJ, Goodwin L.

 

Feinstein Institute for Medical Research, Manhasset, NY 11030, USA.

 

Diindolylmethane (DIM), a biologically active congener of indole-3-carbinol (I3C)

derived from cruciferous vegetables, is a promising agent for the prevention of

estrogen-sensitive cancers. Both DIM and estrogen affect transcription of genes

by binding receptors, such as aryl hydrocarbon receptor (AhR) or estrogen

receptors (ER). Gene regulation by DIM and estradiol (E2) can be very complex.

While DIM typically binds the AhR, this complex can directly associate with the

ER, recruit co-activators that bind to estrogen-responsive promoters, and

activate transcription. Alternately, DIM can bind the ER directly. In this study,

we have analyzed gene expression using microarray profiling and quantitative real

time-polymerase chain reaction in MCF7 breast cancer cells treated with E2 (1 nM)

or DIM (25 microM) alone or in combination for 16 h. The interplay of E2 and DIM

was reflected in the expression of a subset of genes (<90) in which the

combination of E2 and DIM acted either additively or antagonistically to alter

gene expression.

 

PMID: 17515958

 

Cancer Res. 2007 Apr 1;67(7):3310-9.

 

Inhibition of angiogenesis and invasion by 3,3'-diindolylmethane (DIM) is mediated by

the nuclear factor-kappaB downstream target genes MMP-9 and uPA that regulated

bioavailability of vascular endothelial growth factor in prostate cancer.

 

Kong D, Li Y, Wang Z, Banerjee S, Sarkar FH.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, Michigan, USA.

 

Progression of prostate cancer is believed to be dependent on angiogenesis

induced by tumor cells. 3,3'-Diindolylmethane (DIM) has been shown to repress

neovascularization in a Matrigel plug assay and inhibit cell proliferation,

migration, invasion, and capillary tube formation of cultured human umbilical

vein endothelial cells. However, the molecular mechanism, by which DIM inhibits

angiogenesis and invasion, has not been fully elucidated. Therefore, we sought to

explore the molecular mechanism by which DIM inhibits angiogenesis and invasion,

specifically by investigating the role of angiogenic factors secreted by prostate

cancer cells which control all steps of angiogenesis. We found that DIM inhibited

angiogenesis and invasion by reducing the bioavailability of vascular endothelial growth

factor (VEGF) via repressing extracellular matrix-degrading proteases, such as

matrix metalloproteinase (MMP)-9 and urokinase-type plasminogen activator (uPA),

in human prostate cancer cells and reduced vascularity (angiogenesis) in vivo

using Matrigel plug assay. We also found that DIM treatment inhibited DNA

binding activity of nuclear factor-kappaB (NF-kappaB), which is known to mediate

the expression of many NF-kappaB downstream target genes, including VEGF, IL-8,

uPA, and MMP-9, all of which are involved in angiogenesis, invasion, and

metastasis. Our data suggest that inhibition of NF-kappaB DNA binding activity by

DIM contributes to the regulated bioavailability of VEGF by MMP-9 and uPA and,

in turn, inhibits invasion and angiogenesis, which could be mechanistically

linked with the antitumor activity of DIM as observed previously by our

laboratory in a prostate cancer animal model.

 

PMID: 17409440

 

Carcinogenesis. 2007 Jul;28(7):1471-7. Epub 2007 Feb 28.

 

Quantitative combination effects between sulforaphane and 3,3'-diindolylmethane (DIM)

on proliferation of human colon cancer cells in vitro.

 

Pappa G, Strathmann J, L÷winger M, Bartsch H, Gerhńuser C.

 

Division of Toxicology and Cancer Risk Factors, German Cancer Research Center

(DKFZ), C010-2 Chemoprevention, Im Neuenheimer Feld 280, 69120 Heidelberg,

Germany.

 

Isothiocyanates (ITCs) and indoles derived from cruciferous vegetables possess

growth-inhibiting and apoptosis-inducing activities in cancer cell lines in

vitro. ITCs like sulforaphane (SFN) are cytotoxic, whereas indoles including

indole-3-carbinol or its condensation product 3,3'-diindolylmethane (DIM) are

acting by cytostatic mechanisms in human colon cancer cell lines. In the present

study, we have investigated the impact of defined combinations of SFN and DIM

(ratio 1:4, 1:2, 1:1, 2:1 and 4:1) on cell proliferation, cell-cycle progression

and apoptosis induction in cultured 40-16 colon carcinoma cells. Calculations of

combination effects were based on the method of Chou et al. (1984) Adv. Enzyme

Regul., 22, 27-55, and were expressed as a combination index (CI) with CI < 1, CI

= 1 or CI > 1 representing synergism, additivity or antagonism, respectively.

Interestingly, at a total drug concentration of 2.5 microM, all combinations of

SFN and DIM were antagonistic. With increasing concentrations, the antagonistic

effect gradually turned into a synergistic interaction at the highest combined

cytotoxic concentration of 40 microM. Cell-cycle analyses with SFN:DIM ratios of

1:1, 1:2 and 1:4 and total concentrations between 10 and 25 microM confirmed

antagonism at low and additive effects at higher doses. SFN (10 microM) in

combination with DIM (10 microM) resulted in strong G(2)/M cell-cycle arrest,

which was not observed with either compound alone. Our results indicate that

cytotoxic concentrations of SFN:DIM combinations affect cell proliferation

synergistically. At low total concentrations (below 20 microM), which are

physiologically more relevant, the combined broccoli compounds showed

antagonistic interactions in terms of cell growth inhibition. These data stress

the need for elucidating mechanistic interactions for better predicting

beneficial health effects of bioactive food components.

 

PMID: 17331956

   

J Nutr. 2007 Jan;137(1):31-6.

 

Activation of caspase-8 contributes to 3,3'-Diindolylmethane-induced apoptosis in

colon cancer cells.

 

Kim EJ, Park SY, Shin HK, Kwon DY, Surh YJ, Park JH.

 

Center for Efficacy Assessment and Development of Functional Foods and Drugs,

Hallym University, Chuncheon, 200-702, Korea.

 

3,3'-Diindolylmethane (DIM) is the major in vivo product of acid-catalyzed

oligomerization of indole-3-carbinol, which is a promising anticancer agent

present in cruciferous vegetables and has itself been reported to have

anticarcinogenic properties. This study examined DIM-mediated regulation of

apoptosis in the HCT116 (wild-type p53) and HT-29 (mutant p53) human colon cancer

cell lines. DIM (0-30 micromol/L) substantially decreased the number of viable

cells and induced apoptosis of HCT116 and HT-29 cells in a

concentration-dependent manner. Western-blot analyses of total cell lysates

revealed that DIM increased the activation of caspase-3, -7, -8, and -9 and

enhanced poly(ADP-ribose) polymerase cleavage in both HCT116 and HT-29 cells. In

addition, DIM increased the translocation of cytochrome c and Smac/Diablo from

the mitochondria to the cytoplasm. In concert with the caspase-8 activation by

DIM, increased levels of Fas and truncated Bid were observed. DIM did not affect

the protein levels of p53, Bcl-2, Bax, or Fas ligand (FasL) in HCT116 cells. In

HT-29 cells, however, DIM decreased Bcl-2 levels, although the protein levels of

Bax or FasL were not affected. The caspase-8 inhibitor Z-IETD-FMK attenuated the

DIM-induced apoptosis, indicating that increased activation of this enzyme

contributed to the increase in p53-independent apoptosis that was observed in

colon cancer cells. We have demonstrated that DIM induces apoptosis in colon

cancer cells, providing insights into the mechanisms underlying its

antitumorigenic activities.

 

PMID: 17182797

  

Mol Pharmacol. 2007 Feb;71(2):558-69. Epub 2006 Nov 8.

 

1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes inhibit growth, induce

apoptosis, and decrease the androgen receptor in LNCaP prostate cancer cells

through peroxisome proliferator-activated receptor gamma-independent pathways.

 

Chintharlapalli S, Papineni S, Safe S.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466

TAMU, Vet. Res. Bldg. 409, College Station, TX 77843-4466, USA.

 

1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes (C-DIMs) containing

para-trifluoromethyl, t-butyl, and phenyl groups are a novel class of peroxisome

proliferator-activated receptor (PPAR)gamma agonists. In LNCaP prostate cancer

cells, these compounds induce PPARgamma-dependent transactivation, inhibit cell

proliferation, and induce apoptosis. In addition, these PPARgamma agonists

modulate a number of antiproliferative and proapoptotic responses, including

induction of p27, activating transcription factor 3, and nonsteroidal

anti-inflammatory drug-activated gene-1 and down-regulation of cyclin D1 and

caveolin-1. Moreover, the PPARgamma antagonist 2-chloro-5-nitrobenzanilide

(GW9662) does not inhibit these effects. The C-DIM compounds also abrogate

androgen receptor (AR)-mediated signaling and decrease prostate-specific antigen

(PSA) and AR protein expression, and these responses were PPARgamma-independent.

The effects of C-DIMs on AR and PSA were due to decreased AR and PSA mRNA

expression in LNCaP cells. Thus, this series of methylene-substituted

diindolylmethane derivatives simultaneously activate multiple pathways in LNCaP

cells, including ablation of androgen-responsiveness and down-regulation of

caveolin-1. Both of these responses are associated with activation of

proapoptotic pathways in this cell line.

 

PMID: 17093136

 

Am J Pathol. 2006 Nov;169(5):1833-42.

 

Fas-mediated apoptosis in cholangiocarcinoma cells is enhanced by

3,3'-diindolylmethane (DIM) through inhibition of AKT signaling and FLICE-like

inhibitory protein.

 

Chen Y, Xu J, Jhala N, Pawar P, Zhu ZB, Ma L, Byon CH, McDonald JM.

 

Department of Pathology, University of Alabama at Birmingham, LHRB 511, 1530 3rd

Ave. South, Birmingham, AL 35294, USA.

 

Stimulation of Fas-mediated apoptosis has been promoted as a potential therapy

for many cancers, including cholangiocarcinoma. We have previously reported that

Fas-resistant, but not Fas-sensitive, cholangiocarcinoma cells are tumorigenic in

nude mice. The present studies sought to identify molecular targets that promote

Fas-mediated apoptosis in cholangiocarcinoma. We found that Fas-resistant

cholangiocarcinoma cells exhibited increased constitutive phosphorylation of AKT

compared with Fas-sensitive cells. Increased phosphorylation of AKT was also

demonstrated in human cholangiocarcinoma tumors and was evident in a mouse

xenograft cholangiocarcinoma model. Furthermore, we found that

3,3'-diindolylmethane (DIM), a vegetable autolysis product, promoted Fas-mediated

apoptosis of cholangiocarcinoma cells. DIM inhibited phosphorylation of AKT and

activation of FLICE-like-inhibitory-protein (FLIP). Inhibition of

phosphatidylinositol 3-kinase/AKT decreased FLIP activation and promoted

Fas-mediated apoptosis. By contrast, adenovirus-mediated constitutively activated

AKT protected cholangiocarcinoma cells from Fas-mediated apoptosis. Decreased

activation of extracellular signal-regulated kinase and nuclear factor-kappaB and

increased activation of caspase-3, -8, and -9 were associated with inhibition of

AKT and FLIP. These results support AKT and FLIP as potential molecular targets

and DIM as a potent compound for cholangiocarcinoma intervention.

 

PMID: 17071604

 

Cancer Res. 2006 Oct 15;66(20):10064-72.

 

Down-regulation of androgen receptor by 3,3'-diindolylmethane (DIM) contributes to

inhibition of cell proliferation and induction of apoptosis in both

hormone-sensitive LNCaP and insensitive C4-2B prostate cancer cells.

 

Bhuiyan MM, Li Y, Banerjee S, Ahmed F, Wang Z, Ali S, Sarkar FH.

 

Departments of Pathology and Internal Medicine, Karmanos Cancer Institute, Wayne

State University School of Medicine, Detroit, Michigan 48201, USA.

 

Despite the initial efficacy of androgen deprivation therapy, most patients with

advanced prostate cancer eventually progress to hormone-refractory prostate

cancer, for which there is no curative therapy. Previous studies from our

laboratory and others have shown the antiproliferative and proapoptotic effects

of 3,3'-diindolylmethane (DIM) in prostate cancer cells. However, the molecular

mechanism of action of DIM has not been investigated in androgen receptor

(AR)-positive hormone-responsive and -nonresponsive prostate cancer cells.

Therefore, we investigated the effects of DIM, a formulated DIM with greater

bioavailability, on AR, Akt, and nuclear factor kappaB (NF-kappaB) signaling in

hormone-sensitive LNCaP (AR+) and hormone-insensitive C4-2B (AR+) prostate cancer

cells. We found that DIM significantly inhibited cell proliferation and induced

apoptosis in both cell lines. By Akt gene transfection, reverse

transcription-PCR, Western blot analysis, and electrophoretic mobility shift

assay, we found a potential crosstalk between Akt, NF-kappaB, and AR.

Importantly, DIM significantly inhibited Akt activation, NF-kappaB DNA binding

activity, AR phosphorylation, and the expressions of AR and prostate-specific

antigen, suggesting that DIM could interrupt the crosstalk. Confocal studies

revealed that DIM inhibited AR nuclear translocation, leading to the

down-regulation of AR target genes. Moreover, DIM significantly inhibited C4-2B

cell growth in a severe combined immunodeficiency-human model of experimental

prostate cancer bone metastasis. These results suggest that DIM-induced cell

proliferation inhibition and apoptosis induction are partly mediated through the

down-regulation of AR, Akt, and NF-kappaB signaling. These observations provide a

rationale for devising novel therapeutic approaches for the treatment of

hormone-sensitive, but more importantly, hormone-refractory prostate cancer by

using DIM alone or in combination with other therapeutics.

 

PMID: 17047070

   

Cancer Res. 2006 May 1;66(9):4952-60.

 

Gene expression profiling revealed survivin as a target of

3,3'-diindolylmethane-induced cell growth inhibition and apoptosis in breast

cancer cells.

 

Rahman KW, Li Y, Wang Z, Sarkar SH, Sarkar FH.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, Michigan 48201, USA.

 

The phytochemical indole-3-carbinol (I3C), found in cruciferous vegetables, and

its major acid-catalyzed reaction product 3,3'-diindolylmethane (DIM) showed

anticancer activity mediated by its pleiotropic effects on cell cycle

progression, apoptosis, carcinogen bioactivation, and DNA repair. To further

elucidate the molecular mechanism(s) by which 3,3'-diindolylmethane exerts its

effects on breast cancer cells, we have used microarray gene expression profiling

analysis. We found a total of 1,238 genes altered in

3,3'-diindolylmethane-treated cells, among which 550 genes were down-regulated

and 688 genes were up-regulated. Clustering analysis showed significant

alterations in some genes that are critically involved in the regulation of cell

growth, cell cycle, apoptosis, and signal transduction, including down-regulation

of survivin. Previous studies have shown that antiapoptotic protein survivin is

overexpressed in many human cancers, including breast cancer. However, very

little or no information is available regarding the consequence of

down-regulation of survivin for cancer therapy. We, therefore, hypothesized that

down-regulation of survivin as observed by 3,3'-diindolylmethane could be an

important approach for the treatment of breast cancer. We have tested our

hypothesis using multiple molecular approaches and found that

3,3'-diindolylmethane inhibited cell growth and induced apoptosis in MDA-MB-231

breast cancer cells by down-regulating survivin, Bcl-2, and cdc25A expression and

also caused up-regulation of p21(WAF1) expression, which could be responsible for

cell cycle arrest. Down-regulation of survivin by small interfering RNA before

3,3'-diindolylmethane treatment resulted in enhanced cell growth inhibition and

apoptosis, whereas overexpression of survivin by cDNA transfection abrogated

3,3'-diindolylmethane-induced cell growth inhibition and apoptosis. These results

suggest that targeting survivin by 3,3'-diindolylmethane could be a new and novel

approach for the prevention and/or treatment of breast cancer.

 

PMID: 16651453

 

Cancer Res. 2006 May 1;66(9):4880-7.

 

3,3'-Diindolylmethane (DIM) is a novel mitochondrial H(+)-ATP synthase inhibitor that

can induce p21(Cip1/Waf1) expression by induction of oxidative stress in human

breast cancer cells.

 

Gong Y, Sohn H, Xue L, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, California 94720, USA.

 

Epidemiologic evidence suggests that high dietary intake of Brassica vegetables,

such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis

in multiple organs. 3,3'-Diindolylmethane, one of the active products derived

from Brassica vegetables, is a promising antitumor agent. Previous studies in our

laboratory showed that 3,3'-diindolylmethane induced a G(1) cell cycle arrest in

human breast cancer MCF-7 cells by a mechanism that included increased expression

of p21. In the present study, the upstream events leading to p21 overexpression

were further investigated. We show for the first time that 3,3'-diindolylmethane

is a strong mitochondrial H(+)-ATPase inhibitor (IC(50) approximately 20

micromol/L). 3,3'-Diindolylmethane treatment induced hyperpolarization of

mitochondrial inner membrane, decreased cellular ATP level, and significantly

stimulated mitochondrial reactive oxygen species (ROS) production. ROS

production, in turn, led to the activation of stress-activated pathways involving

p38 and c-Jun NH(2)-terminal kinase. Using specific kinase inhibitors (SB203580

and SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal kinase

(JNK) pathways in 3,3'-diindolylmethane-induced p21 mRNA transcription. In

addition, antioxidants significantly attenuated 3,3'-diindolylmethane-induced

activation of p38 and JNK and induction of p21, indicating that oxidative stress

is the major trigger of these events. To further support the role of ROS in

3,3'-diindolylmethane-induced p21 overexpression, we showed that

3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial

respiratory chain deficient rho(0) MCF-7 cells, in which 3,3'-diindolylmethane

did not stimulate ROS production. Thus, we have established the critical role of

enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21

up-regulation in human breast cancer cells.

 

PMID: 16651444

 

J Surg Res. 2006 May 15;132(2):208-13. Epub 2006 Mar 31.

 

3,3'-diindolylmethane (DIM) and paclitaxel act synergistically to promote apoptosis in

HER2/Neu human breast cancer cells.

 

McGuire KP, Ngoubilly N, Neavyn M, Lanza-Jacoby S.

 

Department of Surgery, Jefferson Medical College, Thomas Jefferson University,

Philadelphia, Pennsylvania 19107, USA.

