This section is updated on a monthly
basis. Paper abstracts are presented in chronological order from most recent.
Important note for readers:
while many of the scientific discoveries and clinical developments listed in
this section are extremely exciting, they are stepping stones in the studies of
Diindolylmethane (DIM) and some of the other key natural ingredients in
ActivaMune. Only the US FDA has the authority to recognize a compound as a drug
or therapeutic for a particular condition in the US and that only occurs after
the compound has been thoroughly studied and its efficacy established in four
consecutive human clinical trials. At this point in time, Diindolylmethane and
the other natural ingredients in ActivaMune are regarded as dietary supplements
and not therapeutics for any specific condition by the US FDA. ActivaMune is a
nutritional supplement.
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 ΅M 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 ΅M. 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, Universitι du Quιbec, 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 ΅M. 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 ΅M) 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, Gerhδuser 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
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.. [Epub ahead of
print]
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 B-DIM could be
mediated via inactivation of uPA-uPAR system. We found that B-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
B-DIM indicating an important role of uPA-uPAR in B-DIM-mediated inhibition of
cell growth and migration. We also found similar effects of B-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 B-DIM. Our results,
therefore, suggest that B-DIM down-regulates uPA-uPAR in aggressive breast
cancers but in the absence of uPA-uPAR, B-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 (DIM) 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 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 (DIM), 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 (DIM) 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 (DIM) 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 (DIM) 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 (DIM) 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 (DIM) 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 (DIM) 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 B-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
B-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.
Wihlιn 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 (DIM) 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 (DIM) 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 (DIM) 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 (DIM) 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) 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 (DIM), 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 (DIM) 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 (DIM) 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 B-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
(DIM)
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 (DIM) 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 (DIM) 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 (DIM) 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 (DIM) 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
Mol Pharmacol. 2008 Nov;74(5):1292-307. Epub 2008 Aug 14.
Mitochondria-dependent reactive oxygen species-mediated programmed cell death
induced by 3,3'-Diindolylmethane (DIM) through inhibition of F0F1-ATP synthase in
unicellular protozoan parasite Leishmania donovani.
Roy A, Ganguly A, BoseDasgupta S, Das BB, Pal C, Jaisankar P, Majumder HK.
Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4, Raja
S.C. Mullick Rd., Kolkata-700032, India.
Mitochondria are the principal site for the generation of cellular ATP by
oxidative phosphorylation. F0F1-ATP synthase, a complex V of the electron
transport chain, is an important constituent of mitochondria-dependent signaling
pathways involved in apoptosis. In the present study, we have shown for the
first
time that 3,3'-diindolylmethane (DIM), a DNA topoisomerase I poison, inhibits
mitochondrial F0F1-ATP synthase of Leishmania donovani and induces programmed
cell death (PCD), which is a novel insight into the mechanism in protozoan
parasites. DIM-induced inhibition of F0F1-ATP synthase activity causes depletion
of mitochondrial ATP levels and significant stimulation of mitochondrial
reactive
oxygen species (ROS) production, followed by depolarization of mitochondrial
membrane potential (DeltaPsi(m)). Because DeltaPsi(m) is the driving force for
mitochondrial ATP synthesis, loss of DeltaPsi(m) results in depletion of
cellular
ATP level. The loss of DeltaPsi(m) causes the cellular ROS generation and in
turn
leads to the oxidative DNA lesions followed by DNA fragmentation. In contrast,
loss of DeltaPsi(m) leads to release of cytochrome c into the cytosol and
subsequently activates the caspase-like proteases, which lead to
oligonucleosomal
DNA cleavage. We have also shown that mitochondrial DNA-depleted cells are
insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for
cytotoxicity of DIM in kinetoplastid parasites. Taken together, our study
indicates for the first time that DIM-induced mitochondrial dysfunction by
inhibition of F0F1-ATP synthase activity leads to PCD in Leishmania spp.
parasites, which could be exploited to develop newer potential therapeutic
targets.
