Diindolylmethane (DIM) Information Resource Center

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

 

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 µM 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 µM. 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 

Arch Toxicol. 2011 Mar 16.

Effect of diindolylmethane on Ca(2+) movement and viability in HA59T human
hepatoma cells.

Cheng JS, Shu SS, Kuo CC, Chou CT, Tsai WL, Fang YC, Kuo LN, Yeh JH, Chen WC,
Chien JM, Lu T, Pan CC, Cheng HH, Chai KL, Jan CR.

Department of Medicine, Yongkang Veterans Hospital, Tainan, 710, Taiwan.

The effect of diindolylmethane, a natural compound derived from indole-3-carbinol
in cruciferous vegetables, on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and
viability in HA59T human hepatoma cells is unclear. This study explored whether
diindolylmethane changed [Ca(2+)](i) in HA59T cells. The Ca(2+)-sensitive
fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at
concentrations of 1-50 μM evoked a [Ca(2+)](i) rise in a concentration-dependent 
manner. The signal was reduced by removing Ca(2+). Diindolylmethane-induced
Ca(2+) influx was not inhibited by nifedipine, econazole, SK&F96365, and protein 
kinase C modulators but was inhibited by aristolochic acid. In Ca(2+)-free
medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitors
thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished
diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane
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 10-75 μM, diindolylmethane killed cells in a
concentration-dependent manner. The cytotoxic effect of diindolylmethane was not 
reversed by chelating cytosolic Ca(2+) with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Propidium iodide
staining data suggest that diindolylmethane (25-50 μM) induced apoptosis in a
concentration-dependent manner. Collectively, in HA59T cells, diindolylmethane
induced a [Ca(2+)](i) rise by causing phospholipase C-dependent Ca(2+) release
from the endoplasmic reticulum and Ca(2+) influx via phospholipase A(2)-sensitive
channels. Diindolylmethane induced cell death that may involve apoptosis.

PMID: 21409406 

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, Université du Québec, 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 µM. 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 µM) 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, Gerhäuser 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.

Wihlén 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, Löwinger M, Bartsch H, Gerhäuser 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, Gerhäuser 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 t