 

BACKGROUND: HER2/neu positive breast tumors are difficult to treat. About 25 to

30% of invasive breast tumors overexpress the HER2/neu oncogene. These tumors are

aggressive and become resistant to chemotherapeutic drugs. 3'3'-diindolylmethane

(DIM), the active metabolite of indole-3-carbinol, a naturally occurring compound

found in cruciferous vegetables, has been found to have anti-cancer properties in

both humans and animals. DIM has been shown to induce cell cycle arrest and

apoptosis in animal breast cancer models. Because HER2/neu overexpression confers

resistance to paclitaxel, and DIM has anti-tumor effects, we hypothesized that

DIM will enhance the cytotoxic effects of paclitaxel, a common taxane drug, on

human Her2/neu breast cancer cells by potentiating its effect on cell cycle and

stimulating apoptosis. METHODS: The MDA-MB-435eB1 human Her2/neu breast

cancer cells were treated with varying concentrations of DIM and paclitaxel. The cells

were analyzed at different time points (24, 48, and 72 h). Proliferation was

measured by a commercial cell proliferation assay (Promega Procheck Assay).

Cell-cycle analysis and apoptosis were determined by flow cytometry. Western blot

analysis was performed on to determine the effect of DIM and/or paclitaxel on the

proteins involved in apoptosis, and epidermal growth factor-induced activation of

HER2/neu and ERK1/2 signaling proteins. RESULTS: Both DIM and paclitaxel

exhibited time and concentration dependent inhibition of cell proliferation.

TUNEL assay indicated that the combination also increased the number of apoptotic

cells more than either agent alone. The presence of cleaved poly (ADP-Ribose)

polymerase (PARP) significantly increased in the combination treatment, whereas

Bcl-2 is decreased. DIM alone decreased the activation of the Her2/neu receptor;

the combination decreased the activation of ERK1/ERK2. CONCLUSIONS: DIM in

combination with paclitaxel synergistically inhibits growth of Her2/neu human

breast cancer cells through G2M phase cell-cycle arrest and induction of

apoptosis/necrosis. The Her2/neu receptor and its downstream signaling protein

ERK1/2 appear to be involved in DIM's affect on cell growth and differentiation,

whereas apoptosis appears to be mediated through the mitochondrial pathway

(Bcl-2/PARP). It appears DIM, a naturally occurring, nontoxic compound, may be a

beneficial addition to a traditional (taxane-based) chemotherapy regimen.

 

PMID: 16580691

 

Mol Cancer Ther. 2006 Mar;5(3):556-63.

 

Multiple, disparate roles for calcium signaling in apoptosis of human prostate

and cervical cancer cells exposed to diindolylmethane (DIM).

 

Savino JA 3rd, Evans JF, Rabinowitz D, Auborn KJ, Carter TH.

 

Department of Biological Sciences, St. John's University, Jamaica, New York, USA.

 

Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables,

causes growth arrest and apoptosis of cancer cells in vitro. DIM also induces

endoplasmic reticulum (ER) stress, and thapsigargin, a specific inhibitor of the

sarcoplasmic reticulum/ER calcium-dependent ATPase, enhances this effect. We

asked whether elevated cytosolic free calcium [Ca2+]i is required for

cytotoxicity of DIM and thapsigargin in two cancer cells lines (C33A, from

cervix, and DU145, from prostate). [Ca2+]i was measured in real-time by FURA-2

fluorescence. We tested whether DIM, thapsigargin, and DIM + thapsigargin cause

apoptosis, measured by nucleosome release, under conditions that prevented

elevation of [Ca2+]i, using both cell-permeable and cell-impermeable forms of the

specific calcium chelator BAPTA. DIM, like thapsigargin, rapidly mobilized ER

calcium. C33A and DU145 responded differently to perturbations in Ca2+

homeostasis, suggesting that DIM induces apoptosis by different mechanisms in

these two cell lines and/or that calcium mobilization also activates different

survival pathways in C33A and DU145. Apoptosis in C33A was independent of

increased [Ca2+]i, suggesting that depletion of ER Ca2+ stores may be sufficient

for cell killing, whereas apoptosis in DU145 required elevated [Ca2+]i for full

response. Inhibitor studies using cyclosporin A and KN93 showed that Ca2+

signaling is important for cell survival but the characteristics of this response

also differed in the two cell lines. Our results underscore the complex and

variable nature of cellular responses to disrupted Ca2+ homeostasis and suggest

that alteration Ca2+ homeostasis in the ER can induce cellular apoptosis by both

calcium-dependent and calcium-independent mechanisms.

 

PMID: 16546969

 

Chem Res Toxicol. 2006 Mar;19(3):436-42.

 

Fate of 3,3'-diindolylmethane (DIM) in cultured MCF-7 human breast cancer cells.

 

Staub RE, Onisko B, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, California 94720, USA.

 

3,3'-Diindolylmethane (DIM) is a major in vivo product of the cancer preventative

agent indole-3-carbinol that is found in vegetables of the genus Brassica. Here,

we report on the metabolic fate of radiolabeled DIM in MCF-7 cells. DIM was

slowly metabolized to several sulfate conjugates of oxidized DIM products that

were primarily detected in the medium. The radioactivity detected in cells was

predominantly unmodified DIM (81-93%) at all time intervals up to 72 h treatment.

Co-treatment of MCF-7 cells with quercetin slowed the rate that oxidized DIM

products accumulated in the medium, while indole[3,2-b]carbazole (ICZ)

co-treatment accelerated their production. ICZ is an inducer of P450 1A2, while

quercetin is a specific inhibitor of this isoform, suggesting that P450 1A2 is

primarily responsible for the oxidation of DIM, probably through 2,3-epoxidation

similar to 3-methylindole. Sulfate conjugates of oxidized DIM metabolites were

cleaved by sulfatase digestion and identified by LC/MS as

3-(1H-indole-3-ylmethyl)-2-oxindole (2-ox-DIM), bis(1H-indol-3-yl)methanol

(3-methylenehydroxy-DIM), 3-[hydroxy-(1H-indol-3-yl)-methyl]-1,3-dihydro-2-oxindole

(3-methylenehydroxy-2-ox-DIM), and 3-hydroxy-3-(1H-indole-3-ylmethyl)-2-oxindole

(3-hydroxy-2-ox-DIM). Derivatives of 2-ox-DIM represented greater than 30% of the

radioactivity in the sulfatase-digested medium. Although oxindole formation was

the primary metabolic pathway in MCF-7 cells, synthetic 2-ox-DIM was inactive in

a 4-ERE-luciferase reporter assay and, therefore, probably not responsible for

the estrogenic activity previously observed for DIM. Unmodified DIM rapidly

accumulated in the nuclear membranes representing approximately 35-40% of the

radioactivity after 0.5-2 h treatment. Uptake of radiolabeled DIM appeared to be

a passive partitioning into the nuclear membranes and was not dependent upon the

cell cytosol. The nuclear uptake of DIM was not saturable and could not be

blocked by pretreatment with unlabeled DIM (100 microM). Further, treatments in

serum-free medium increased the uptake of radiolabeled DIM by the MCF-7 cells.

These findings show that the uptake of DIM by membranes significantly increases

its localized concentration, which may contribute to its biological activities.

 

PMID: 16544949

  

Mutat Res. 2006 Jul 25;599(1-2):76-87. Epub 2006 Feb 24.

 

Comparison of growth inhibition profiles and mechanisms of apoptosis induction in

human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae.

 

Pappa G, Lichtenberg M, Iori R, Barillari J, Bartsch H, Gerhńuser C.

 

Division of Toxicology and Cancer Risk Factors, German Cancer Research Center,

DKFZ, 69120 Heidelberg, Germany.

 

The isothiocyanates sulforaphane and PEITC (beta-phenethyl isothiocyanate) as

well as the indoles indole-3-carbinol and its condensation product

3,3'-diindolylmethane are known to inhibit cancer cell proliferation and induce

apoptosis. In this study, we compared the cell growth inhibitory potential of the

four compounds on the p53 wild type human colon cancer cell line 40-16 (p53(+/+))

and its p53 knockout derivative 379.2 (p53(-/-)) (both derived from HCT116).

Using sulforhodamin B staining to assess cell proliferation, we found that the

isothiocyanates were strongly cytotoxic, whereas the indoles inhibited cell

growth in a cytostatic manner. Half-maximal inhibitory concentrations of all four

compounds in both cell lines ranged from 5-15 microM after 24, 48 and 72 h of

treatment. Apoptosis induction was analyzed by immunoblotting of

poly(ADP-ribose)polymerase (PARP). Treatment with sulforaphane (15 microM), PEITC

(10 microM), indole-3-carbinol (10 microM) and 3,3'-diindolylmethane (10 microM)

induced PARP cleavage after 24 and 48 h in both 40-16 and the 379.2 cell lines,

suggestive of a p53-independent mechanism of apoptosis induction. In cultured

40-16 cells, activation of caspase-9 and -7 detected by Western blotting

indicated involvement of the mitochondrial pathway. We detected time- and

concentration-dependent changes in protein expression of anti-apoptotic Bcl-x(L)

as well as pro-apoptotic Bax and Bak proteins. Of note is that for sulforaphane

only, ratios of pro- to anti-apoptotic Bcl-2 family protein levels directly

correlated with apoptosis induction measured by PARP cleavage. Taken together, we

demonstrated that the glucosinolate breakdown products investigated in this study

have distinct profiles of cell growth inhibition, potential to induce

p53-independent apoptosis and to modulate Bcl-2 family protein expression in

human colon cancer cell lines.

 

PMID: 16500682

   

Mol Pharmacol. 2006 Apr;69(4):1320-7. Epub 2005 Dec 29.

 

3,3'-diindolylmethane (DIM) is a novel topoisomerase II alpha catalytic inhibitor that

induces S-phase retardation and mitotic delay in human hepatoma HepG2 cells.

 

Gong Y, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, 119 Morgan Hall, University of

California, Berkeley, CA 94720-3104, USA.

 

Epidemiological evidence suggests that high consumption of Brassica genus

vegetables, such as broccoli, cabbage, and Brussels sprouts, is very effective in

reducing the risks of several types of cancers. 3,3'-Diindolylmethane (DIM), one

of the most abundant and biologically active dietary compounds derived from

Brassica genus vegetables, displays remarkable antitumor activity against several

experimental tumors. In the present study, we demonstrate for the first time that

DIM is a novel catalytic topoisomerase IIalpha inhibitor. In supercoiled DNA

relaxation assay and kinetoplast DNA decatenation assay, DIM strongly inhibited

DNA topoisomerase IIalpha and also partially inhibited DNA topoisomerases I and

IIbeta. DIM did not stabilize DNA cleavage complex and did not prevent

etoposide-induced DNA cleavage complex formation. Further experiments showed that

DIM inhibited topoisomerase IIalpha-catalyzed ATP hydrolysis, which is a

necessary step for the enzyme turnover. In cultured human hepatoma HepG2 cells,

DIM blocked DNA synthesis and mitosis in a concentration-dependent manner, which

was consistent with the outcome of topoisomerase inhibition in these cell-cycle

phases. Our results identified a new mode of action for this intriguing dietary

component that might be exploited for therapeutic development.

 

PMID: 16385077

 

Biochem Biophys Res Commun. 2006 Feb 10;340(2):718-25. Epub 2005 Dec 20.

 

3,3'-Diindolylmethane (DIM) downregulates pro-survival pathway in hormone independent

prostate cancer.

 

Garikapaty VP, Ashok BT, Tadi K, Mittelman A, Tiwari RK.

 

Department of Microbiology and Immunology, New York Medical College,

Valhalla, NY, 10595, USA.

 

Epidemiological evidences suggest that the progression and promotion of prostate

cancer (CaP) can be modulated by diet. Since all men die with prostate cancer

rather than of the disease, it is of particular interest to prevent or delay the

progression of the disease by chemopreventive strategies. We have been studying

the anticancer properties of compounds present in cruciferous vegetables such as

indole-3-carbinol (I3C). Diindolylmethane (DIM) is a dimer of I3C that is formed

under acidic conditions and unlike I3C is more stable with higher anti-cancer

effects. In the present report, we demonstrate that DIM is a potent

anti-proliferative agent compared to I3C in the hormone independent DU 145 CaP

cells. The anti-prostate cancer effect is mediated by the inhibition of the Akt

signal transduction pathway as DIM, in sharp contrast to I3C, induces the

downregulation of Akt, p-Akt, and PI3 kinase. DIM also induced a G1 arrest in DU

145 cells by flow cytometry and downstream concurrent inhibition of cell cycle

parameters such as cyclin D1, cdk4, and cdk6. Our data suggest a need for further

development of DIM, as a chemopreventive agent for CaP, which justifies

epidemiological evidences and molecular targets that are determinants for CaP

dissemination/progression. The ingestion of DIM may benefit CaP patients and

reduce disease recurrence by eliminating micro-metastases that may be present in

patients who undergo radical prostatectomy.

 

PMID: 16380095

 

Mol Cancer Ther. 2005 Dec;4(12):1972-81.

 

Down-regulation of c-FLIP contributes to the sensitization effect of

3,3'-diindolylmethane (DIM) on TRAIL-induced apoptosis in cancer cells.

 

Zhang S, Shen HM, Ong CN.

 

Department of Community, Occupational and Family Medicine, Faculty of Medicine,

National University of Singapore, 16 Medical Drive, Singapore 117597, Singapore.

 

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of

the tumor necrosis factor superfamily, which has been shown to preferentially

induce apoptosis in cancer cells without adverse effects on normal cells.

However, there are still some cancer cells, especially those with high

malignancy, resistant to TRAIL-induced apoptosis, impeding the clinical

anticancer efficiency of TRAIL. In this report, we showed that

3,3'-diindolylmethane, an indole compound derived from cruciferous vegetables, is

capable of overcoming TRAIL resistance by sensitizing TRAIL-induced apoptosis in

human cancer cells. Noncytotoxic concentrations of 3,3'-diindolylmethane

significantly enhanced TRAIL-resistant cancer cells to TRAIL-induced apoptosis

via promoting the caspase cascade, a process independent of nuclear factor-kappaB

activation and cell surface TRAIL receptor expression. In the search of the

molecular mechanisms involved in the sensitization activity of

3,3'-diindolylmethane, we found that combined treatment of 3,3'-diindolylmethane

and TRAIL led to significant down-regulation of the cellular FLICE inhibitory

protein expression (c-FLIP). Furthermore, we provided evidence showing that the

reduced c-FLIP level is predominately mediated by the ubiquitin-proteasome

degradation system. These findings reveal a novel anticancer property of

3,3'-diindolylmethane and suggest that this compound could have potential use in

cancer therapy to overcome TRAIL resistance.

 

PMID: 16373712

 

Prostate. 2006 Apr 1;66(5):453-62.

 

Synthetic dimer of indole-3-carbinol: second generation diet derived anti-cancer

agent in hormone sensitive prostate cancer.

 

Garikapaty VP, Ashok BT, Tadi K, Mittelman A, Tiwari RK.

 

Department of Microbiology & Immunology, New York Medical College, Valhalla, New

York 10595, USA.

 

BACKGROUND: Cruciferous vegetables have been found to have anti-prostate cancer

effects. The active compounds mediating these effects include indoles such as

indole-3-carbinol (I3C) and isothiocyanates. I3C is unstable having tissue tropic

effects and clinical utility has been partly addressed by the synthesis of a more

stable dimer diindolylmethane (DIM). METHODS: Anti-proliferative activity was

measured by XTT assay and cytosolic proteins quantitated by Western blot

analysis. RESULTS: DIM (IC(50) 50 microM) is a better anti-proliferative agent

than I3C (IC(50) 150 microM) in androgen dependent LNCaP cells, inhibits DNA

synthesis, and growth of R1881 stimulated LNCaP cells. Androgen receptor (AR),

cyclin D1, and cdk4, induced by R1881, are downregulated by DIM. DIM

downregulates phosphorylated Akt and phosphatidyl inositol 3-kinase and

downstream inhibition of cyclin D1 and cdk4. CONCLUSION: These studies provide

evidence that DIM is a second-generation chemopreventive agent with a viable

cellular target and has clinical potential as an anti-prostate cancer

chemopreventive. (c) 2005 Wiley-Liss, Inc.

 

PMID: 16353249

 

Carcinogenesis. 2006 Apr;27(4):717-28. Epub 2005 Dec 6.

 

3,3'-diindolylmethane (DIM) and its derivatives induce apoptosis in pancreatic

cancer cells through endoplasmic reticulum stress-dependent upregulation of DR5.

 

Abdelrahim M, Newman K, Vanderlaag K, Samudio I, Safe S.

 

Institute of Biosciences and Technology, The Texas A&M University System Health

Science Center, 2121 W. Holcombe Boulevard, Houston, TX 77030, USA.

 

3,3'-Diindolylmethane (DIM), ring-substituted DIMs and

1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes (C-DIMs) inhibit growth of

Panc-1 and Panc-28 pancreatic cancer cells. Although DIMs (diarylmethanes) and

selected C-DIMs (triarylmethanes), such as the p-t-butyl derivative

(DIM-C-pPhtBu), activate the aryl hydrocarbon receptor and peroxisome

proliferator-activated receptor gamma, respectively, this study shows that both

DIM and DIM-C-pPhtBu induce common receptor-independent pathways. Both DIM and

DIM-C-pPhtBu increased endoplasmic reticulum (ER) staining and ER calcium release

in Panc-1 cells, and this was accompanied by increased expression of glucose

related protein 78 and C/EBP homologous transcription factor (CHOP/GADD153)

proteins. Similar results were observed after treatment with thapsigargin (Tg), a

prototypical inducer of ER stress. The subsequent downstream effects of

DIM/DIM-C-pPhtBu- and Tg-induced ER stress included CHOP-dependent induction of

death receptor DR5 and subsequent cleavage of caspase 8, caspase 3, Bid and PARP.

Activation of both receptor-dependent and receptor-independent (ER stress)

pathways by DIM and DIM-C-pPhtBu in pancreatic cancer cells enhances the efficacy

and potential clinical importance of these compounds for cancer chemotherapeutic

applications.

 

PMID: 16332727

  

Mol Pharmacol. 2006 Feb;69(2):430-9. Epub 2005 Nov 2.

 

Activation and potentiation of interferon-gamma signaling by

3,3'-diindolylmethane in MCF-7 breast cancer cells.

 

Riby JE, Xue L, Chatterji U, Bjeldanes EL, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, 94720-3104, USA.

 

3,3'-Diindolylmethane (DIM), a natural autolytic product in plants of the

Brassica genus, including broccoli, cauliflower, and Brussels sprouts, exhibits

promising cancer protective activities, especially against mammary neoplasia in

animal models. We observed previously that DIM induced a G(1) cell-cycle arrest

and strong induction of cell-cycle inhibitor p21 expression and promoter activity

in both estrogen-responsive and -independent breast cancer cell lines. We showed

recently that DIM up-regulates the expression of interferon gamma (IFNgamma) in

human MCF-7 breast cancer cells. This novel effect may contribute to the

anticancer effects of DIM because IFNgamma plays an important role in preventing

the development of primary and transplanted tumors. In this study, we observed

that DIM activated the IFNgamma signaling pathway in human breast cancer cells.