PMID: 18703668
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, B-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 (DIM) 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 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,
Lφwinger M, Bartsch H, Gerhδuser 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 (DIM)
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 (DIM) 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 (DIM) 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 (DIM)
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 (DIM)
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 (DIM) 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 (DIM), one of the active products derived
from Brassica vegetables,
is a promising antitumor agent. Previous studies in our
laboratory showed that
3,3'-Diindolylmethane (DIM) 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 (DIM)
is a strong mitochondrial
H(+)-ATPase inhibitor (IC(50) approximately 20
micromol/L).
3,3'-Diindolylmethane (DIM) 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 (DIM)
failed to induce p21 overexpression in mitochondrial
respiratory chain deficient rho(0) MCF-7 cells, in which
3,3'-Diindolylmethane (DIM)
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, Gerhδuser 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 (DIM) 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 (DIM) (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 (DIM), 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 (DIM)
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 (DIM), we found that combined treatment of
3,3'-Diindolylmethane (DIM) 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 (DIM) 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 (DIM) 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 (DIM). 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 (DIM) 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 (DIM)
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 (DIM) 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 (DIM)
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 (DIM) 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 (DIM) 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 (DIM) 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 (DIM)
is mediated by cross talk
between the protein kinase A and mitogen-activated
protein kinase signaling
pathways.
Leong H, Riby JE,
Firestone GL, Bjeldanes LF.
Department of Nutritional
Sciences and Toxicology, University of California,
Berkeley, California
94720, USA.
We investigated the
mechanism of ligand-independent activation of the estrogen
receptor (ER) by
3,3'-diindolylmethane (DIM), a promising anticancer agent
derived from vegetables of
the Brassica genus, in Ishikawa and HEC-1B human
endometrial cancer cells.
DIM stimulated the activity of an ER-responsive
reporter by over 40-fold,
equivalent to the maximum induction produced by
estradiol (E2), whereas
cotreatment of cells with the ER antagonist, ICI-182,780
(ICI), abolished the
stimulatory effect of DIM. DIM also induced the expressions
of the endogenous genes,
TGF-alpha, alkaline phosphatase, and progesterone
receptor similar to levels
induced by E2. Induction of gene expression by DIM was
inhibited by the protein
synthesis inhibitor, cycloheximide. In addition,
cotreatment of cells with
the protein kinase A (PKA) inhibitor, H89, or the MAPK
inhibitor, PD98059,
reduced DIM activation of the ER by 75% and 50%,
respectively. Simultaneous
treatment of cells with both inhibitors completely
abolished the effect of
DIM. DIM stimulated MAPK activity and induced
phosphorylation of the
endogenous PKA target, cAMP response element binding
protein (CREB), in a PKA-dependent
manner. Expression of MCREB, a
nonphosphorylatable CREB
mutant, partially abolished activation of the ER by DIM.
These results demonstrate
that DIM is a mechanistically novel activator of the ER
that requires PKA-dependent
phosphorylation of CREB.
PMID: 14645498
Curr Opin Otolaryngol Head
Neck Surg. 2003 Dec;11(6):433-41.
Overview of recurrent
respiratory papillomatosis.
Wiatrak BJ.
Department of Pediatrics
and Surgery, University of Alabama at Birmingham, USA.
PURPOSE OF REVIEW: The
purpose of this article is to review recent literature
regarding pediatric
recurrent respiratory papillomatosis (RRP) published within
the last year. By
reviewing and assessing these articles, a more clear
understanding regarding
the etiology and management of pediatric RRP can be
obtained, allowing
physicians to better care for their pediatric RRP patients.
RECENT FINDINGS: Pediatric
RRP continues to be an extremely difficult management
problem for
otolaryngologists. This disease process continues to be a significant
burden on the health care
system and is a significant cause of morbidity in
affected patients and
their families. The incidence of RRP continues to be
approximately 3.96 per
100,000 in the pediatric population. It has been noted
recently that
approximately 7 of every 1000 children born to mothers with vaginal
condyloma develop
pediatric RRP. Although the mainstay of surgical management has
traditionally been the CO2
laser, newer surgical techniques have demonstrated
efficacy in the management
of pediatric RRP patients, including powered
instrumentation and the
pulse-dye laser. The traditional adjuvant medical
therapies used for
pediatric RRP continue to be commonly used, including
interferon-alpha2a,
retinoic acid, and indol-3-carbinol/diindolylmethane
(I3C/DIM). Recently
cidofovir has demonstrated efficacy in selected patients. In
addition, current research
regarding vaccine therapy for pediatric RRP has shown
promise. Basic science
research in the field of immunology has demonstrated
multiple defects in
cell-mediated immunity, which has shed further light on the
etiology of pediatric RRP.