DIM activated the expression of the IFNgamma receptor (IFNGR1) and

IFNgamma-responsive genes p56- and p69-oligoadenylate synthase (OAS). In

cotreatments with IFNgamma, DIM produced an additive activation of endogenous

p69-OAS and of an OAS-Luc reporter and a synergistic activation of a GAS-Luc

reporter. DIM synergistically augmented the IFNgamma induced phosphorylation of

signal transducer and activator of transcription factor 1, further evidence of

DIM activation of the IFNgamma pathway. DIM and IFNgamma produced an additive

inhibition of cell proliferation and a synergistic increase in levels of major

histocompatibility complex class-1 (MHC-1) expression, accompanied by increased

levels of mRNAs of MHC-1-associated proteins and transporters. These results

reveal novel immune activating and potentiating activities of DIM in human tumor

cells that may contribute to the established effectiveness of this dietary indole

against various tumors types.

 

PMID: 16267208

 

Biochem Biophys Res Commun. 2005 Nov 25;337(3):1019-25. Epub 2005 Oct 3.

 

3,3'-Diindolylmethane (DIM), a cruciferous vegetable derived synthetic

anti-proliferative compound in thyroid disease.

 

Tadi K, Chang Y, Ashok BT, Chen Y, Moscatello A, Schaefer SD, Schantz SP,

Policastro AJ, Geliebter J, Tiwari RK.

 

Department of Microbiology and Immunology, New York Medical College, Valhalla,

NY, USA.

 

Considerable epidemiological evidence exists to link thyroid disease with

differing patterns of dietary consumption, in particular, cruciferous vegetables.

We have been studying the anti-thyroid cancer (TCa) activity of indole-3-carbinol

(I3C) found in cruciferous vegetables and its acid catalyzed dimer,

3,3'-diindolylmethane (DIM). There are no studies as yet to elucidate the effect

of these compounds on the altered proliferative patterns in goiter or thyroid

neoplasia. In this study, we tested the anti-proliferative effects of I3C and DIM

on four different thyroid cancer cell lines representative of papillary (B-CPAP

and 8505-C) and follicular carcinoma of the thyroid (CGTH-W-1 and ML-1), and

primary human goiter cells. Cell survival and IC50 values for I3C and DIM were

calculated by the XTT assay and cell cycle distribution analysis was done by flow

cytometry. DIM was found to be a better anti-proliferative agent than I3C in both

papillary and follicular TCa resulting in a greater cytotoxic effect at a

concentration over three fold lower than predicted by the molar ratio of DIM and

I3C. The anti-proliferative activity of DIM in follicular TCa was mediated by a

G1 arrest followed by induction of apoptosis. DIM also inhibited the growth of

primary goiter cells by 70% compared to untreated controls. Contrary to

traditional belief that cruciferous vegetables are "goitrogenic", DIM has

anti-proliferative effects in glandular thyroid proliferative disease. Our

preclinical studies provide a strong rationale for the clinical exploration of

DIM as an adjuvant to surgery in thyroid proliferative disease.

 

PMID: 16219298

 

Carcinogenesis. 2006 Mar;27(3):541-50. Epub 2005 Sep 30.

 

Inhibition of growth factor-induced Ras signaling in vascular endothelial cells

and angiogenesis by 3,3'-diindolylmethane (DIM).

 

Chang X, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720, USA.

 

3,3'-Diindolylmethane (DIM), an indole derivative produced on consumption of

broccoli and other cruciferous vegetables, has been shown to have multiple

anticancer effects in both in vivo and in vitro models. The present study was

carried out to clarify the mechanism of DIM's antiangiogenic activity. We found

that DIM can inhibit vascular endothelial growth factor (VEGF)-induced cell

proliferation and DNA synthesis in human umbilical vascular endothelial cells

(HUVECs). Consistent with this inhibition, VEGF-induced extracellular

signal-regulated kinase (ERK1/2) phosphorylation was greatly reduced. However,

VEGF receptor phosphorylation induced by VEGF was not affected by DIM, indicating

that DIM does not exert a direct and specific effect on the tyrosine kinase

activity of this receptor. Further studies showed that DIM had a similar

inhibitory effect on ERK1/2 phosphorylation induced by a variety of growth

factors. Furthermore, Ras-GTP content, which dramatically increased after HUVECs

were challenged by either individual growth factors or serum, was reduced by

approximately 80% with 25 muM DIM treatment, which in turn resulted in the

reduced activities of Raf and MEK, culminating in the drop of ERK1/2 activation.

Overexpression of constitutively active GTPase mutant, Ras G12V, in HUVECs

reversed the inhibitory effect of DIM on ERK1/2 activation. In a rodent Matrigel

plug model, the presence of DIM strongly reduced VEGF-induced neovascularization,

indicating that DIM is active in vivo. These data provide evidence that DIM

inhibits Ras signaling induced by VEGF and other growth factors, which interferes

with its downstream biological effects necessary for angiogenesis.

 

PMID: 16199440  

  

Toxicol Appl Pharmacol. 2006 Mar 1;211(2):115-23. Epub 2005 Jul 25.

 

Indole-3-carbinol, but not its major digestive product 3,3'-diindolylmethane (DIM),

induces reversible hepatocyte hypertrophy and cytochromes P450.

 

Crowell JA, Page JG, Levine BS, Tomlinson MJ, Hebert CD.

 

Division of Cancer Prevention, National Cancer Institute, Bethesda, MD

20892-7322, USA. jc94@nih.gov

 

Indole-3-carbinol (I-3-C) and 3,3'-diindolylmethane (DIM) have been shown to

reduce the incidence and multiplicity of cancers in laboratory animal models.

Based on the observation that I-3-C induced hepatocyte hypertrophy when

administered orally for 13 weeks to rats, a treatment and recovery study was

undertaken to test the hypothesis that the induction of hepatocyte hypertrophy

and cytochrome P450 (CYP) activity by I-3-C are adaptive, reversible responses.

Additionally, we directly compared the effects of I-3-C to those of its principle

metabolite DIM. Rats were treated orally for 28 days with 2 doses of I-3-C (5 and

50 mg I-3-C/kg body weight/day) and DIM (7.5 and 75 mg DIM/kg body weight/day)

and then one-half of the animals were not treated for an additional 28 days.

Organ weights, histopathology, and the CYP enzyme activities of 1A1/2, 2B1/2,

2C9, 2D6, 2E1, 3A4, and 19 A were measured both after treatment and after

recovery. Oral administration of 50 mg I-3-C/kg body weight/day to rats for 28

days significantly increased liver weights and CYP enzyme activities. The effects

in males were more pronounced and persistent after recovery than the effects in

females. The increased organ weights returned to control values after treatment.

Conversely, DIM did not alter liver weights and had no effect on CYP activities

after the 28-day treatment. Some changes in CYP activities were measured after

the DIM recovery period but the magnitudes of the changes were considered

biologically insignificant. The results show that I-3-C, but not DIM, induces

reversible adaptive responses in the liver.

 

PMID: 16043203

  

J Agric Food Chem. 2005 May 18;53(10):3895-901.

 

Effects of Brussels sprout juice on the cell cycle and adhesion of human

colorectal carcinoma cells (HT29) in vitro.

 

Smith TK, Lund EK, Clarke RG, Bennett RN, Johnson IT.

 

Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA,

United Kingdom.

 

Consumption of Brassica vegetables is associated with a reduced risk of cancer of

the alimentary tract in animal models and human populations. We used raw juice

extracted from Brussels sprouts rich in the glucosinolate sinigrin to explore the

effect of naturally occurring glucosinolate breakdown products on cell cycle

progression and apoptosis in human colorectal carcinoma cells (HT29). Juice was

prepared from sprout tissue immediately before use, and the glucosinolate

breakdown products were determined by gas chromatography mass spectrometry and

liquid chromatography mass spectrometry. The cell cycle was analyzed by flow

cytometry on detached and adherent cells, and apoptosis was measured in the

detached population by annexin V staining. Twenty-four hours after challenge with

juice (10 microL/mL), 7-13% of adherent cells had detached from the substratum

but the majority (82%) of these cells had not entered apoptosis, whereas only 33%

of detached control cells were not apoptotic (p < 0.05). The main glucosinolate

breakdown products were as follows: the sinigrin breakdown product,

1-cyano-2,3-epithiopropane (ca. 38 mM); the gluconapin hydrolysis product,

3-butenyl isothiocyanate (ca. 2.2.mM); the glucobrassicin metabolite, ascorbigen

(ca. 8 mM); and low concentrations of other indole glucosinolate-derived

hydrolysis products such as neoascorbigen and 3,3'-diindolylmethane. A variety of

biologically active glucosinolate breakdown products are released by mechanical

disruption of raw Brussels sprout tissue, but contrary to previous assumptions,

allyl isothiocyanate is not the main compound responsible for the inhibition of

cell proliferation.

 

PMID: 15884814

   

Oncogene. 2005 Mar 31;24(14):2343-53.

 

DIM stimulates IFNgamma gene expression in human breast cancer cells via the

specific activation of JNK and p38 pathways.

 

Xue L, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California, 119

Morgan Hall, Berkeley, CA 94720-3104, USA.

 

3,3'-Diindolylmethane (DIM) is a promising anticancer agent derived from Brassica

vegetables, but the mechanisms of DIM action are largely unknown. We have shown

that DIM can upregulate the expression and stimulate the secretion of

interferon-gamma (IFNgamma) in the human MCF-7 breast cancer cell line. This

novel effect may provide important clues to explain the anticancer effects of DIM

because it is well known that IFNgamma plays an important role in preventing the

development of primary and transplanted tumors. Utilizing promoter deletions, we

show here that the region between -108 and -36 bp in the IFNgamma promoter, which

contains two conserved and essential regulatory elements, is required for

DIM-induced IFNgamma expression. DIM activates both JNK and p38 pathways, induces

the phosphorylation of c-Jun and ATF-2, and increases the binding of the

homodimer or heterodimer of c-Jun/ATF-2 to the proximal AP-1.CREB-ATF-binding

element. Moreover, studies with specific enzyme inhibitors showed that up-stream

Ca2+-dependent kinase(s) is required for the inducing effects of DIM in MCF-7

cells. These results establish that DIM-induced IFNgamma expression in human

breast tumor cells is mediated by activation of both JNK and p38 pathways, which

is ultimately dependent on intracellular calcium signaling.

 

PMID: 15735741

  

Xenobiotica. 2004 Jul;34(7):619-32.

 

Phytochemical-induced changes in gene expression of carcinogen-metabolizing

enzymes in cultured human primary hepatocytes.

 

Gross-Steinmeyer K, Stapleton PL, Liu F, Tracy JH, Bammler TK, Quigley SD, Farin

FM, Buhler DR, Safe SH, Strom SC, Eaton DL.

 

Department of Environmental and Occupational Health Sciences, University of

Washington, Seattle, WA 98105, USA.

 

The naturally occurring compounds curcumin (CUR), 3,3'-diindolylmethane (DIM),

isoxanthohumol (IXN), 8-prenylnaringenin (8PN), phenethyl isothiocyanate (PEITC)

and sulforaphane (SFN) protect animals against chemically induced tumours.

Putative chemoprotective mechanisms include modulated expression of hepatic

biotransformation enzymes. However, few, if any, studies have used human primary

cells as test models. 2. The present study investigated the effects of these

phytochemicals on the expression of four carcinogenesis-relevant

enzymes--cytochrome P450 (CYP)1A1 and 1A2, NAD(P)H:quinone oxidoreductase (NQO1)

and glutathione S-transferase A1 (GSTA1)--in primary cultures of freshly isolated

human hepatocytes. 3. Quantitative RT-PCR analyses demonstrated that CYP1A1 was

up-regulated by PEITC and DIM in a dose-dependent manner. CYP1A2 transcription

was significantly activated following DIM, IXN, 8PN and PEITC treatments. DIM

exhibited a remarkably effective induction response of CYP1A1 (474-, 239- and

87-fold at 50, 25 and 10 microM, respectively) and CYP1A2 (113-, 70- and 31-fold

at 50, 25 and 10 microM, respectively), that was semiquantitatively reflected in

protein levels. NQO1 expression responded to PEITC (11 x at 25 microM), DIM (4.5

x at 50 microM) and SFN (5 x at 10 microM) treatments. No significant effects on

GSTA1 transcription were seen. 4. The findings show novel and unexpected effects

of these phytochemicals on the expression of human hepatic biotransformation

enzymes that play key roles in chemical-induced carcinogenesis.

 

PMID: 15672752

 

Biochem Biophys Res Commun. 2005 Mar 4;328(1):63-9.

 

Indole-3-carbinol and 3,3'-diindolylmethane induce expression of NAG-1 in a

p53-independent manner.

 

Lee SH, Kim JS, Yamaguchi K, Eling TE, Baek SJ.

 

Laboratory of Environmental Carcinogenesis, Department of Pathobiology, College

of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.

 

Indole-3-carbinol (I3C), present in cruciferous vegetables, and its major in vivo

product 3,3'-diindolylmethane (DIM), have been reported to suppress cancer

development. However, the responsible molecular mechanisms are not fully

understood. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a

TGF-beta superfamily gene associated with pro-apoptotic and anti-tumorigenic

activities. The present study was performed to investigate whether I3C and DIM

influence NAG-1 expression and to provide the potential molecular mechanism of

their effects on anti-tumorigenesis. The I3C repressed cell proliferation and

induced NAG-1 expression in a concentration-dependent manner. In addition, DIM

increased the expression of NAG-1 as well as activating transcription factor 3

(ATF3), and the induction of ATF3 was earlier than that of NAG-1. The DIM

treatment increased luciferase activity of NAG-1 in HCT-116 cells transfected

with NAG-1 promoter construct. The results suggest that I3C represses cell

proliferation through up-regulation of NAG-1 and that ATF3 may play a pivotal

role in DIM-induced NAG-1 expression in human colorectal cancer cells.

Furthermore, the mixture of I3C with resveratrol enhances NAG-1 expression,

suggesting the synergistic effect of these two unrelated compounds on NAG-1

expression.

 

PMID: 15670751

 

Cancer Res. 2005 Jan 1;65(1):364-71.

 

Inhibition of nuclear translocation of nuclear factor-{kappa}B contributes to

3,3'-diindolylmethane-induced apoptosis in breast cancer cells.

 

Rahman KW, Sarkar FH.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, MI 48201, USA.

 

Dietary indole-3-carbinol (I3C), a natural compound present in vegetables of the

genus Brassica, showed clinical benefits and caused apoptosis in breast cancer

cells. Our laboratory and others have shown that I3C induces apoptosis in breast

cancer cells mediated by inactivation of Akt and nuclear factor-kappaB

(NF-kappaB) pathway. 3,3'-Diindolylmethane (DIM), a major in vivo acid-catalyzed

condensation product of I3C, also showed some benefit in breast cancer. However,

the precise molecular mechanism(s) by which DIM induces apoptosis in breast

cancer cells has not been fully elucidated. Hence, we investigated whether

DIM-induced apoptosis of breast cancer cells could also be mediated by

inactivation of Akt and NF-kappaB. We found that DIM induces apoptotic processes

in MCF10A derived malignant (MCF10CA1a) cell lines but not in nontumorigenic

parental MCF10A cells. DIM specifically inhibits Akt kinase activity and

abrogates the epidermal growth factor-induced activation of Akt in breast cancer

cells, similar to those observed for I3C. We also found that DIM reduces

phosphorylation of IkappaBalpha, an inhibitor of NF-kappaB. Our confocal

microscopy study clearly showed that DIM blocks the translocation of p65, a

subunit of NF-kappaB to the nucleus. DNA binding analysis and transfection

studies with IkappaB kinase cDNA revealed that overexpression of IkappaB kinase

mediates IkappaBalpha phosphorylation, which activates NF-kappaB, and this

activation was completely abrogated by DIM treatment. Taken together, these

results showed for the first time that the inactivation of Akt and NF-kappaB

activity also plays important roles in DIM-induced apoptosis in breast cancer

cells, which seems to be more relevant to in vivo situations.

 

PMID: 15665315

 

Carcinogenesis. 2005 Apr;26(4):771-8. Epub 2005 Jan 20.

 

3,3'-Diindolylmethane inhibits angiogenesis and the growth of transplantable

human breast carcinoma in athymic mice.

 

Chang X, Tou JC, Hong C, Kim HA, Riby JE, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720, USA.

 

Studies have linked the consumption of broccoli and other cruciferous vegetables

to a reduced risk of breast cancer. The phytochemical indole-3-carbinol (I3C),

present in cruciferous vegetables, and its major acid-catalyzed reaction product

3,3'-diindolylmethane (DIM) have bioactivities relevant to the inhibition of

carcinogenesis. In this study, the effect of DIM on angiogenesis and

tumorigenesis in a rodent model was investigated. We found that DIM produced a

concentration-dependent decrease in proliferation, migration, invasion and

capillary tube formation of cultured human umbilical vein endothelial cells

(HUVECs). Consistent with its antiproliferative effect, which was significant at

only 5 microM DIM, this indole caused a G1 cell cycle arrest in actively

proliferating HUVECs. Furthermore, DIM downregulated the expression of

cyclin-dependent kinases 2 and 6 (CDK2, CDK6), and upregulated the expression of

CDK inhibitor, p27(Kip1), in HUVECs. We observed further in a complementary in

vivo Matrigel plug angiogenesis assay that, compared with vehicle control,

neovascularization was inhibited up to 76% following the administration of 5

mg/kg DIM to female C57BL/6 mice. Finally, this dose of DIM also inhibited the

growth of human MCF-7 cell tumor xenografts by up to 64% in female athymic

(nu/nu) mice, compared with the vehicle control. This is the first study to show

that DIM can strongly inhibit the development of human breast tumor in a

xenograft model and to provide evidence for the antiangiogenic properties of this

dietary indole.

 

PMID: 15661811

 

Nutr Cancer. 2004;50(2):161-7.

 

Pilot study: effect of 3,3'-diindolylmethane supplements on urinary hormone

metabolites in postmenopausal women with a history of early-stage breast cancer.

 

Dalessandri KM, Firestone GL, Fitch MD, Bradlow HL, Bjeldanes LF.

 

Department of Molecular and Cell Biology, University of California, Berkeley,

94720-3200, USA.