SUMMARY: Pediatric RRP continues to be a highly morbid
disease process. New
surgical and medical therapies offer hope for better control
of this disease in
affected patients. Recent advances in immunologic research
offer the hope of immune
system modulation and augmentation as potential future
treatment modalities to
better control this disease process.
PMID: 14631175
Mol Cell Biol. 2003
Nov;23(21):7920-5.
Agonist and
chemopreventative ligands induce differential transcriptional
cofactor recruitment by
aryl hydrocarbon receptor.
Hestermann EV, Brown M.
Department of Molecular
Oncology, Dana-Farber Cancer Institute, Brigham and
Women's Hospital and
Harvard Medical School, Boston, Massachusetts, USA.
Aryl hydrocarbon receptor
(AHR) is a transcription factor whose activity is
regulated by environmental
agents, including several carcinogenic agonists. We
measured recruitment of
AHR and associated proteins to the human cytochrome
P4501A1 gene promoter in
vivo. Upon treatment with the agonist
beta-naphthoflavone, AHR
is rapidly associated with the promoter and recruits the
three members of the p160
family of coactivators as well as the p300 histone
acetyltransferase, leading
to recruitment of RNA polymerase II (Pol II) and
induction of gene
transcription. AHR, coactivators, and Pol II cycle on and off
the promoter, with a
period of approximately 60 min. In contrast, the
chemopreventative AHR
ligand 3,3'-Diindolylmethane (DIM) promotes AHR nuclear
translocation and p160
coactivator recruitment but, remarkably, fails to recruit
Pol II or cause histone
acetylation. This novel mechanism of receptor antagonism
may account for the
antitumor properties of chemopreventative compounds targeting
the AHR.
PMID: 14560034
J Nutr. 2003 Jul;133(7
Suppl):2470S-2475S.
Indole-3-carbinol and 3-3'-Diindolylmethane (DIM) antiproliferative signaling pathways
control cell-cycle gene
transcription in human breast cancer cells by regulating
promoter-Sp1 transcription
factor interactions.
Firestone GL, Bjeldanes
LF.
Department of Molecular
and Cell Biology and Cancer Research Laboratory,
University of California
at Berkeley, Berkeley, CA 94720-3200, USA.
Indole-3-carbinol (I3C), a
compound that occurs naturally in Brassica vegetables
such as cabbage and
broccoli, can induce a G1 cell-cycle arrest of human MCF-7
breast cancer cells that
is accompanied by the selective inhibition of
cyclin-dependent kinase 6
(Cdk6) expression and stimulation of p21(Waf1/Cip1)
gene expression.
Construction and transfection of a series of promoter-reporter
plasmids demonstrate that
the indole-regulated changes in Cdk6 and p21(Waf1/Cip1)
levels are due to specific
effects on their corresponding promoters. Mutagenic
analysis reveals that I3C
signaling targets a composite transcriptional element
in the Cdk6 promoter that
requires both Sp1 and Ets transcription factors for
transactivation function.
Analysis of protein-DNA complexes formed with nuclear
proteins isolated from
I3C-treated and -untreated cells demonstrates that the Sp1
DNA element in the Cdk6
promoter interacts with an I3C-inhibited protein-protein
complex that contains the
Sp1 transcription factor. In indole-treated cells, a
fraction of [(3)H]I3C was
converted into its natural diindole product
(3)H-labeled
3-3'-diindolylmethane ([(3)H]DIM), which accumulates in the nucleus;
this suggests that DIM may
have a role in the transcriptional activities of I3C.
Mutagenic analysis of the
p21(Waf1/Cip1) promoter reveals that in transfected
breast cancer cells, DIM
(as well as I3C) stimulates p21(Waf1/Cip1) transcription
through an indole-responsive
region of the promoter that contains multiple Sp1
consensus sequences.