 

Dietary indoles, present in Brassica plants such as cabbage, broccoli, and

Brussels sprouts, have been shown to provide potential protection against

hormone-dependent cancers. 3,3'-Diindolylmethane (DIM) is under study as one of

the main protective indole metabolites. Postmenopausal women aged 50-70 yr from

Marin County, California, with a history of early-stage breast cancer, were

screened for interest and eligibility in this pilot study on the effect of DIM supplements

on urinary hormone metabolites. The treatment group received daily DIM (108 mg

DIM/day) supplements for 30 days, and the control group received a placebo capsule

daily for 30 days. Urinary metabolite analysis included 2-hydroxyestrone (2-OHE1),

16-alpha hydroxyestrone (16alpha-OHE1), DIM, estrone (El), estradiol(E2), estriol (E3),

6beta-hydroxycortisol (6beta-OHC), and cortisol in the first morning urine sample

before intervention and 31 days after intervention. Nineteen women completed the

study,for a total of 10 in the treatment group and 9 in the placebo group.

DIM-treated subjects, relative to placebo, showed a significant increase in

levels of2-OHE1 (P=0. 020), DIM (P =0. 045), and cortisol (P = 0.039), and a

nonsignificant increase of 47% in the 2-OHE1/16alpha-OHE1 ratio from 1.46 to 2.14

(P=0.059). In this pilot study, DIM increased the 2-hydroxylation of estrogen

urinary metabolites.

 

PMID: 15623462

 

J Nutr. 2004 Dec;134(12 Suppl):3493S-3498S.

 

Indole-3-carbinol and prostate cancer.

 

Sarkar FH, Li Y.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, MI, 48201, USA.

 

Epidemiological and dietary studies have revealed an association between high

dietary intake of cruciferous vegetables and decreased prostate cancer risk. Our

studies have shown that indole-3-carbinol (I3C), a common phytochemical in

cruciferous vegetables, and its in vivo dimeric product 3,3'-diindolylmethane

(DIM) upregulate the expression of phase I and phase II enzymes, suggesting

increased capacity for detoxification and inhibition of carcinogens. Studies from

our laboratory and others have found that I3C can induce G1 cell-cycle arrest and

apoptosis in prostate cancer cells. In addition, we found, by microarray gene

expression profiling, that I3C and DIM regulate many genes that are important for

the control of cell cycle, cell proliferation, signal transduction, and other

cellular processes, suggesting the pleiotropic effects of I3C and DIM on prostate

cancer cells. We recently found that I3C functions as an inhibitor of Akt and

nuclear factor kappaB (NF-kappaB), which play important roles in cell survival

and which are believed to be potential targets in cancer therapy. Studies have

already shown that the inactivation of Akt and NF-kappaB is responsible for

chemosensitization of chemoresistant cancer cells. Because there is no effective

treatment strategy for hormone-dependent and, most importantly,

hormone-independent and metastatic prostate cancer, our strategies to sensitize

prostate cancer cells to a chemotherapeutic agent by I3C and DIM is a novel

breakthrough that could be used for devising novel therapies for prostate cancer.

In conclusion, the results from our laboratory and from others provide ample

evidence for the benefit of I3C and DIM for the prevention and the treatment of

prostate cancer.

 

PMID: 15570059

 

Chem Biol Interact. 2004 Nov 20;150(2):161-70.

 

Lack of antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin's (TCDDs) induction of

cytochrome P4501A1 (CYP1A1) by the putative selective aryl hydrocarbon receptor

modulator 6-alkyl-1,3,8-trichlorodibenzofuran (6-MCDF) in the mouse hepatoma cell

line Hepa-1c1c7.

 

Fretland AJ, Safe S, Hankinson O.

 

Department of Pathology and Laboratory Medicine, Jonsson Comprehensive Cancer

Center, Center for Health Sciences, University of California, Los Angeles, CA

90095-1732, USA.

 

Regulation of gene expression by the aryl hydrocarbon (AHR) receptor is a

much-studied pathway of molecular toxicology. Activation of AHR by the xenobiotic

ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is hypothesized as the

mechanism by which TCDD exerts its toxic and carcinogenic effects. Paradoxically,

some studies have shown that TCDD acts as an antiestrogen. This has led to the

hypothesis that so-called selective aryl hydrocarbon receptor modulators

(SAhRMs), AHR ligands that retain the antiestrogenic effects but lack the

transcriptional effects of TCDD associated with toxicity, may be utilized as

cancer chemotherapeutics in conjunction with other antiestrogenic compounds such

as tamoxifen. The present study attempts to further define the molecular

mechanism of action of the putative SAhRMs, 6-alkyl-1,3,8-trichlorodibenzofuran

(6-MCDF), and diindolylmethane (DIM), focusing particularly on the former. We

tested 6-MCDF and DIM for the recruitment of AHR and RNA polymerase II (pol II)

to the regulatory region of the AHR responsive gene, cytochrome P4501A1 (CYP1A1),

using the chromatin immunoprecipitation (ChIP) assay in the mouse hepatoma cell

line Hepa-1c1c7 (Hepa-1). We also tested the level of CYP1A1 induction in Hepa-1

cells using quantitative real-time PCR. We show no difference in the recruitment

of AHR or pol II to the regulatory region of CYP1A1 in response to TCDD, 6-MCDF,

or co-treatment with both TCDD and 6-MCDF. Our results also show no antagonism of

CYP1A1 induction with co-treatment of Hepa-1 cells with TCDD and 6-MCDF. These

data suggest that 6-MCDF exhibits agonist activity with respect to induction of

CYP1A1 in the Hepa-1 cell line.

 

PMID: 15535986

 

Mutat Res. 2004 Nov 2;555(1-2):53-64.

 

Cell signaling pathways altered by natural chemopreventive agents.

 

Sarkar FH, Li Y.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, 715 Hudson Webber Cancer Research Center, 110 E Warren, Detroit, MI

48201, USA

 

Epidemiological studies have indicated a significant difference in the incidence

of cancers among ethnic groups, who have different lifestyles and have been

exposed to different environmental factors. It has been estimated that more than

two-thirds of human cancers, which are contributed by mutations in multiple

genes, could be prevented by modification of lifestyle including dietary

modification. The consumption of fruits, soybean and vegetables has been

associated with reduced risk of several types of cancers. The in vitro and in

vivo studies have demonstrated that some dietary components such as isoflavones,

indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM), curcumin,

(-)-epigallocatechin-3-gallate (EGCG), apigenin, etc., have shown inhibitory

effects on human and animal cancers, suggesting that they may serve as

chemopreventive agents. Experimental studies have also revealed that these

components regulate the molecules in the cell signal transduction pathways

including NF-kappaB, Akt, MAPK, p53, AR, and ER pathways. By modulating cell

signaling pathways, these components, among other mechanisms, activate cell death

signals and induce apoptosis in precancerous or cancer cells, resulting in the

inhibition of cancer development and/or progression. This article reviews current

studies regarding the effects of natural chemopreventive agents on cancer-related

cell signaling pathways and provides comprehensive knowledge of the biological

and molecular roles of chemopreventive agents in cancer cells.

 

PMID: 15476851

  

Br J Cancer. 2004 Oct 4;91(7):1358-63.

 

Induction of apoptosis in human prostate cancer cell line, PC3, by

3,3'-diindolylmethane through the mitochondrial pathway.

 

Nachshon-Kedmi M, Yannai S, Fares FA.

 

Faculty of Food Engineering and Biotechnology, Technion-Israel Institute of

Technology, Haifa 32000, Israel.

 

Prostate cancer is the most common malignancy and the second leading cause of

male death in Western countries. Prostate cancer mortality results from

metastases to the bones and lymph nodes and progression from androgen-dependent

to androgen-independent disease. Although androgen ablation was found to be

effective in treating androgen-dependent prostate cancer, no effective

life-prolonging therapy is available for androgen-independent cancer.

Epidemiological studies have shown a strong correlation between consumption of

cruciferous vegetables and a lower risk of prostate cancer. These vegetables

contain glucosinolates, which during metabolism give rise to several breakdown

products, mainly indole-3-carbinol (I3C), which may be condensed to polymeric

products, especially 3,3'-diindolylmethane (DIM). It was previously shown that

these indole derivatives have significant inhibitory effects in several human

cancer cell lines, which are exerted through induction of apoptosis. We have

previously reported that I3C and DIM induce apoptosis in prostate cancer cell

lines through p53-, bax-, bcl-2- and fasL-independent pathways. The objective of

this study was examination of the apoptotic pathways that may be involved in the

effect of DIM in the androgen-independent prostate cancer cell line, PC3, in

vitro. Our results suggest that DIM induces apoptosis in PC3 cells, through the

mitochondrial pathway, which involves the translocation of cytochrome c from the

mitochondria to the cytosol and the activation of initiator caspase, 9, and

effector caspases, 3 and 6, leading to poly ADP-ribose polymerase (PARP) cleavage

and induction of apoptosis. Our findings may lead to the development of new

therapeutic strategies for the treatment of androgen-independent prostate cancer.

 

PMID: 15328526

 

Prostate. 2004 Oct 1;61(2):153-60.

 

Therapeutic activity of 3,3'-diindolylmethane on prostate cancer in an in vivo

model.

 

Nachshon-Kedmi M, Fares FA, Yannai S.

 

Faculty of Food Engineering and Biotechnology, Technion-Israel Institute of

Technology, Haifa, Israel.

 

BACKGROUND: Prostate cancer (PC) is the second leading cancer-related death in

men in Western countries. Hence, efficient anti-carcinogenic and therapeutic

compounds against PC are badly needed. We have previously shown that

3,3'-diindolylmethane (DIM) has a suppressive effect on the growth of human

breast and PC cell lines. The objective of this study was examination of the

potential therapeutic effects of DIM in an in vivo model. METHODS: TRAMP-C2, a

mouse PC cell line, was injected into the flank of male C57BL/6 mice. When tumors

appeared, mice were injected intraperitoneally with either corn oil (vehicle) or

DIM (2.5, 5, or 10 mg per kg body weight) 3-times a week, for 3 weeks, and tumor

volumes were measured bi-weekly with calibermeters. Later, the tumors were

removed, their final weights and volumes were measured, and tumor sections were

tested for histological studies. RESULTS: DIM had a significant inhibitory

effect, caused by diminished tumor growth. Histological examination of tumors

from treated groups revealed apoptosis and decreased cell proliferation, compared

with the controls. DIM didn't affect body weights or kidney and liver

functioning. CONCLUSIONS: The inhibitory action of DIM on tumor growth was

demonstrated in vivo. Hence, this compound at the concentrations tested may offer

an effective and non toxic therapeutic means against tumor growth in rodents, and

may serve as a potential natural anti-carconigenic compound in humans. Copyright

2004 Wiley-Liss, Inc

 

PMID: 15305338

 

Cell Stress Chaperones. 2004 Mar;9(1):76-87.

 

Endoplasmic reticulum stress as a correlate of cytotoxicity in human tumor cells

exposed to diindolylmethane in vitro.

 

Sun S, Han J, Ralph WM Jr, Chandrasekaran A, Liu K, Auborn KJ, Carter TH.

 

North Shore-Long Island Jewish Research Institute, Manhasset, NY 11030, USA.

 

The dietary phytochemical indole-3-carbinol (I3C) protects against cervical

cancer in animal model studies and in human clinical trials. I3C and its

physiologic condensation product diindolylmethane (DIM) also induce apoptosis of

tumor cells in vitro and in vivo, suggesting that these phytochemicals might be

useful as therapeutic agents as well as for cancer prevention. Deoxyribonucleic

acid microarray studies on transformed keratinocytes and tumor cell lines exposed

to pharmacologic concentrations of DIM in vitro are consistent with a cellular

response to nutritional deprivation or disruptions in protein homeostasis such as

endoplasmic reticulum (ER) stress. In this report we investigate whether specific

stress response pathways are activated in tumor cells exposed to DIM and whether

the ER stress response might contribute to DIM's cytotoxicity. Induction of the

stress response genes GADD153, GADD34 and GADD45A, XBP-1, GRP78,

GRP94, and asparagine synthase was documented by Western blot and real-time reverse

transcriptase-polymerase chain reaction in C33A cervical cancer cells, and

induction of a subset of these was also observed in cancer cell lines from breast

(MCF-7) and prostate (DU145). The results are consistent with activation of more

than 1 stress response pathway in C33A cells exposed to 75 microM DIM.

Phosphorylation elF2alpha was rapidly and transiently increased, followed by

elevated levels of ATF4 protein. Activation of IRE1alpha was indicated by a rapid

increase in the stress-specific spliced form of XBP-1 messenger ribonucleic acid

and a rapid and persistent phosphorylation of JNK1 and JNK2. Transcriptional

activation dependent on an ATF6-XBP-1 binding site was detected by transient

expression in MCF-7, C33A, and a transformed epithelial cell line (HaCaT);

induction of the GADD153 (CHOP) promoter was also confirmed by transient

expression. Cleavage of caspase 12 was observed in both DIM-treated and untreated

C33A cells but did not correlate with cytotoxicity, whereas caspase 7 was cleaved

at later times, coinciding with the onset of apoptosis. The results support the

hypothesis that cytotoxic concentrations of DIM can activate cellular stress

response pathways in vitro, including the ER stress response. Conversely, DIM was

especially cytotoxic to stressed cells. Thapsigargin and tunicamycin, agents that

induce ER stress, sensitized cells to the cytotoxic effects of DIM to differing

degrees; nutrient limitation had a similar, but even more pronounced, effect.

Because DIM toxicity in vitro is enhanced in cells undergoing nutritional

deprivation and ER stress, it is possible that stressed cells in vivo, such as

those within developing solid tumors, also have increased sensitivity to killing

by DIM.

 

PMID: 15270080

 

Cancer Detect Prev. 2004;28(1):72-9.

 

Differences in the hepatic P450-dependent metabolism of estrogen and tamoxifen in

response to treatment of rats with 3,3'-diindolylmethane and its parent compound

indole-3-carbinol.

 

Parkin DR, Malejka-Giganti D.

 

Veterans Affairs Medical Center, Minneapolis, MN 55417, USA.

 

Indole-3-carbinol (I3C), present in cruciferous vegetables, and its major in vivo

product 3,3'-diindolylmethane (DIM), have been reported to suppress

estrogen-responsive cancers. This effect may be mediated through the modification

of cytochrome P450 (CYP) complement and activities leading to estrogen

detoxification. We examined the effects of a 4-day treatment of female

Sprague-Dawley rats with DIM at 8.4 and 42 mg/kg body weight (bwt), on the

hepatic CYP protein level, CYP1A1, 1A2, 2B1/2 and 3A1/2 probe activities and

CYP-dependent metabolism of 17beta-estradiol (E2) and estrone (E1). At 42 mg/kg

bwt, DIM effected a small increase (2.8-fold) in CYP1A1 activity, and at both

dose levels it reduced CYP3A1/2 activity by approximately 40%. At the higher dose

level, DIM decreased the rates of oxidation of E2 to 4-OH-E2, 4-OH-E1,

6alpha-OH-E2 and 6(alpha+beta)-OH-E1 by 39, 44, 71 and 60%, respectively, and E1

to 6(alpha+beta)-OH-E1 by 39%. These effects were considerably different from

those of I3C reported by us previously. We also examined the effects of DIM and

I3C on the hepatic microsomal metabolism of tamoxifen (TAM). Whereas metabolism

of TAM was unaffected by DIM, formation of N-desmethyl-TAM (and its presumed

derivative) was increased approximately 3-fold by I3C at 250 mg/kg bwt. Since

N-desmethyl-TAM is transformed to a genotoxic metabolite, dietary exposure to I3C

may enhance hepatic carcinogenicity of TAM in the rat. The differences between

I3C and DIM in CYP-mediated activities and metabolism indicate that DIM is not a

proximate intermediate in the mechanism of action of I3C.

 

PMID: 15041081

  

Mol Endocrinol. 2004 Feb;18(2):291-302. Epub 2003 Nov 26.

 

Potent ligand-independent estrogen receptor activation by 3,3'-diindolylmethane

is mediated by cross talk between the protein kinase A and mitogen-activated

protein kinase signaling pathways.

 

Leong H, Riby JE, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, California 94720, USA.

 

We investigated the mechanism of ligand-independent activation of the estrogen

receptor (ER) by 3,3'-diindolylmethane (DIM), a promising anticancer agent

derived from vegetables of the Brassica genus, in Ishikawa and HEC-1B human

endometrial cancer cells. DIM stimulated the activity of an ER-responsive

reporter by over 40-fold, equivalent to the maximum induction produced by

estradiol (E2), whereas cotreatment of cells with the ER antagonist, ICI-182,780

(ICI), abolished the stimulatory effect of DIM. DIM also induced the expressions

of the endogenous genes, TGF-alpha, alkaline phosphatase, and progesterone

receptor similar to levels induced by E2. Induction of gene expression by DIM was

inhibited by the protein synthesis inhibitor, cycloheximide. In addition,

cotreatment of cells with the protein kinase A (PKA) inhibitor, H89, or the MAPK

inhibitor, PD98059, reduced DIM activation of the ER by 75% and 50%,

respectively. Simultaneous treatment of cells with both inhibitors completely

abolished the effect of DIM. DIM stimulated MAPK activity and induced

phosphorylation of the endogenous PKA target, cAMP response element binding

protein (CREB), in a PKA-dependent manner. Expression of MCREB, a

nonphosphorylatable CREB mutant, partially abolished activation of the ER by DIM.

These results demonstrate that DIM is a mechanistically novel activator of the ER

that requires PKA-dependent phosphorylation of CREB.

 

PMID: 14645498

 

Curr Opin Otolaryngol Head Neck Surg. 2003 Dec;11(6):433-41.

 

Overview of recurrent respiratory papillomatosis.

 

Wiatrak BJ.

 

Department of Pediatrics and Surgery, University of Alabama at Birmingham, USA.

 

PURPOSE OF REVIEW: The purpose of this article is to review recent literature

regarding pediatric recurrent respiratory papillomatosis (RRP) published within

the last year. By reviewing and assessing these articles, a more clear

understanding regarding the etiology and management of pediatric RRP can be

obtained, allowing physicians to better care for their pediatric RRP patients.