Furthermore, DIM treatment regulates the presence of a
nuclear Sp1 DNA-binding
activity. Our results demonstrate that both the Cdk6 and
p21(Waf1/Cip1) promoters
are newly defined downstream targets of the
indole-signaling pathway,
and that the observed transcriptional effects are due
to a combination of the
cellular activities of I3C and DIM.
PMID: 12840223
Food Chem Toxicol. 2003
Jun;41(6):745-52.
Indole-3-carbinol and
3,3'-Diindolylmethane (DIM) induce apoptosis in human prostate
cancer cells.
Nachshon-Kedmi M, Yannai
S, Haj A, Fares FA.
Faculty of Food
Engineering and Biotechnology, Technion- Israel Institute of
Technology, 32000, Haifa,
Israel.
Cruciferous vegetables
contain glucobrassicin which, during metabolism, yields
indole-3-carbinol (I3C).
In a low pH environment I3C is converted into polymeric
products, among which
3,3'-diindolylmethane (DIM) is the main one. The apoptotic
effects of I3C and DIM
were exhibited in human breast cancer cells. The
objectives of this study
were: (a) examination of the potential effects of I3C
and DIM on the
proliferation and induction of apoptosis in human prostate cancer
cell lines with different
p53 status; (b) to try to characterise the mechanism(s)
involved in these effects.
Our results indicate that both indole derivatives
suppress the growth of
these cells in a dose- and time-dependent manner, by
inducing apoptosis. It
appears that these indolic compounds may offer effective
means against prostate
cancer. Induction of apoptosis was p53-independent.
Moreover, the indole
derivatives employed did not affect the levels of bcl-2, bax
and fasL.
PMID: 12738179
J Nutr. 2003
Apr;133(4):1011-9.
Gene expression profiles
of I3C- and DIM-treated PC3 human prostate cancer cells
determined by cDNA
microarray analysis.
Li Y, Li X, Sarkar FH.
Department of Pathology,
Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI
48201, USA.
Studies from our
laboratory and others have shown that indole-3-carbinol (I3C)
and its in vivo dimeric
product, 3,3'-diindolylmethane (DIM), inhibit the growth
of PC3 prostate cancer
cells and induce apoptosis by inhibiting nuclear factor
(NF)-kappaB and Akt
pathways. To obtain comprehensive gene expression profiles
altered by I3C- and
DIM-treated PC3 cells, we utilized cDNA microarray to
interrogate the expression
of 22,215 known genes using the Affymetrix Human
Genome U133A Array. We
found a total of 738 genes that showed a greater than
twofold change after 24 h
of DIM treatment. Among these genes, 677 genes were
down-regulated and 61 were
up-regulated. Similarly, 727 genes showed a greater
than twofold change in
expression, with down-regulation of 685 genes and
up-regulation of 42 genes
in I3C-treated cells. The altered expressions of genes
were observed as early as
6 h and were more evident with longer treatment. Upon
cluster analysis, we found
that both I3C and DIM up-regulated the expression of
genes that are related to
the Phase I and Phase II enzymes, suggesting their
increased capacity for
detoxification of carcinogens or chemicals. We also found
that I3C and DIM
down-regulated the expression of genes that are critically
involved in the regulation
of cell growth, cell cycle, apoptosis, signal
transduction, Pol II
transcription factor and oncogenesis. Real-time reverse
transcription-polymerase
chain reaction analysis was conducted to confirm the
cDNA microarray data, and
the results were consistent. We conclude that I3C and
DIM affected the
expression of a large number of genes that are related to the
control of carcinogenesis,
cell survival and physiologic behaviors. This may help
determine the molecular
mechanism(s) by which I3C and DIM exert their pleiotropic
effects on PC3 prostate
cancer cells; in addition, this information could be
further exploited for
devising chemopreventive and/or therapeutic strategies for
prostate cancer.
PMID: 12672912
J Biol Chem. 2003 Jun
6;278(23):21136-45. Epub 2003 Mar 27.
Plant-derived
3,3'-Diindolylmethane (DIM) is a strong androgen antagonist in human
prostate cancer cells.
Le HT, Schaldach CM,
Firestone GL, Bjeldanes LF.
Department of Nutritional
Sciences and Toxicology, The University of California,
Berkeley, California
94720-3104, USA.