RECENT FINDINGS: Pediatric RRP continues to be an extremely difficult management

problem for otolaryngologists. This disease process continues to be a significant

burden on the health care system and is a significant cause of morbidity in

affected patients and their families. The incidence of RRP continues to be

approximately 3.96 per 100,000 in the pediatric population. It has been noted

recently that approximately 7 of every 1000 children born to mothers with vaginal

condyloma develop pediatric RRP. Although the mainstay of surgical management has

traditionally been the CO2 laser, newer surgical techniques have demonstrated

efficacy in the management of pediatric RRP patients, including powered

instrumentation and the pulse-dye laser. The traditional adjuvant medical

therapies used for pediatric RRP continue to be commonly used, including

interferon-alpha2a, retinoic acid, and indol-3-carbinol/diindolylmethane

(I3C/DIM). Recently cidofovir has demonstrated efficacy in selected patients. In

addition, current research regarding vaccine therapy for pediatric RRP has shown

promise. Basic science research in the field of immunology has demonstrated

multiple defects in cell-mediated immunity, which has shed further light on the

etiology of pediatric RRP. SUMMARY: Pediatric RRP continues to be a highly morbid

disease process. New surgical and medical therapies offer hope for better control

of this disease in affected patients. Recent advances in immunologic research

offer the hope of immune system modulation and augmentation as potential future

treatment modalities to better control this disease process.

 

PMID: 14631175

 

Mol Cell Biol. 2003 Nov;23(21):7920-5.

 

Agonist and chemopreventative ligands induce differential transcriptional

cofactor recruitment by aryl hydrocarbon receptor.

 

Hestermann EV, Brown M.

 

Department of Molecular Oncology, Dana-Farber Cancer Institute, Brigham and

Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

 

Aryl hydrocarbon receptor (AHR) is a transcription factor whose activity is

regulated by environmental agents, including several carcinogenic agonists. We

measured recruitment of AHR and associated proteins to the human cytochrome

P4501A1 gene promoter in vivo. Upon treatment with the agonist

beta-naphthoflavone, AHR is rapidly associated with the promoter and recruits the

three members of the p160 family of coactivators as well as the p300 histone

acetyltransferase, leading to recruitment of RNA polymerase II (Pol II) and

induction of gene transcription. AHR, coactivators, and Pol II cycle on and off

the promoter, with a period of approximately 60 min. In contrast, the

chemopreventative AHR ligand 3,3'-diindolylmethane promotes AHR nuclear

translocation and p160 coactivator recruitment but, remarkably, fails to recruit

Pol II or cause histone acetylation. This novel mechanism of receptor antagonism

may account for the antitumor properties of chemopreventative compounds targeting

the AHR.

 

PMID: 14560034

  

J Nutr. 2003 Jul;133(7 Suppl):2470S-2475S.

 

Indole-3-carbinol and 3-3'-diindolylmethane antiproliferative signaling pathways

control cell-cycle gene transcription in human breast cancer cells by regulating

promoter-Sp1 transcription factor interactions.

 

Firestone GL, Bjeldanes LF.

 

Department of Molecular and Cell Biology and Cancer Research Laboratory,

University of California at Berkeley, Berkeley, CA 94720-3200, USA.

 

Indole-3-carbinol (I3C), a compound that occurs naturally in Brassica vegetables

such as cabbage and broccoli, can induce a G1 cell-cycle arrest of human MCF-7

breast cancer cells that is accompanied by the selective inhibition of

cyclin-dependent kinase 6 (Cdk6) expression and stimulation of p21(Waf1/Cip1)

gene expression. Construction and transfection of a series of promoter-reporter

plasmids demonstrate that the indole-regulated changes in Cdk6 and p21(Waf1/Cip1)

levels are due to specific effects on their corresponding promoters. Mutagenic

analysis reveals that I3C signaling targets a composite transcriptional element

in the Cdk6 promoter that requires both Sp1 and Ets transcription factors for

transactivation function. Analysis of protein-DNA complexes formed with nuclear

proteins isolated from I3C-treated and -untreated cells demonstrates that the Sp1

DNA element in the Cdk6 promoter interacts with an I3C-inhibited protein-protein

complex that contains the Sp1 transcription factor. In indole-treated cells, a

fraction of [(3)H]I3C was converted into its natural diindole product

(3)H-labeled 3-3'-diindolylmethane ([(3)H]DIM), which accumulates in the nucleus;

this suggests that DIM may have a role in the transcriptional activities of I3C.

Mutagenic analysis of the p21(Waf1/Cip1) promoter reveals that in transfected

breast cancer cells, DIM (as well as I3C) stimulates p21(Waf1/Cip1) transcription

through an indole-responsive region of the promoter that contains multiple Sp1

consensus sequences. Furthermore, DIM treatment regulates the presence of a

nuclear Sp1 DNA-binding activity. Our results demonstrate that both the Cdk6 and

p21(Waf1/Cip1) promoters are newly defined downstream targets of the

indole-signaling pathway, and that the observed transcriptional effects are due

to a combination of the cellular activities of I3C and DIM.

 

PMID: 12840223

 

Food Chem Toxicol. 2003 Jun;41(6):745-52.

 

Indole-3-carbinol and 3,3'-diindolylmethane induce apoptosis in human prostate

cancer cells.

 

Nachshon-Kedmi M, Yannai S, Haj A, Fares FA.

 

Faculty of Food Engineering and Biotechnology, Technion- Israel Institute of

Technology, 32000, Haifa, Israel.

 

Cruciferous vegetables contain glucobrassicin which, during metabolism, yields

indole-3-carbinol (I3C). In a low pH environment I3C is converted into polymeric

products, among which 3,3'-diindolylmethane (DIM) is the main one. The apoptotic

effects of I3C and DIM were exhibited in human breast cancer cells. The

objectives of this study were: (a) examination of the potential effects of I3C

and DIM on the proliferation and induction of apoptosis in human prostate cancer

cell lines with different p53 status; (b) to try to characterise the mechanism(s)

involved in these effects. Our results indicate that both indole derivatives

suppress the growth of these cells in a dose- and time-dependent manner, by

inducing apoptosis. It appears that these indolic compounds may offer effective

means against prostate cancer. Induction of apoptosis was p53-independent.

Moreover, the indole derivatives employed did not affect the levels of bcl-2, bax

and fasL.

 

PMID: 12738179

  

J Nutr. 2003 Apr;133(4):1011-9.

 

Gene expression profiles of I3C- and DIM-treated PC3 human prostate cancer cells

determined by cDNA microarray analysis.

 

Li Y, Li X, Sarkar FH.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, MI 48201, USA.

 

Studies from our laboratory and others have shown that indole-3-carbinol (I3C)

and its in vivo dimeric product, 3,3'-diindolylmethane (DIM), inhibit the growth

of PC3 prostate cancer cells and induce apoptosis by inhibiting nuclear factor

(NF)-kappaB and Akt pathways. To obtain comprehensive gene expression profiles

altered by I3C- and DIM-treated PC3 cells, we utilized cDNA microarray to

interrogate the expression of 22,215 known genes using the Affymetrix Human

Genome U133A Array. We found a total of 738 genes that showed a greater than

twofold change after 24 h of DIM treatment. Among these genes, 677 genes were

down-regulated and 61 were up-regulated. Similarly, 727 genes showed a greater

than twofold change in expression, with down-regulation of 685 genes and

up-regulation of 42 genes in I3C-treated cells. The altered expressions of genes

were observed as early as 6 h and were more evident with longer treatment. Upon

cluster analysis, we found that both I3C and DIM up-regulated the expression of

genes that are related to the Phase I and Phase II enzymes, suggesting their

increased capacity for detoxification of carcinogens or chemicals. We also found

that I3C and DIM down-regulated the expression of genes that are critically

involved in the regulation of cell growth, cell cycle, apoptosis, signal

transduction, Pol II transcription factor and oncogenesis. Real-time reverse

transcription-polymerase chain reaction analysis was conducted to confirm the

cDNA microarray data, and the results were consistent. We conclude that I3C and

DIM affected the expression of a large number of genes that are related to the

control of carcinogenesis, cell survival and physiologic behaviors. This may help

determine the molecular mechanism(s) by which I3C and DIM exert their pleiotropic

effects on PC3 prostate cancer cells; in addition, this information could be

further exploited for devising chemopreventive and/or therapeutic strategies for

prostate cancer.

 

PMID: 12672912

 

J Biol Chem. 2003 Jun 6;278(23):21136-45. Epub 2003 Mar 27.

 

Plant-derived 3,3'-Diindolylmethane is a strong androgen antagonist in human

prostate cancer cells.

 

Le HT, Schaldach CM, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, The University of California,

Berkeley, California 94720-3104, USA.

 

3,3'-Diindolylmethane (DIM) is a major digestive product of indole-3-carbinol, a

potential anticancer component of cruciferous vegetables. Our results indicate

that DIM exhibits potent antiproliferative and antiandrogenic properties in

androgen-dependent human prostate cancer cells. DIM suppresses cell proliferation

of LNCaP cells and inhibits dihydrotestosterone (DHT) stimulation of DNA

synthesis. These activities were not produced in androgen-independent PC-3 cells.

Moreover, DIM inhibited endogenous PSA transcription and reduced intracellular

and secreted PSA protein levels induced by DHT in LNCaP cells. Also, DIM

inhibited, in a concentration-dependent manner, the DHT-induced expression of a

prostate-specific antigen promoter-regulated reporter gene construct in

transiently transfected LNCaP cells. Similar effects of DIM were observed in PC-3

cells only when these cells were co-transfected with a wild-type androgen

receptor expression plasmid. Using fluorescence imaging with green fluorescent

protein androgen receptor and Western blot analysis, we demonstrated that DIM

inhibited androgen-induced androgen receptor (AR) translocation into the nucleus.

Results of receptor binding assays indicated further that DIM is a strong

competitive inhibitor of DHT binding to the AR. Results of structural modeling

studies showed that DIM is remarkably similar in conformational geometry and

surface charge distribution to an established synthetic AR antagonist, although

the atomic compositions of the two substances are quite different. Taken together

with our published reports of the estrogen agonist activities of DIM, the present

results establish DIM as a unique bifunctional hormone disrupter. To our

knowledge, DIM is the first example of a pure androgen receptor antagonist from

plants.

 

PMID: 12665522

   

Carcinogenesis. 2002 Aug;23(8):1297-305.

 

3,3'-Diindolylmethane (DIM) induces a G(1) cell cycle arrest in human breast

cancer cells that is accompanied by Sp1-mediated activation of p21(WAF1/CIP1)

expression.

 

Hong C, Kim HA, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720, USA.

 

3,3'-Diindolylmethane (DIM) is a promising cancer chemopreventive agent derived

from Brassica food plants. To determine whether this natural indole has a direct

growth inhibitory effect on human breast cancer cells, we examined the cell cycle

regulatory effects of DIM in estrogen-dependent (MCF-7) and estrogen-independent

(MDA-MB-231) human breast cancer cell lines. Results of flow cytometry studies

showed that DIM treatment produced a marked increase (from 51 to 79%) in the

proportion of cells in the G(1) phase of the cell cycle, regardless of

estrogen-receptor status. Analyses of G(1)-acting cell cycle components indicated

that the enzymatic activity of cyclin-dependent kinase (CDK) 2 was also strongly

reduced. Western blot analyses showed that, concurrent with the DIM-induced cell

cycle arrest, DIM stimulated a rapid and pronounced increase in levels of the CDK

inhibitor, p21(WAF1/CIP1) (p21). Northern blot analysis demonstrated that DIM

increased p21 mRNA expression with a maximal 6-7-fold induction, and exposure to

cycloheximide did not block the response. Similar increases in expression of p21

protein and mRNA were observed in both MCF-7 and MDA-MB-231 human

breast cancer cells, suggesting that DIM induction of p21 expression is independent of

estrogen-receptor signaling and p53. Transient transfection of 5'-deletion

constructs of the p21 promoter demonstrated that the first 291 bp segment of the

proximal promoter, which contains six promoter specific transcription factor 1

(Sp1) elements, maintained DIM responsiveness. Consistent with a role for Sp1 in

this response, a reporter construct driven by three consensus Sp1 binding sites

was responsive to DIM. In addition, electrophoretic mobility shift assays showed

that DIM induced the binding of Sp1 and Sp3 to the consensus Sp1 responsive

element. Thus, our observations have uncovered an antiproliferative pathway for

DIM that implicates Sp1/Sp3-induced expression of p21 as a target for cell cycle

control in human breast cancer cells.

 

PMID: 12151347

 

Nutr Cancer. 2001;41(1-2):57-63.

 

Quantitative determination of 3,3'-diindolylmethane in urine of individuals

receiving indole-3-carbinol.

 

Sepkovic DW, Bradlow HL, Bell M.

 

Institute for Biomedical Research, Hackensack University Medical Center,

Hackensack, NJ 07601, USA.

 

Indole-3-carbinol (I3C) or, more correctly, its acid condensation products act as

chemoprotective agents via several mechanisms. It induces the expression of

cytochrome P-450 1A1, which shifts the estrogen metabolic pathway in favor of C-2

hydroxylation and away from the formation of 16 alpha-hydroxyestrone, a suspected

endogenous carcinogen. Increased 16 alpha-hydroxylation of estrogen is associated

with greater risk of cancer of the cervix, breast, endometrium, and larynx. The

production of 4-hydroxyestrone is also inhibited by I3C. I3C can induce a G1 cell

cycle arrest in human MCF-7 breast cancer cells. It can suppress aberrant crypt

foci. I3C significantly inhibits the cell adhesion, spreading, and invasion

associated with an upregulation of PTEN (a tumor suppressor gene) and E-cadherin

(a regulator of cell-cell adhesion) expression in T47-D human breast cancer

cells. Thus I3C exhibits anticancer activities by suppressing breast tumor cell

growth and metastatic spread. A gas chromatography-mass spectrometry method for

the quantitation of diindolylmethane, the principal acid condensation product of

I3C, has been developed for use in determining compliance in subjects who have

been treated with I3C. The method utilizes a 1-ml urine sample. We have used this

method to correlate I3C ingestion with regression of cervical intraepithelial

neoplasia in a population of women at risk for cervical cancer. The assay

provides an objective marker of consumption using a noninvasive biological fluid

and illustrates that diindolylmethane may be used as a marker of compliance in

I3C dietary intervention studies.

 

PMID: 12094629

 

Antivir Ther. 2002 Mar;7(1):1-9.

 

Therapy for recurrent respiratory papillomatosis.

 

Auborn KJ.

 

Department of Otolaryngology, Long Island Jewish Medical Center, Albert Einstein

College of Medicine, New Hyde Park, NY, USA.

 

Human papillomaviruses types 6 or 11 are aetiological agents of recurrent

respiratory papillomatosis, a disease characterized by benign exophytic tumours

usually on the vocal cords. Surgery debulks the tumours, but these growths

generally recur at regular intervals. Adjunct medical treatments, aimed at

containing the virus and growth of tumours, include indole-3-carbinol or its

dimer diindolylmethane, interferon, photodynamic therapy and others. Preventive

and therapeutic vaccines hold promise for eliminating the virus.

 

PMID: 12008782

 

Altern Med Rev. 2002 Apr;7(2):112-29.

 

Estrogen metabolism and the diet-cancer connection: rationale for assessing the

ratio of urinary hydroxylated estrogen metabolites.

 

Lord RS, Bongiovanni B, Bralley JA.

 

MetaMetrix Clinical Laboratory, 4855 Peachtree Industrial Boulevard, Suite 201,

Norcross, GA, 30092, USA.

 

Estrogens are known for their proliferative effects on estrogen-sensitive tissues

resulting in tumorigenesis. Results of experiments in multiple laboratories over

the last 20 years have shown that a large part of the cancer-inducing effect of

estrogen involves the formation of agonistic metabolites of estrogen, especially

16-alpha-hydroxyestrone. Other metabolites, such as 2-hydroxyestrone and

2-hydroxyestradiol, offer protection against the estrogen-agonist effects of

16-alpha-hydroxyestrone. An ELISA method for measuring 2- and

16-alpha-hydroxylated estrogen (OHE) metabolites in urine is available and the

ratio of urinary 2-OHE/16-alpha-OHE (2/16-alpha ratio) is a useful biomarker for

estrogen-related cancer risk. The CYP1A1 enzyme that catalyzes 2-hydroxyestrone

(2-OHE1) formation is inducible by dietary modification and supplementation with

the active components of cruciferous vegetables, indole-3-carbinol (I-3-C), or

diindolylmethane (DIM). Other dietary components, especially omega-3

polyunsaturated fatty acids and lignans in foods like flax seed, also exert

favorable effects on estrogen metabolism. Thus, there appear to be effective

dietary means for reducing cancer risk by improving estrogen metabolism. This

review presents the accumulated evidence to help clinicians evaluate the merit of

using tests that measure estrogen metabolites and using interventions to modify

estrogen metabolism.

 

PMID: 11991791

 

Biochem Pharmacol. 2002 Mar 15;63(6):1085-97.

 

Bcl-2 family-mediated apoptotic effects of 3,3'-diindolylmethane (DIM) in human

breast cancer cells.

 

Hong C, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley 94720-3200, USA.

 

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of the putative

anticancer agent indole-3-carbinol (I3C), which is present in vegetables of the

Brassica genus. At concentrations above 10 microM, DIM inhibited DNA synthesis

and cell proliferation in both estrogen receptor replete (MCF-7) and deficient

(MDA-MB-231) human breast cancer cells in a concentration- and time-dependent

manner. These antiproliferative effects were accompanied by characteristic

indications of programmed cell death in both cell lines, including

externalization of phosphatidylserine, chromatin condensation, and DNA

fragmentation. Furthermore, Western and Northern blot analyses, as well as

coimmunoprecipitation assays, revealed that in both MCF-7 and MDA-MB-231 cells,

DIM treatment decreased total transcript and protein levels of the apoptosis

inhibitory protein Bcl-2, and the amount of Bcl-2 bound to the pro-apoptotic

protein Bax. DIM treatment also caused an increase in Bax protein levels, but did

not affect the level of Bax that was bound to Bcl-2. As a functional test of the

role of Bcl-2 down-regulation in the DIM-induced apoptotic response, ectopic

expression of Bcl-2 in MCF-7 cells was shown to attenuate the apoptotic effect of

DIM. These results demonstrate that DIM can induce apoptosis in breast cancer

cells independent of estrogen receptor status by a process that is mediated by

the modulated expression of the Bax/Bcl-2 family of apoptotic regulatory factors.

 

PMID: 11931841

 

Chem Res Toxicol. 2002 Feb;15(2):101-9.

 

Fate of indole-3-carbinol in cultured human breast tumor cells.

 

Staub RE, Feng C, Onisko B, Bailey GS, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, California 94720, USA.

 

Indole-3-carbinol (I3C), a natural component of Brassica vegetables, is a

promising cancer preventive agent that can reduce the incidence of tumors in

reproductive organs when administered in the diet. Here we report on the

metabolic fate of radiolabeled I3C in MCF-7 cells. I3C was surprisingly inert to

metabolism by these cells with a half-life in medium of approximately 40 h.

[(3)H]I3C levels in media declined at a similar rate whether incubation was with

cultured cells or in cell-free medium. Neither [(3)H]I3C nor its modified

products accumulated in MCF-7 cells and only low levels of intact I3C were

detected in cellular fractions. In contrast, I3C represented over 30% of the

radioactivity in media even after 72 h. In cytosolic fractions, the

3-(cystein-S-ylmethyl) and 3-(glutathion-S-ylmethyl) conjugates of [(3)H]I3C were

the primary conversion products identified after 16 h, representing approximately

50% and approximately 15% of the radioactivity in these fractions, respectively.

The reaction of I3C with thiols appears to be nonenzymatic since the cysteine

conjugate is produced when I3C is incubated in cell-free medium containing

additional cysteine. Both cellular and extracellular proteins were

nonspecifically modified with [(3)H]I3C. In medium, proteins are radiolabeled

even in the absence of cells, indicating again that enzymatic activation was not

required. I3C was also oxidized to indole-3-carboxaldehyde and

indole-3-carboxylic acid in culture medium independent of cells. Unexpectedly,

3,3'-diindolylmethane (DIM), an I3C product with in vitro and in vivo biological

activity, was detected in cellular fractions and appeared to accumulate in the

nucleus, representing approximately 40% of this fraction after 72 h treatment.

These findings suggest that MCF-7 cells do not vigorously metabolize I3C and that

the major route of reaction is with cellular thiols such as glutathione and

proteins. The accumulation of DIM in the nucleus suggests that this product may

have a role in the cellular biological activities of I3C.3

 

PMID: 11849035

   

Carcinogenesis. 2001 Nov;22(11):1809-17.

 

Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells

result from an estrogen receptor-mediated increase in transforming growth

factor-alpha expression.

 

Leong H, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of

California-Berkeley, Berkeley, CA 94720, USA.

 

3,3'-Diindolylmethane (DIM), a major in vivo product of indole-3-carbinol (I3C),

is a promising anticancer agent derived from vegetables of the Brassica genus

including broccoli, Brussels sprouts and cabbage. We report here that DIM has a

potent cytostatic effect in cultured human Ishikawa endometrial cancer cells. A

combination of northern blot and quantitative PCR analyses revealed that DIM

induced the level of TGF-alpha transcripts by approximately 4-fold within 24 h of

indole treatment. DIM also induced a 4-fold increase in the activity of the

estrogen response marker, alkaline phosphatase (AP). Co-treatment of cells with

the estrogen receptor (ER) antagonist ICI, or with the inhibitor of PKA-mediated

activation of the ER, H89, ablated the DIM induction of both TGF-alpha expression

and AP activity. Furthermore, DIM increased the maximum stimulatory effect of

estrogen on TGF-alpha expression. Co-treatment with the protein synthesis

inhibitor, cycloheximide, abolished the inductive effects of DIM, indicating

differences in the mechanistic requirements of DIM and estrogen. DIM treatment

also stimulated levels of secreted TGF-alpha protein by >10-fold. The ectopic

addition of TGF-alpha inhibited the growth of Ishikawa cells, whereas incubation

with a TGF-alpha antibody partially reversed the growth inhibitory effects of

DIM. Taken together, these results extend our previous findings of the ligand

independent estrogen receptor agonist activity of DIM, and uncover an essential

role for the stimulation in TGF-alpha expression and the TGF-alpha activated

signal transduction pathway in the potent cytostatic effects of DIM in

endometrial cancer cells.

 

PMID: 11698343

 

Cancer Res. 2001 Aug 15;61(16):6120-30.

 

Dietary indoles and isothiocyanates that are generated from cruciferous

vegetables can both stimulate apoptosis and confer protection against DNA damage

in human colon cell lines.

 

Bonnesen C, Eggleston IM, Hayes JD.

 

Biomedical Research Centre, Ninewells Hospital and Medical School, University of

Dundee, Dundee DD1 9SY, Scotland, United Kingdom.

 

The natural indoles 3,3'-diindolylmethane (DIM), ascorbigen (ASG),

indole-3-carbinol (I3C), and indolo[3,2-b]carbazole (ICZ), as well as the natural

isothiocyanates sulforaphane (SUL), benzyl isothiocyanate (BITC) and phenethyl

isothiocyanate (PEITC), all possess cancer chemopreventive properties. It is now

shown that DIM, ICZ, SUL, and BITC can each stimulate apoptosis in human colon

adenocarcinoma LS-174 and Caco-2 cells. Treatment of LS-174 cells with nontoxic

doses of DIM, ASG, I3C, or ICZ affected an increase of up to 21-fold in

cytochrome P450 1A1 (CYP1A1). None of these indoles caused an elevation in either

aldo-keto reductase 1C1 (AKR1C1) or the gamma-glutamylcysteine synthetase heavy

subunit (GCS(h)), but DIM, I3C, and ICZ produced a very modest increase in

NAD(P)H:quinone oxidoreductase 1 (NQO1). By contrast, nontoxic doses of SUL,

BITC, or PEITC failed to induce expression of CYP1A1 in LS-174 cells, but caused

an increase of between 11- and 17-fold in the protein levels of AKR1C1, NQO1, and

GCS(h). Treatment of the colon cell line with ICZ or SUL caused increases in the

levels of mRNA for CYP1A1, AKR1C1, and NQO1 that were consistent with the enzyme

data. Exposure of Caco-2 cells to media containing indoles or isothiocyanates

gave similar results to those obtained using LS-174 cells. Evidence is presented

that the ability of indoles and isothiocyanates to stimulate either xenobiotic

response element- or antioxidant response element-driven gene expression accounts

for the two groups of phytochemicals inducing different gene batteries.

Pretreatment of LS-174 cells for 24 h with ICZ and SUL before exposure for 24 h

to benzo(a)pyrene (BaP) reduced to <20% the number of single-strand DNA breaks

produced by the carcinogen. Neither ICZ alone nor SUL alone were able to confer

the same degree of protection against DNA damage produced by BaP as they achieved

in combination. Similar results were obtained with H(2)O(2) as the genotoxic

agent. Together, these phytochemicals may prevent colon tumorigenesis by both

stimulating apoptosis and enhancing intracellular defenses against genotoxic

agents.

 

PMID: 11507062

 

Breast Cancer Res Treat. 2001 Mar;66(2):147-57.

 

Methyl-substituted diindolylmethanes as inhibitors of estrogen-induced growth of

T47D cells and mammary tumors in rats.

 

McDougal A, Gupta MS, Morrow D, Ramamoorthy K, Lee JE, Safe SH.

 

Department of Veterinary Physiology & Pharmacology, Texas A &M University,

College Station 77843-4466, USA.

 

Diindolylmethane (DIM) is formed by acid catalyzed dimerization of the

phytochemical indole-3-carbinol, and both compounds inhibit formation and/or

growth of mammary tumors in rodents. In this study, we have investigated the aryl

hydrocarbon receptor (AhR) agonist activity and inhibitory AhR-estrogen receptor

crosstalk induced by the following methyl-substituted DIMs: 1,1'-dimethyl-,

2,2'-dimethyl-, 5,5'-dimethyl-, 6,6'-dimethyl-, and 7,7'-dimethylDIM and

1,1',2,2'-tetramethylDIM. The six compounds bound to the rat cytosolic AhR in a

transformation assay but, at concentrations < or = 10 microM, exhibited minimal

to non-detectable AhR agonist or antagonist activities associated with CYP1A1

induction. In contrast, the methyl-substituted DIMs inhibited estrogen-induced

T47D human breast cancer cell growth and the four most active compounds (1,1'-,

2,2'-, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM) inhibited one or more

estrogen-induced responses in the 21-day-old female B6C3F1 mice at a dose of 100

mg/kg/day (X3). Induction of hepatic CYP1A1-dependent activity was not observed

at this high dose. The antitumorigenic activity of these compounds was examined

in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor model in which the

DIM analogs were orally administered (by gavage in corn oil) at a dose of 1

mg/kg/day (X10). 1,1'-DimethylDIM, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM

significantly inhibited mammary tumor growth, and this was not accompanied by

changes in organ/body weights or histopathology. These studies demonstrate that

methyl-substituted DIMs are selective AhR modulators (SAhRMs) with potential for

clinical treatment of breast cancer.

 

PMID: 11437101

 

Toxicol Sci. 2001 May;61(1):40-8.

 

2,3,7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce

cytochrome P450 1A1, 1B1, and 19 in H295R human adrenocortical carcinoma cells.

 

Sanderson JT, Slobbe L, Lansbergen GW, Safe S, van den Berg M.

 

Research Institute for Toxicology, Utrecht University, P.O. Box 80176, 3508 TD

Utrecht, The Netherlands.

 

Diindolylmethane (DIM) is an acid-catalyzed condensation product of

indole-3-carbinol, a constituent of cruciferous vegetables, and is formed in the

stomach. DIM alters estrogen metabolism and inhibits carcinogen-induced mammary

tumor growth in rodents. DIM is a weak agonist for the aryl hydrocarbon (Ah)

receptor and blocks the effects of estrogens via inhibitory Ah receptor-estrogen

receptor cross-talk. DIM and various structural analogs were examined in H295R

cells for effects on 3 cytochrome P450 (CYP) enzymes involved in estrogen

synthesis and/or metabolism: CYP1A1, CYP1B1, and CYP19 (aromatase). Aromatase

activity was measured by conversion of 1 beta-(3)H-androstenedione to estrone and

(3)H(2)O. H295R cells were exposed to the test chemicals dissolved in dimethyl

sulfoxide for 24 h prior to analyses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)

(0--30 nM) and DIM (0--10 microM) induced ethoxyresorufin-O-deethylase (EROD)

activity, as a measure of CYP1A1 and possibly 1B1 activity, with EC(50) values of

about 0.3 nM and 3 microM, respectively. DIM, but not TCDD, induced aromatase

activity with an apparently maximal 2-fold increase at 10 microM; higher

concentrations of DIM and many of its analogs were cytotoxic. TCDD (30 nM)

significantly increased CYP1A1 and 1B1 mRNA levels, but had no effect on mRNA for

CYP19. DIM (3 microM) significantly increased mRNA levels for all three CYPS: DIM

analogs with substitutions on the 5 and 5' position (3 microM) induced aromatase

and EROD activity, together with mRNA levels of CYP1A1, 1B1, and 19; analogs that

were substituted on the central carbon of the methane group showed little or no

inductive activity toward the CYPS: In conclusion, DIM and several of its analogs

appear to induce CYPs via multiple yet distinct pathways in H295R human

adrenocortical carcinoma cells.

 

PMID: 11294972

 

Gene. 2001 Jan 10;262(1-2):207-14.

 

Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7

breast cancer cells using suppression subtractive hybridization.

 

Chen I, Hsieh T, Thomas T, Safe S.

 

Department of Veterinary Physiology and Pharmacology, A&M University, College

Station, TX 77843, USA.

 

Aryl hydrocarbon receptor (AhR) agonists inhibit 17beta-estradiol (E2) induced

growth of MCF-7 human breast cancer cells in vitro and rodent mammary tumor

growth in vivo. Genes associated with inhibitory AhR-estrogen receptor (ER)

crosstalk were investigated in MCF-7 human breast cancer cells using

poly(A)(+)RNA from cells treated with either 1 nM E2 (target) or E2 plus 1 nM

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (reference) or 25 microM

diindolylmethane (DIM) as AhR agonists in MCF-7 cells. Suppression subtractive

hybridization (SSH) was subsequently used to identify 33 genes with sequence

homology to known human genes that are induced by E2 and inhibited by AhR

agonists in MCF-7 cells; two unknown genes were also identified. Many of these

genes are involved in cell proliferation and these include cell cycle regulators

(cdc28/cdc2-associated protein), nucleotide synthases (thymidylate synthase),

early intermediate genes (early growth response alpha, EGRalpha) and other

proteins involved in signaling pathways (calmodulin, ATP synthase alpha subunit).

Thus SSH has identified a diverse spectrum of new genes that are affected by

inhibitory AhR-ER crosstalk and among this group are a subset of genes that may

be critical for the in vivo antitumorigenic effects of AhR agonists.

 

PMID: 11179685

 

Drug Metab Dispos. 2000 Aug;28(8):930-6.

 

Concurrent flavin-containing monooxygenase down-regulation and cytochrome P-450

induction by dietary indoles in rat: implications for drug-drug interaction.

 

Katchamart S, Stresser DM, Dehal SS, Kupfer D, Williams DE.

 

Department of Environmental and Molecular Toxicology, Oregon State University,

Corvallis, Oregon, USA.

 

Our laboratory has previously shown that dietary administration of

indole-3-carbinol (I3C) to male Fischer 344 rats has the very unusual property of

inducing hepatic levels of a number of cytochrome P450s (CYPs), especially

CYP1A1, while markedly inhibiting the levels of flavin-containing monooxygenase

(FMO) 1 protein and its catalytic activity. We hypothesized that rats fed I3C or

3,3'-diindolylmethane (DIM), one of its major acid condensation products formed

in vivo, should exhibit a marked shift in the metabolic profiles of drugs or

xenobiotics that are substrates for both monooxygenase systems. Male rats were

fed AIN-76A powdered diets containing 0, 1000, or 2500 ppm I3C or DIM for 4

weeks. Dietary I3C and DIM reduced FMO1 protein levels (8% reduction with I3C and

84% with DIM at 1000 ppm, and 90% reduction with I3C and 97% with DIM at 2500

ppm) in hepatic microsomes. The ratio of FMO (N-oxygenation)- to CYP

(N-demethylation)-mediated metabolism of N,N-dimethylaniline decreased in liver

microsomes from I3C- or DIM-fed rats from near unity to 0.02 at the highest

dietary doses. FMO-mediated N-oxygenation (nicotine N-1'-oxide) was decreased,

whereas CYP-mediated (nornicotine and nicotine delta (1,5)-iminium ion)

metabolism of nicotine was unchanged in liver microsomes from rats fed I3C or

DIM. Similarly, the ratio of FMO to CYP metabolites of tamoxifen decreased due to

a reduction in N-oxygenation. This study demonstrates alteration of FMO- and

CYP-mediated drug metabolism in vitro by dietary I3C or DIM and suggests the

potential for altered toxicity of tamoxifen and nicotine in vivo.

 

PMID: 10901703

 

Biochem Pharmacol. 2000 Jul 15;60(2):167-77.

 

Ligand-independent activation of estrogen receptor function by 3,

3'-diindolylmethane in human breast cancer cells.

 

Riby JE, Chang GH, Firestone GL, Bjeldanes LF.

 

Division of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720, USA.

 

3,3'-Diindolylmethane (DIM), a major in vivo product of acid-catalyzed

oligomerization of indole-3-carbinol (I3C), is a promising anticancer agent

present in vegetables of the Brassica genus. We investigated the effects of DIM

on estrogen-regulated events in human breast cancer cells and found that DIM was

a promoter-specific activator of estrogen receptor (ER) function in the absence

of 17beta-estradiol (E(2)). DIM weakly inhibited the E(2)-induced proliferation

of ER-containing MCF-7 cells and induced proliferation of these cells in the

absence of steroid, by approximately 60% of the E(2) response. DIM had little

effect on proliferation of ER-deficient MDA-MB-231 cells, suggesting that it is

not generally toxic at these concentrations. Although DIM did not bind to the ER

in this concentration range, as shown by a competitive ER binding assay, it

activated the ER to a DNA-binding species. DIM increased the level of transcripts

for the endogenous pS2 gene and activated the estrogen-responsive pERE-vit-CAT

and pS2-tk-CAT reporter plasmids in transiently transfected MCF-7 cells. In

contrast, DIM failed to activate transcription of the simple E(2)- and

diethylstilbesterol-responsive reporter construct pATC2. The estrogen antagonist

ICI 182780 (7alpha-[9-[(4,4,5,5,

5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3, 17beta-diol) was

effective against DIM-induced transcriptional activity of the pERE-vit-CAT

reporter, which further supports the hypothesis that DIM is acting through the

ER. We demonstrated that ligand-independent activation of the ER in MCF-7 cells

could be produced following treatment with the D1 dopamine receptor agonist

SKF-82958 [(+/-)6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,

5-tetrahydro-1H-3-benzazepinehydrobromide]. We also demonstrated that the agonist

effects of SKF-82958 and DIM, but not of E(2), could be blocked by co-treatment

with the protein kinase A (PKA) inhibitor H-89

(N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide). These results have

uncovered a promoter-specific, ligand-independent activation of ER signaling for

DIM that may require activation by PKA, and suggest that this major I3C product

may be a selective activator of ER function.

 

PMID: 10825461

 

Cancer Lett. 2000 Apr 14;151(2):169-79.

 

Inhibition of carcinogen-induced rat mammary tumor growth and other

estrogen-dependent responses by symmetrical dihalo-substituted analogs of

diindolylmethane.

 

McDougal A, Sethi Gupta M, Ramamoorthy K, Sun G, Safe SH.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University,

College Station, TX 77843-4466, USA.

 

90%) by the haloDIMs at concentrations of 5 or 10 microM, and only 4,

4'-dichloroDIM alone increased cell proliferation. With the exception of

5,5'-difluoroDIM, the remaining compounds also inhibited E2-induced growth of

MCF-7 human breast cancer cells. DihaloDIMs (100 mg/kg/dayx3) were not estrogenic

in the immature female B6C3F1 mouse uterus; however, in animals co-treated with

E2 (0.02 microg/mouse), 5,5'-dichloro- and 6,6'-dichloroDIM inhibited uterine

progesterone receptor (PR) binding and uterine peroxidase activity, whereas

5,5'-dichloro- and 5,5'-dichloro-2,2'-dimethylDIM inhibited only the latter

response. The antitumorigenic activities of the dihaloDIMs were determined by

their inhibition of carcinogen-induced mammary tumor growth in female

Sprague-Dawley rats. 4,4'-Dichloro-, 5,5'-dibromo- and 6,6'-dichloroDIM,

significantly inhibited mammary tumor growth at doses of 1 mg/kg every second

day, and no significant changes in organ weights or liver and kidney

histopathology were observed. These three compounds were more active than DIM in

the same in vivo assay.

 

PMID: 10738111

   

Toxicol Sci. 1999 Dec;52(2):178-88.

 

The anti-estrogenicity of Ah receptor agonists in carp (Cyprinus carpio)

hepatocytes.

 

Smeets JM, van Holsteijn I, Giesy JP, van den Berg M.

 

Research Institute of Toxicology, Utrecht University, The Netherlands.