3,3'-Diindolylmethane
(DIM) is a major digestive product of indole-3-carbinol, a
potential anticancer
component of cruciferous vegetables. Our results indicate
that DIM exhibits potent
antiproliferative and antiandrogenic properties in
androgen-dependent human
prostate cancer cells. DIM suppresses cell proliferation
of LNCaP cells and
inhibits dihydrotestosterone (DHT) stimulation of DNA
synthesis. These
activities were not produced in androgen-independent PC-3 cells.
Moreover, DIM inhibited
endogenous PSA transcription and reduced intracellular
and secreted PSA protein
levels induced by DHT in LNCaP cells. Also, DIM
inhibited, in a
concentration-dependent manner, the DHT-induced expression of a
prostate-specific antigen
promoter-regulated reporter gene construct in
transiently transfected
LNCaP cells. Similar effects of DIM were observed in PC-3
cells only when these
cells were co-transfected with a wild-type androgen
receptor expression
plasmid. Using fluorescence imaging with green fluorescent
protein androgen receptor
and Western blot analysis, we demonstrated that DIM
inhibited androgen-induced
androgen receptor (AR) translocation into the nucleus.
Results of receptor
binding assays indicated further that DIM is a strong
competitive inhibitor of
DHT binding to the AR. Results of structural modeling
studies showed that DIM is
remarkably similar in conformational geometry and
surface charge
distribution to an established synthetic AR antagonist, although
the atomic compositions of
the two substances are quite different. Taken together
with our published reports
of the estrogen agonist activities of DIM, the present
results establish DIM as a
unique bifunctional hormone disrupter. To our
knowledge, DIM is the
first example of a pure androgen receptor antagonist from
plants.
PMID: 12665522
Carcinogenesis. 2002
Aug;23(8):1297-305.
3,3'-Diindolylmethane
(DIM) induces a G(1) cell cycle arrest in human breast
cancer cells that is
accompanied by Sp1-mediated activation of p21(WAF1/CIP1)
expression.
Hong C, Kim HA, Firestone
GL, Bjeldanes LF.
Department of Nutritional
Sciences and Toxicology, University of California,
Berkeley, CA 94720, USA.
3,3'-Diindolylmethane
(DIM) is a promising cancer chemopreventive agent derived
from Brassica food plants.
To determine whether this natural indole has a direct
growth inhibitory effect
on human breast cancer cells, we examined the cell cycle
regulatory effects of DIM
in estrogen-dependent (MCF-7) and estrogen-independent
(MDA-MB-231) human breast
cancer cell lines. Results of flow cytometry studies
showed that DIM treatment
produced a marked increase (from 51 to 79%) in the
proportion of cells in the
G(1) phase of the cell cycle, regardless of
estrogen-receptor status.
Analyses of G(1)-acting cell cycle components indicated
that the enzymatic
activity of cyclin-dependent kinase (CDK) 2 was also strongly
reduced. Western blot
analyses showed that, concurrent with the DIM-induced cell
cycle arrest, DIM
stimulated a rapid and pronounced increase in levels of the CDK
inhibitor, p21(WAF1/CIP1)
(p21). Northern blot analysis demonstrated that DIM
increased p21 mRNA
expression with a maximal 6-7-fold induction, and exposure to
cycloheximide did not
block the response. Similar increases in expression of p21
protein and mRNA were
observed in both MCF-7 and MDA-MB-231 human
breast cancer cells,
suggesting that DIM induction of p21 expression is independent of
estrogen-receptor
signaling and p53. Transient transfection of 5'-deletion
constructs of the p21
promoter demonstrated that the first 291 bp segment of the
proximal promoter, which
contains six promoter specific transcription factor 1
(Sp1) elements, maintained
DIM responsiveness. Consistent with a role for Sp1 in
this response, a reporter
construct driven by three consensus Sp1 binding sites
was responsive to DIM. In
addition, electrophoretic mobility shift assays showed
that DIM induced the
binding of Sp1 and Sp3 to the consensus Sp1 responsive
element. Thus, our
observations have uncovered an antiproliferative pathway for
DIM that implicates
Sp1/Sp3-induced expression of p21 as a target for cell cycle
control in human breast
cancer cells.
PMID: 12151347
Nutr Cancer.
2001;41(1-2):57-63.
Quantitative determination
of 3,3'-Diindolylmethane (DIM) in urine of individuals
receiving
indole-3-carbinol.
Sepkovic DW, Bradlow HL,
Bell M.
Institute for Biomedical
Research, Hackensack University Medical Center,
Hackensack, NJ 07601, USA.
Indole-3-carbinol (I3C)
or, more correctly, its acid condensation products act as
chemoprotective agents via
several mechanisms. It induces the expression of
cytochrome P-450 1A1,
which shifts the estrogen metabolic pathway in favor of C-2
hydroxylation and away
from the formation of 16 alpha-hydroxyestrone, a suspected
endogenous carcinogen.
Increased 16 alpha-hydroxylation of estrogen is associated
with greater risk of
cancer of the cervix, breast, endometrium, and larynx. The
production of
4-hydroxyestrone is also inhibited by I3C. I3C can induce a G1 cell
cycle arrest in human
MCF-7 breast cancer cells. It can suppress aberrant crypt
foci. I3C significantly
inhibits the cell adhesion, spreading, and invasion
associated with an
upregulation of PTEN (a tumor suppressor gene) and E-cadherin
(a regulator of cell-cell
adhesion) expression in T47-D human breast cancer
cells. Thus I3C exhibits
anticancer activities by suppressing breast tumor cell
growth and metastatic
spread. A gas chromatography-mass spectrometry method for
the quantitation of
Diindolylmethane (DIM), the principal acid condensation product of
I3C, has been developed
for use in determining compliance in subjects who have
been treated with I3C. The
method utilizes a 1-ml urine sample. We have used this
method to correlate I3C
ingestion with regression of cervical intraepithelial
neoplasia in a population
of women at risk for cervical cancer. The assay
provides an objective
marker of consumption using a noninvasive biological fluid
and illustrates that
Diindolylmethane (DIM) may be used as a marker of compliance in
I3C dietary intervention
studies.
PMID: 12094629
Antivir Ther. 2002
Mar;7(1):1-9.
Therapy for recurrent
respiratory papillomatosis.
Auborn KJ.
Department of
Otolaryngology, Long Island Jewish Medical Center, Albert Einstein
College of Medicine, New
Hyde Park, NY, USA.
Human papillomaviruses
types 6 or 11 are aetiological agents of recurrent
respiratory papillomatosis,
a disease characterized by benign exophytic tumours
usually on the vocal
cords. Surgery debulks the tumours, but these growths
generally recur at regular
intervals. Adjunct medical treatments, aimed at
containing the virus and
growth of tumours, include indole-3-carbinol or its
dimer Diindolylmethane (DIM),
interferon, photodynamic therapy and others. Preventive
and therapeutic vaccines
hold promise for eliminating the virus.
PMID: 12008782
Altern Med Rev. 2002
Apr;7(2):112-29.
Estrogen metabolism and
the diet-cancer connection: rationale for assessing the
ratio of urinary
hydroxylated estrogen metabolites.
Lord RS, Bongiovanni B,
Bralley JA.
MetaMetrix Clinical
Laboratory, 4855 Peachtree Industrial Boulevard, Suite 201,
Norcross, GA, 30092, USA.
Estrogens are known for
their proliferative effects on estrogen-sensitive tissues
resulting in tumorigenesis.
Results of experiments in multiple laboratories over
the last 20 years have
shown that a large part of the cancer-inducing effect of
estrogen involves the
formation of agonistic metabolites of estrogen, especially
16-alpha-hydroxyestrone.
Other metabolites, such as 2-hydroxyestrone and
2-hydroxyestradiol, offer
protection against the estrogen-agonist effects of
16-alpha-hydroxyestrone.
An ELISA method for measuring 2- and
16-alpha-hydroxylated
estrogen (OHE) metabolites in urine is available and the
ratio of urinary
2-OHE/16-alpha-OHE (2/16-alpha ratio) is a useful biomarker for
estrogen-related cancer
risk. The CYP1A1 enzyme that catalyzes 2-hydroxyestrone
(2-OHE1) formation is
inducible by dietary modification and supplementation with
the active components of
cruciferous vegetables, indole-3-carbinol (I-3-C), or
diindolylmethane (DIM).