 

Cultured hepatocytes of female carp (Cyprinus carpio) were coexposed for 4 days

to 200 nM 17beta-estradiol (E2), and concentration ranges of nine known Ah

receptor (AhR) agonists: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),

3,3'4,4'5-pentachlorobiphenyl (PCB 126), 2,3'4,4'5-pentachlorobiphenyl (PCB 118),

beta-naphthoflavone (BNF), benzo(a)pyrene (BaP), benzo(a)anthracene (BaA),

diindolylmethane (DIM), 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) and

hexachlorobenzene (HCB). TCDD caused a greater than 100-fold induction of

cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylase

(EROD), with an EC50 of 6 pM. Based on EC50 values, the order of potency as CYP1A

inducers was TCDD > PCB 126 > BNF > BaP > BaA > PCB 118. DIM and MCDF

caused a lower maximum CYP1A induction (< 9-fold), whereas HCB caused no EROD

induction at concentrations up to 6 microM. TCDD, PCB 126, BNF, BaP, and DIM also

caused a concentration-dependent suppression of the secretion of the yolk protein

vitellogenin (Vtg), relative to E2-treated hepatocytes. Suppression of Vtg

secretion was not directly correlated with EROD activity, and the antiestrogenic

effects occurred at higher concentrations than the induction of CYP1A. This

indicates that the anti-estrogenicity was not caused by increased metabolism of

E2 due to induction of CYP1A. Nevertheless, the order of potency of the tested

compounds for suppression of Vtg secretion was comparable to the order of potency

for CYP1A induction. This concurrence suggests that the anti-estrogenicity of

these compounds is AhR-mediated, but does not involve CYP1A. This could be

relevant for feral fish populations, as they are frequently exposed to AhR

agonists, to an extent that AhR-mediated effects are observed.

 

PMID: 10630570

 

Toxicol Sci. 1999 Nov;52(1):1-8.

 

Symposium on mechanisms of action of naturally occurring anticarcinogens.

 

Safe S, Wargovich MJ, Lamartiniere CA, Mukhtar H.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University,

College Station 77843-4466, USA.

 

PMID: 10568692

 

Food Chem Toxicol. 1999 Jun;37(6):609-18.

 

Effect of some indole derivatives on xenobiotic metabolism and xenobiotic-induced

toxicity in cultured rat liver slices.

 

Renwick AB, Mistry H, Barton PT, Mallet F, Price RJ, Beamand JA, Lake BG.

 

BIBRA International, Carshalton, Surrey, UK.

 

In this study the effect of some indole derivatives on xenobiotic metabolizing

enzymes and xenobiotic-induced toxicity has been examined in cultured

precision-cut liver slices from male Sprague-Dawley rats. While treatment of rat

liver slices for 72 hours with 2-200 microM of either indole-3-carbinol (I3C) or

indole-3-acetonitrile (3-ICN) had little effect on cytochrome P-450

(CYP)-dependent enzyme activities, enzyme induction was observed after in vivo

administration of I3C. The treatment of rat liver slices with 50 microM

3,3'-diindolylmethane (DIM; a dimer derived from I3C under acidic conditions) for

72 hours resulted in a marked induction of CYP-dependent enzyme activities. DIM

appears to be a mixed inducer of CYP in rat liver slices having effects on CYP1A,

CYP2B and CYP3A subfamily isoforms. Small increases in liver slice reduced

glutathione levels and glutathione S-transferase activity were also observed

after DIM treatment. While aflatoxin B1 and monocrotaline produced a

concentration-dependent inhibition of protein synthesis in 72-hour-cultured rat

liver slices, cytotoxicity was markedly reduced in liver slices cultured with 50

microM DIM. These results demonstrate that cultured rat liver slices may be

employed to evaluate the effects of chemicals derived from cruciferous and other

vegetables on CYP isoforms. In addition, liver slices can also be utilized to

examine the ability of such chemicals to modulate xenobiotic-induced toxicity.

 

PMID: 10478829

 

Biochem Pharmacol. 1999 Sep 1;58(5):825-34.

 

Cytostatic and antiestrogenic effects of

2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major in vivo product of dietary

indole-3-carbinol.

 

Chang YC, Riby J, Chang GH, Peng BC, Firestone G, Bjeldanes LF.

 

Division of Nutritional Sciences and Toxicology, University of California,

Berkeley 94720, USA.

 

Under acidic conditions, indole-3-carbinol (13C) is converted to a series of

oligomeric products thought to be responsible for the biological effects of

dietary 13C. Chromatographic separation of the crude acid mixture of 13C, guided

by cell proliferation assay in human MCF-7 cells, resulted in the isolation of

2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr-1) as a major antiproliferative

component. LTr-1 inhibited the growth of both estrogen-dependent (MCF-7) and

-independent (MDA-MB-231) breast cancer cells by approximately 60% at a

non-lethal concentration of 25 microM. LTr-1 had no apparent effect on the

proliferation of MCF-7 cells in the absence of estrogen. LTr-1 was a weak ligand

for the estrogen receptor (ER) (IC50 70 microM) and efficiently inhibited the

estradiol (E2)-induced binding of the ER to its cognate DNA responsive element.

The antagonist effects of LTr-1 also were exhibited in assays of endogenous pS2

gene expression and in cells transiently transfected with an estrogen-responsive

reporter construct (pERE-vit-CAT). LTr-1 activated both binding of the aryl

hydrocarbon (Ah) receptor to its cognate DNA responsive element and expression of

the Ah receptor-responsive gene CYP1A1. LTr-1 was a competitive inhibitor of

CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity. In summary, these

results demonstrated that LTr-1, a major in vivo product of I3C, could inhibit

the proliferation of both estrogen-dependent and -independent breast tumor cells

and that LTr-1 is an antagonist of estrogen receptor function and a weak agonist

of Ah receptor function.

 

PMID: 10449193

 

Carcinogenesis. 1998 Sep;19(9):1631-9.

 

Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of

diindolylmethane.

 

Chen I, McDougal A, Wang F, Safe S.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University,

College Station 77843-4466, USA.

 

Phytochemicals such as indole-3-carbinol (I3C) and sulforaphane are components of

cruciferous vegetables which exhibit antitumorigenic activity associated with

altered carcinogen metabolism and detoxification. Diindolylmethane (DIM) is a

major acid-catalyzed metabolite of I3C formed in the gut that binds to the aryl

hydrocarbon receptor (AhR) and treatment of MCF-7 human breast cancer cells with

10-50 microM DIM resulted in rapid formation of the nuclear AhR complex and

induction of CYP1A1 gene expression was observed at concentrations >50 microM.

Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD), a high affinity AhR ligand, inhibits 17beta-estradiol (E2)-induced

responses in MCF-7 cells and growth of E2-dependent 7,12-dimethylbenzanthracene

(DMBA)-induced mammary tumors in female Sprague-Dawley rats. Results of this

study show that like TCDD, DIM inhibits E2-induced proliferation of MCF-7 cells,

reporter gene activity in cells transiently transfected with an E2-responsive

plasmid (containing a frog vitellogenin A2 gene promoter insert) and

down-regulates the nuclear estrogen receptor. Moreover, DIM (5 mg/kg every other

day) also inhibits DMBA-induced mammary tumor growth in Sprague-Dawley rats and

this was not accompanied by induction of hepatic CYP1A1-dependent activity. Thus,

DIM represents a new class of relatively non-toxic AhR-based antiestrogens that

inhibit E2-dependent tumor growth in rodents and current studies are focused on

development of analogs for clinical treatment of breast cancer.

 

PMID: 9771935

 

Xenobiotica. 1998 Aug;28(8):803-11.

 

3,3'-Diindolylmethane induces CYP1A2 in cultured precision-cut human liver

slices.

 

Lake BG, Tredger JM, Renwick AB, Barton PT, Price RJ.

 

BIBRA International, Carshalton, UK.

 

1. The effect of 3,3'-diindolylmethane (DIM), an indole derivative derived from

cruciferous vegetables, on cytochrome P450 (CYP) isoforms in the CYP1A and CYP3A

subfamilies has been studied in 72-h cultured human liver slices. 2. In cultured

human liver slices 50 microM DIM induced 7-ethoxyresorufin O-deethylase and to a

lesser extent 7-methoxyresorufin O-demethylase activities. 3. Western

immunoblotting of liver slice microsomes was performed with antibodies to rat

CYP1A2 and human CYP3A4. Compared with control liver slice microsomes (dimethyl

sulphoxide-only treated), DIM induced levels of CYP1A2 but had little effect on

levels of CYP3A4. The treatment of human liver slices with 2 microg/ml of the

polycholorinated biphenyl mixture Aroclor 1254 also resulted in an induction of

levels of CYP1A2, but had no effect on CYP3A4. 4. These results demonstrate that

DIM induces CYP1A isoforms in cultured human liver slices. Some variability in

the magnitude of induction of enzyme activities by DIM was observed in four human

liver samples examined. For 7-ethoxyresorufin O-deethylase, the magnitude of

induction by 50 microM DIM ranged from 2.3- to 19.3-fold. 5. These results

demonstrate that cultured human liver slices can be used to evaluate the effect

of chemicals derived from cruciferous and other vegetables on CYP isoforms.

 

PMID: 9741959

 

Anticancer Drugs. 1998 Feb;9(2):141-8.

 

Selective cytostatic and cytotoxic effects of glucosinolates hydrolysis products

on human colon cancer cells in vitro.

 

Gamet-Payrastre L, Lumeau S, Gasc N, Cassar G, Rollin P, Tulliez J.

 

INRA, Laboratoire des XÚnobiotiques, Toulouse, France.

 

Glucosinolates hydrolysis products are attracting increasing attention since many

studies have suggested that they may be involved in the anticarcinogenic property

of cruciferous vegetables. In this study, we show that diindolylmethane (DIM) and

sulforaphane, produced during the hydrolysis of glucobrassicin and glucoraphanin,

respectively, exert a dose-dependent cytotoxicity on human colon adenocarcinoma

HT29 cells. Moreover, these products are able to inhibit quiescent cells to

re-enter the cell cycle. Interestingly, our results clearly show that low doses

of DIM and sulforaphane, although very effective on undifferentiated intestinal

HT29 cells, do not affect the viability of the differentiated CaCo2 cells. The

reversibility of their effects has also been tested and is discussed.

 

PMID: 9510500

 

Biochem Biophys Res Commun. 1996 Nov 1;228(1):153-8.

 

3,3'-Diindolylmethane induces apoptosis in human cancer cells.

 

Ge X, Yannai S, Rennert G, Gruener N, Fares FA.

 

Department of Food Engineering and Biotechnology, Technion-Israel Institute of

Technology, Haifa, Israel.

 

3,3'-Diindolylmethane is a dimer of indole-3-carbinol formed both in vivo and in

vitro. In this study, human cancer cells MCF-7 (with wild-type p53), T47-D

(mutant p53), and Saos-2 (deficient in p53 gene), were used to examine the

anticancer activities of 3,3'-diindolylmethane. The dose-dependent growth

inhibitory effect was found in all these cell lines. Exposure of the cells to 50

microM solution of 3,3'-diindolylmethane for 48 h, apoptosis (programmed cell

death) was evidenced by the characteristic morphology of cell nuclei under

fluorescence microscope and the DNA "ladder" in agarose gel electrophoresis. The

percentage of apoptotic cells in each cell line was found to be 12% for MCF-7,

14% for T47D and 13% for Saos2 cells. Exposure of MCF-7 cells to 100 microM

3,3'-diindolylmethane for 24 h, 19% of apoptotic cells were detected by flow

cytometry analysis. The lowest dose required for induction of apoptosis in MCF-7

cells was found to be 10 microM after 72 h incubation. Western blot showed that

wild-type p53 protein was unchanged after MCF-7 cells had been exposed to 50

microM 3,3'-diindolylmethane for 8 h. This study provides evidences that

3,3'-diindolylmethane induces apoptosis in human cancer cells and that the

induction of apoptosis is independent of p53 pathway.

 

PMID: 8912651

  

Biochem Pharmacol. 1996 Apr 26;51(8):1069-76.

 

Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah) receptor agonists

and antagonists in T47D human breast cancer cells.

 

Chen I, Safe S, Bjeldanes L.

 

Veterinary Physiology and Pharmacology, Texas A&M University, College Station

77843-4466, USA.

 

Indole-3-carbinol (I3C) is a major component of Brassica vegetables, and

diindolylmethane (DIM) is the major acid-catalyzed condensation product derived

from I3C. Both compounds competitively bind to the aryl hydrocarbon (Ah) receptor

with relatively low affinity. In Ah-responsive T47D human breast cancer cells,

I3C and DIM did not induce significantly CYP1A1-dependent ethoxyresorufin

O-deethylase (EROD) activity or CYP1A1 mRNA levels at concentrations as high as

125 or 31 microM, respectively. A 1 nM concentration of

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced EROD activity in these cells,

and cotreatment with TCDD plus different concentrations of I3C (1-125 microM) or

DIM (1-31 microM) resulted in a > 90% decrease in the induced response at the

highest concentration of I3C or DIM. I3C or DIM also partially inhibited (< 50%)

induction of CYP1A1 mRNA levels by TCDD and reporter gene activity, using an

Ah-responsive plasmid construct in transient transfection assays. In T47D cells

cotreated with 5 nM [3H]TCDD alone or in combination with 250 microM I3C or 31

microM DIM, there was a 37 and 73% decrease, respectively, in formation of the

nuclear Ah receptor. The more effective inhibition of induced EROD activity by

I3C and DIM was due to in vitro inhibition of enzyme activity. Thus, both I3C and

DIM are partial Ah receptor antagonists in the T47D human breast cancer cell

line.

 

PMID: 8866829

 

Food Chem Toxicol. 1995 Oct;33(10):851-7.

 

Mechanisms of indole-3-carbinol (I3C) anticarcinogenesis: inhibition of aflatoxin

B1-DNA adduction and mutagenesis by I3C acid condensation products.

 

Takahashi N, Dashwood RH, Bjeldanes LF, Williams DE, Bailey GS.

 

Department of Food Science & Technology, Oregon State University, Corvallis

97331, USA.

 

Possible inhibitory mechanisms of indole-3-carbinol (I3C) against aflatoxin B1

(AFB1), a potent hepatocarcinogen, were examined in rainbow trout. In the

Salmonella assay using a trout post-mitochondrial activation system, I3C itself

was not an antimutagen against AFB1. The study also evaluated: the antimutagenic

ability of I3C oligomers; an acid reaction mixture (RXM) of I3C, generated at low

pH to simulate I3C products formed under acidic conditions of the stomach;

3,3-diindolylmethane (I33'), the major derivative of I3C found in trout liver;

and 5,6,11,12,17,18- hexahydrocyclononal [1,2-b:4,5-b':7,8-b"]triindole , the

cyclic trimer of I3C (CT), a derivative of I3C in liver and one of the major

components of RXM. Concentrations of 3.5 microM and greater of I33', CT or RXM

showed about 80% inhibition compared with the control. Higher concentrations (70

microM) of the various I3C oligomers also inhibited (to a maximum of 55%)

mutagenesis of synthetic AFB1-8,9-epoxide added to the Salmonella assay, in the

absence of activating enzymes. I33' inhibited total microsome catalysed AFB1-DNA

binding in vitro in an apparently non-competitive manner (Kis = 27.6 +/- 9.4

microM, Kii = 37.5 +/- 32.2 microM). These results suggest that the

anticarcinogenic effect of I3C against AFB1 in rainbow trout, and perhaps other

species, is due in part to inhibition of AFB1 bioactivation enzymes and to

scavenging of the activated AFB1-8,9-epoxide.

 

PMID: 7590529

  

J Biochem Toxicol. 1995 Aug;10(4):191-201.

 

The anticarcinogen 3,3'-diindolylmethane is an inhibitor of cytochrome P-450.

 

Stresser DM, Bjeldanes LF, Bailey GS, Williams DE.

 

Department of Food Science and Technology, Oregon State University, Corvallis

97331-6602, USA.

 

Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several

mechanisms of inhibition may exist. We reported previously that

3,3'-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent

noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent

ethoxyresorufin O-deethylase with Ki values in the low micromolar range. We now

report a similar potent inhibition by 3,3'-diindolylmethane of rat and human

CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential

activity assays. 3,3'-Diindolylmethane also inhibited in vitro CYP-mediated

metabolism of the ubiquitous food contaminant and potent hepatocarcinogen,

aflatoxin B1. There was no inhibition of cytochrome c reductase. In addition, we

found 3,3'-diindolylmethane to be a substrate for rat hepatic microsomal

monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These

observations indicate that 3,3'-diindolylmethane can inhibit the catalytic

activities of a range of CYP isoforms from lower and higher vertebrates in vitro.

This broadly based inhibition of CYP-mediated activation of procarcinogens may be

an indole-3-carbinol anticarcinogenic mechanism applicable to all species,

including humans.

 

PMID: 8568833

 

Food Chem Toxicol. 1995 Feb;33(2):111-20.

 

Regulation of hepatic cytochrome P4501A by indole-3-carbinol: transient induction

with continuous feeding in rainbow trout.

 

Takahashi N, Dashwood RH, Bjeldanes LF, Bailey GS, Williams DE.

 

Department of Food Science and Technology, Oregon State University, Corvallis

97331.

 

This study investigated the kinetics of hepatic cytochrome P-4501A (CYP1A)

induction in rainbow trout by indole-3-carbinol (I3C), a natural tumour modulator

from cruciferous vegetables, and its low pH reaction products

3,3'-diindolylmethane (I33'),

5,6,11,12,17,18-hexahydrocyclononal[1,2-b:4,5-b':7,8-b"]triindo le cyclic trimer

(CT), and the unresolved I3C acid reaction mixture (RXM). RXM, CT and I33' were

potent inducers of total embryonic CYP1A following direct microinjection, and of

fingerling hepatic CYP1A following ip exposure, whereas I3C itself produced only

a transient and relatively weak induction. It is also reported for the first time

that dietary I3C induced hepatic CYP1A and its associated ethoxyresorufin

O-deethylase (EROD) activity in trout but, again, the induction was weak and

transient even with continuous I3C feeding. Mechanism studies and mixed exposures

with the Ah agonist beta-naphthoflavone indicated that transient induction by I3C

was not due to diet ageing, but appears to involve inactivation of the Ah

inductive pathway and irreversible inactivation of CYP1A-mediated EROD activity

by I3C-derived metabolites. Thus, I3C derivatives exhibit dual capacities for

CYP1A induction and inhibition in trout.

 

PMID: 7867998

  

J Steroid Biochem Mol Biol. 1993 Dec;46(6):791-8.

 

Influence of indole carbinols and growth hormone on the metabolism of

4-androstenedione by rat liver microsomes.

 

Jellinck PH, Makin HL, Sepkovic DW, Bradlow HL.

 

Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.

 

The effect of indole-3-carbinol (IC), an anticarcinogen present in cruciferous

vegetables, to alter the metabolism of 4-androstenedione (AD) by female rat liver

microsomes was investigated and compared to that of its main gastric conversion

product, diindolylmethane (DIM) as well as other specific cytochrome P450

inducers. DIM was a more potent inducer of the hydroxylase which converts

androsterone to its 6 beta-hydroxylated derivative 3 alpha, 6 beta-dihydroxy-5

alpha-androstan-17-one (A) than IC after either oral or intraperitoneal

administration and was also a better in vitro inhibitor. Isosafrole (ISF), which

like IC and DIM, induces CYP1A2 as well as gestodene, were powerful inhibitors of

the in vitro reaction. Naringenin produced only a weak inhibitory effect while

3-methylcholanthrene was inactive. SKF-525A, a prototypic hydroxylase inhibitor,

or 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androst-1-ene-3-one which

inhibits steroid 5 alpha-reductase, also decreased the formation of A from AD by

liver microsomes. The infusion of human growth hormone by osmotic minipump, which

feminizes hepatic steroid metabolism, increased the ability of male rat liver

microsomes to convert AD to A and to respond to induction by IC. The identity of

A, the main polar derivative of AD, induced by IC, DIM and ISF, was tentatively

assigned by a combination of GC-MS and results from metabolic studies with

intermediates in the pathway leading to its formation. It is proposed that the

protective role of indole carbinols against mammary carcinoma due to decreased

formation of 16 alpha-hydroxyestrone from estrone may be further enhanced by the

diminished availability of AD for aromatization to estrone.

 

PMID: 8274413

 

Biochem Pharmacol. 1993 Mar 9;45(5):1129-36.

 

Ah receptor binding properties of indole carbinols and induction of hepatic

estradiol hydroxylation.

 

Jellinck PH, Forkert PG, Riddick DS, Okey AB, Michnovicz JJ, Bradlow HL.

 

Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.

 

The effect of route of administration on the ability of indole-3-carbinol (13C),

an anticarcinogen present in cruciferous vegetables, to induce estradiol

2-hydroxylase (EH) in female rat liver microsomes was investigated and compared

to that of its main gastric conversion product, 3,3'-diindolylmethane (DIM). This

dimer was more potent than 13C after either oral or intraperitoneal

administration and was also a better in vitro inhibitor of EH in control and

13C-induced hepatic microsomes. The induction of both CYP1A1 and 1A2 in about

equal amounts by 13C and DIM as well as of CYP2B1/2 was demonstrated using

monoclonal antibodies. DIM, isosafrole, beta-naphthoflavone, 3-methylcholanthrene

and naringenin added in vitro inhibited EH strongly in induced microsomes but

gestodene was a better inhibitor of estrogen 2-hydroxylation in liver microsomes

from untreated female rats. The binding affinities of 13C and DIM to the Ah

receptor were compared to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by

competition studies, and the IC50 values were shown to be 2.0 x 10(-9) M, 5.0 x

10(-5) M and 2.3 x 10(-3) M for TCDD, DIM and 13C, respectively. The ability of

13C or DIM to cause in vitro transformation of the Ah receptor to a form able to

bind to the dioxin-responsive element-3 (DRE3) was compared to that of TCDD and

shown to parallel their abilities to compete for binding of [3H]TCDD to the Ah

receptor. These experiments confirm and extend the proposals that dietary indoles

induce specific cytochrome P450s in rat liver by a mechanism possibly involving

the Ah receptor. The induced monooxygenases, in turn, increase the synthesis of

2-hydroxylated estrogens in the competing pathways of 2- and 16

alpha-hydroxylation which decreases the levels of 16 alpha-hydroxyestrone able to

form stable covalent adducts with proteins including the estrogen receptor. Such

steroid-protein interaction has been correlated with mammary carcinogenesis.

 

PMID: 8384853

 

Food Chem Toxicol. 1992 Jul;30(7):589-99.

 

Effects of indole-3-carbinol on biotransformation enzymes in the rat: in vivo

changes in liver and small intestinal mucosa in comparison with primary

hepatocyte cultures.

 

Wortelboer HM, van der Linden EC, de Kruif CA, Noordhoek J, Blaauboer BJ, van

Bladeren PJ, Falke HE.

 

UTOX, c/o Research Institute of Toxicology (RITOX), University of Utrecht, The

Netherlands.

 

Groups of male Wistar rats were fed semi-synthetic diets containing 0, 200 or 500

mg indole-3-carbinol (13C)/kg for 2, 7, 14 or 28 days. After 2 days, P-450

activities were already induced, but the isoenzyme pattern induced was different

in the liver and the small intestine. Hepatic P4501A1, P4501A2 and P4502B1

apoprotein levels were dose-relatedly enhanced, whereas in the small intestine

induced levels of P4502B1 and P4501A1 were detected but P4501A2 was not induced.

Pentoxy- and ethoxyresorufin dealkylation (PROD and EROD) were dose-relatedly

enhanced in the liver (5- and 7-fold, respectively, in the higher dose group) as

well as in the small intestine (8- and 13-fold, respectively, at 500 mg 13C/kg

diet). Testosterone 16 alpha- and 16 beta-hydroxylation in the small intestine

were enhanced (6-9-fold) from day 2 onwards, but in the liver these activities

were only slightly enhanced from day 7 onwards. Thus, the major forms induced in

the liver appear to be P4501A1, P4501A2, P4502B1 and, to a lesser extent, P4503A,

whereas in the small intestine all of the effects that were found are associated

with only one cytochrome P-450, P4502B1. After 2 days I3C (500 mg/kg) induced

glutathione S-transferase in the liver (1.3-fold) and small intestine (1.5-fold).

Hepatic glucuronyl transferase (GT1) was induced (about 1.6-fold) after 7, 14 and

28 days. DT-diaphorase was induced in the liver (2.7-fold) and small intestine

(1.5-fold) after 14 days of exposure to 500 mg I3C/kg diet. Treatment of rat

hepatocytes with indole-3-acetonitrile and 3,3'-diindolylmethane, but not I3C and

indole-3-carboxaldehyde, enhanced EROD activity and halved testosterone 16 alpha-

and 2 alpha-hydroxylation. All four indoles slightly induced glutathione

S-transferase in cultured hepatocytes. Thus, the in vitro studies suggest that

the in vivo effects of I3C have to be attributed to indole-condensation products,

such as 3,3'-diindolylmethane, but not to I3C itself.

 

PMID: 1521833

  

Chem Biol Interact. 1991;80(3):303-15.

 

Structure elucidation of acid reaction products of indole-3-carbinol: detection

in vivo and enzyme induction in vitro.

 

De Kruif CA, Marsman JW, Venekamp JC, Falke HE, Noordhoek J, Blaauboer BJ,

Wortelboer HM.

 

UTOX, University of Utrecht, The Netherlands.

 

The potency of indole-3-carbinol (I3C) to form condensation products under acidic

aqueous conditions was studied. After identifying a known dimer,

3,3'-diindolylmethane (DIM), we elucidated the structures of two trimers also

found in acid reaction mixtures:

5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b':7,8-b"]tri-indole (CTI), and

2,3-bis[3-indolylmethyl] indole (BII). The formation of these indole oligomers

was shown to be pH dependent. The highest amounts of DIM and BII were formed in

aqueous solutions having a pH value ranging from 4 to 5. No CTI could be detected

at pH values above 4.5. In rats that received an oral dose of I3C we could detect

DIM and BII in gastric contents, stomach tissue, small intestine and liver. No

CTI could be detected in vivo after oral exposure to I3C. In in vitro

experiments, using rat hepatocytes, the cytochrome P-450IA1 apoprotein level,

7-ethoxyresorufin O-deethylation activity (EROD) and DT-diaphorase activity (DTD)

were markedly enhanced by DIM and CTI as well as BII.

 

PMID: 1954658

 

Food Chem Toxicol. 1989 Jun;27(6):385-92.

 

In vivo disposition of the natural anti-carcinogen indole-3-carbinol after po

administration to rainbow trout.

 

Dashwood RH, Uyetake L, Fong AT, Hendricks JD, Bailey GS.

 

Department of Food Science and Technology, Oregon State University, Corvallis

97331.

 

Indole-3-carbinol (I3C), a compound found naturally as a glucosinolate in

cruciferous vegetables such as broccoli and cabbage, has been shown to modulate

the carcinogenic process in a number of animal species. The lack of detailed

information on the disposition of I3C in vivo provided the main impetus for the

study reported here, in which the distribution and metabolic fate of I3C was

assessed in selected tissues and excreta after po administration to rainbow trout

(Salmo gairdneri). Animals were fasted for 3 days and given [5-3H]I3C either in

the diet or by single oral gavage (40 mg/kg body weight; 15 muCi/kg body weight).

Following administration, 75% of the initial 3H-dose was detected within the

stomach between 0.5 and 12 hr, after which it was released to distal regions of

the gut for subsequent uptake, distribution and elimination. At the end of the

study (72 hr) 25% of the administered dose was recovered from the water which

reflected excretion through the gills and urinary tract. Significant excretion

also occurred in the bile, with approximately 5% of the initial 3H-dose recovered

from the bile sacs at 72 hr. Further analyses of the radioactive components in

the bile indicated that one or more derivatives of I3C, but not the parent

compound itself, are excreted as glucuronide conjugates using this route.

Radioactivity accumulated in the liver throughout most of the study, reaching

levels of 1-1.5% between 48 and 72 hr of the administered dose. High-performance

liquid chromatography analyses indicated the presence of four main radiolabelled

species in these livers, one of which co-eluted with the parent compound, I3C.

The major radiolabelled species recovered from the liver was tentatively

identified as the dimer, 3,3'-diindolylmethane (I33'), which comprised 40% of the

total hepatic radiolabel. This dimer, I33', was also found to accumulate in the

diet containing I3C, which reflected a time-dependent dimerization of the parent

compound in vitro. These findings are discussed in view of recent postulates of a

role for I3C condensation products such as I33' in the mechanism of I3C

anti-carcinogenesis.

  

PMID: 2792968

 

Food Chem Toxicol. 1987 May;25(5):363-8.

 

Differential induction of mixed-function oxidase (MFO) activity in rat liver and

intestine by diets containing processed cabbage: correlation with cabbage levels

of glucosinolates and glucosinolate hydrolysis products.

 

McDanell R, McLean AE, Hanley AB, Heaney RK, Fenwick GR.

 

Both white and Savoy-type cabbage added to a semi-purified diet at 25% dry weight

and fed to rats ad lib. for 5 days significantly induced ethoxyresorufin (ERR)

deethylation in the small and large intestine. Savoy cabbage also induced hepatic

activity and, in general, exhibited a greater inducing effect than white cabbage.

These enzyme-inducing effects were altered when the cabbage had been processed.

The content of intact glucosinolate was greater in Savoy than in white cabbage.

The indole glucosinolate (glucobrassicin) content of both types of cabbage was

approximately halved by cooking but was unaffected by fermentation, whilst

homogenization of Savoy cabbage led to the total disappearance of intact

glucosinolates. Levels of the indole glucosinolate breakdown products ascorbigen

and indole-3-carbinol were highest in homogenized cabbage, and ascorbigen levels

were also higher in cooked than in fresh cabbage of either type. When added to

the semi-purified diet and fed ad lib. to rats for 5 days, indole-3-carbinol was

a potent inducer of hepatic ERR deethylation and cytochrome P-450 activity, but

had much less effect in the intestine. Other glucobrassicin metabolites,

diindolylmethane and indole-3-acetonitrile, also had some inducing effect in the

liver but no effect in the intestine, while ascorbigen significantly induced ERR

deethylation in the small and large intestine but had no effect on hepatic MFO

activity.

  

PMID: 3609976

 

J Toxicol Environ Health. 1987;21(3):311-23.

 

Structure-activity relationships of dietary indoles: a proposed mechanism of

action as modifiers of xenobiotic metabolism.

 

Bradfield CA, Bjeldanes LF.

 

In an effort to understand the mechanism by which dietary indoles inhibit

chemically initiated tumorigenesis in experimental animals, we have investigated

the potency of 3-substituted and 1,3-disubstituted indoles on the induction of

intestinal and hepatic cytochrome P-448-dependent monooxygenases in the rat. Oral

intubation with indole-3-carbinol (13C), 1-methoxyindole-3-carbinol (N13C),

1-methoxyindole-3-carboxaldehyde (NCHO), and 3,3'-diindolylmethane (133') at 31

mumol/animal led to significant increases in hepatic ethoxyresorufin O-deethylase

activity (EROD; 15, 7, 6, and 5-fold over control, respectively), while

intubation with indole (IND), 3-methylindole (3MI), indole-3-carboxaldehyde

(13CHO), and indole-3-acetonitrile (IAN) did not increase this monooxygenase

activity over control levels. For the eight indoles tested, there was a strong

relationship between instability in acidic solution, as indicated by the

generation of insoluble products, and capacity to induce hepatic EROD. Further

experiments indicated that 13C did not induce hepatic EROD when dosed ip (thus

bypassing the acidity of the stomach). Acid treatment of 13C generated a reaction

mixture (RXM) that induced EROD after ip or po dosing. Chromatographic

fractionation of the RXM indicated that there exist at least four different 13C

acid-condensation products in the RXM with the ability to induce EROD. The

results presented strongly support the hypothesis that dietary indoles influence

the levels of monooxygenase activities via a series of acid-condensation products

generated upon introduction of the indole into the acidic environment of the

stomach.

 

PMID: 3495667

 

Cancer Res. 1978 May;38(5):1410-3.

 

Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally

occurring indoles.

 

Wattenberg LW, Loub WD.

 

Indole-3-carbinol, 3,3'-diindolylmethane, and indole-3-acetonitrile, three

indoles occurring in edible cruciferous vegetables, have been studied for their

effects on 7,12-dimethylbenz(a)anthracene-induced mammary tumor formation in

female Sprague-Dawley rats and on benzo(a)pyrene-induced neoplasia of the

forestomach in female ICR/Ha mice. When given by p.o. intubation 20 hr prior to

7,12-dimethylbenz(a)anthracene administration, indole-3-carbinol and

3,3'-diindolylmethane had an inhibitory effect on mammary tumor formation, but

indole-3-acetonitrile was inactive. Indole-3-carbinol when added to the diet for

8 days prior to challenge with 7,12-dimethylbenz(a)anthracene inhibited mammary

tumor formation, whereas indole-3-acetonitrile did not. Dietary administration of

all three indoles inhibited benzo(a)pyrene-induced neoplasia of the forestomach

in ICR/Ha mice. The identification of dietary constituents that can inhibit

chemical carcinogens ultimately may be of value in understanding the balance of

factors that determines the neoplastic response to these cancer-producing agents

in the environment.

 

PMID: 416908

 

Fed Proc. 1976 May 1;35(6):1327-31.

 

Dietary constituents altering the responses to chemical carcinogens.

 

Wattenberg LW, Loub WD, Lam LK, Speier JL.

 

This paper deals with two categories of compounds having the capacity to inhibit

the neoplastic effects of chemical carcinogens on the host. The first are

inducers of increased microsomal mixed function oxidase activity. An increasing

number of these inducers are being found in natural products. Cruciferous

vegetables including brussels sprouts, cabbage, and cauliflower contain such

compounds. Recently indole-3-acetonitrile, indole-3-carbinol and

3,3'-diindolylmethane have been identified as inducers in these three plants.

Other naturally occurring inducers include flavones, safrole, isosafrole,

beta-ionone, and oxidized sterols. Since previous work has shown that synthetic

inducers may protect against chemical carcinogens, the composition of the diet

could play a role in inhibiting the neoplastic response to these carcinogenic

agents. The second category of inhibitors comprises the antioxidants. Several of

these compounds have been found to inhibit the carcinogenic effects of a variety

of chemical carcinogens. Considerable work of this nature has been done with

butylated hydroxyanisole and butylated hydroxytoluene two antioxidants

extensively used as food additives. Other antioxidants having carcinogen

inhibiting capacities include ethoxyquin, disulfiram, and

dimethyldithiocarbamate.

 

PMID: 1261715

 

Cancer Res. 1975 Nov;35(11 Pt. 2):3326-31.

 

Effects of dietary constituents on the metabolism of chemical carcinogens.

 

Wattenberg LW.

 

Dietary constituents of 2 types have been shown to affect the metabolism of

chemical carcinogens by the microsomal mixed-function oxidase system. Naturally

occurring inducers of increased activity of this system are present in plants.

Cruciferous vegetables including Brussels sprouts, cabbage, and cauliflower are

relatively potent in this regard. From these vegetables, three indoles with

inducing activity have been identified. These are indole-3-acetonitrile,

indole-3-carbinol, and 3,3'-diindolylmethane. A 2nd type of dietary constituent

affecting the microsomal mixed-function oxidase system is added phenolic

antioxidant, i.e., butylated hydroxyanisole (BHA) and butylated hydroxytoluene.

Studies of the effect of BHA on metabolism of bezo(a)-pyrene by liver microsomes

have been carried out. BHA feeding results in microsomal changes. The cytochrome

P-450 shows altered spectral characteristics, and the aryl hydrocarbon

hydroxylase system of these microsomes has an increased sensitivity to inhibition

by alpha-naphthoflavone. In addition, there is a decrease in binding of

metabolites of benzo(a)pyrene to DNA upon incubation of these microsomes of

induction of increased mixed function oxidase activity have shown that increased

levels of activity protect against administration of chemical carcinogens. BHA

and butylated hydroxytoluene also have been found to exert a protective effect

against chemical carcinogens. Thus the constituents of the diet could be of

consequence in the neoplastic response to exposure to carcinogens in the

environment.

 

PMID: 1104144

 

J Natl Cancer Inst. 1975 Apr;54(4):985-8.

 

Aryl hydrocarbon hydroxylase induction in rat tissues by naturally occurring

indoles of cruciferous plants.

 

Loub WD, Wattenberg LW, Davis DW.

 

A phytochemical investigation to identify inducers of increased aryl hydrocarbon

hydroxylase (AHH) activity from three cruciferous vegetables, Brussels sprouts,

cabbage, and cauliflower, resulted in the identification of

indole-3-acetonitrile, indole-3-carbinol, and 3,3'-diindolylmethane as naturally

occurring inducers. These compounds are produced during the hydrolysis of

indolyl-methyl glucosinolate by the plant enzyme myrosinase.

 

PMID: 1127728

 

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