Other dietary components, especially omega-3
polyunsaturated fatty
acids and lignans in foods like flax seed, also exert
favorable effects on
estrogen metabolism. Thus, there appear to be effective
dietary means for reducing
cancer risk by improving estrogen metabolism. This
review presents the
accumulated evidence to help clinicians evaluate the merit of
using tests that measure
estrogen metabolites and using interventions to modify
estrogen metabolism.
PMID: 11991791
Biochem Pharmacol. 2002
Mar 15;63(6):1085-97.
Bcl-2 family-mediated
apoptotic effects of 3,3'-diindolylmethane (DIM) in human
breast cancer cells.
Hong C, Firestone GL,
Bjeldanes LF.
Department of Nutritional
Sciences and Toxicology, University of California,
Berkeley 94720-3200, USA.
3,3'-Diindolylmethane
(DIM) is a major in vivo derivative of the putative
anticancer agent
indole-3-carbinol (I3C), which is present in vegetables of the
Brassica genus. At
concentrations above 10 microM, DIM inhibited DNA synthesis
and cell proliferation in
both estrogen receptor replete (MCF-7) and deficient
(MDA-MB-231) human breast
cancer cells in a concentration- and time-dependent
manner. These
antiproliferative effects were accompanied by characteristic
indications of programmed
cell death in both cell lines, including
externalization of
phosphatidylserine, chromatin condensation, and DNA
fragmentation.
Furthermore, Western and Northern blot analyses, as well as
coimmunoprecipitation
assays, revealed that in both MCF-7 and MDA-MB-231 cells,
DIM treatment decreased
total transcript and protein levels of the apoptosis
inhibitory protein Bcl-2,
and the amount of Bcl-2 bound to the pro-apoptotic
protein Bax. DIM treatment
also caused an increase in Bax protein levels, but did
not affect the level of
Bax that was bound to Bcl-2. As a functional test of the
role of Bcl-2
down-regulation in the DIM-induced apoptotic response, ectopic
expression of Bcl-2 in
MCF-7 cells was shown to attenuate the apoptotic effect of
DIM. These results
demonstrate that DIM can induce apoptosis in breast cancer
cells independent of
estrogen receptor status by a process that is mediated by
the modulated expression
of the Bax/Bcl-2 family of apoptotic regulatory factors.
PMID: 11931841
Chem Res Toxicol. 2002
Feb;15(2):101-9.
Fate of indole-3-carbinol
in cultured human breast tumor cells.
Staub RE, Feng C, Onisko
B, Bailey GS, Firestone GL, Bjeldanes LF.
Department of Nutritional
Sciences and Toxicology, University of California,
Berkeley, California
94720, USA.
Indole-3-carbinol (I3C), a
natural component of Brassica vegetables, is a
promising cancer
preventive agent that can reduce the incidence of tumors in
reproductive organs when
administered in the diet. Here we report on the
metabolic fate of
radiolabeled I3C in MCF-7 cells. I3C was surprisingly inert to
metabolism by these cells
with a half-life in medium of approximately 40 h.
[(3)H]I3C levels in media
declined at a similar rate whether incubation was with
cultured cells or in
cell-free medium. Neither [(3)H]I3C nor its modified
products accumulated in
MCF-7 cells and only low levels of intact I3C were
detected in cellular
fractions. In contrast, I3C represented over 30% of the
radioactivity in media
even after 72 h. In cytosolic fractions, the
3-(cystein-S-ylmethyl) and
3-(glutathion-S-ylmethyl) conjugates of [(3)H]I3C were
the primary conversion
products identified after 16 h, representing approximately
50% and approximately 15%
of the radioactivity in these fractions, respectively.
The reaction of I3C with
thiols appears to be nonenzymatic since the cysteine
conjugate is produced when
I3C is incubated in cell-free medium containing
additional cysteine. Both
cellular and extracellular proteins were
nonspecifically modified
with [(3)H]I3C. In medium, proteins are radiolabeled
even in the absence of
cells, indicating again that enzymatic activation was not
required. I3C was also
oxidized to indole-3-carboxaldehyde and
indole-3-carboxylic acid
in culture medium independent of cells. Unexpectedly,
3,3'-diindolylmethane
(DIM), an I3C product with in vitro and in vivo biological
activity, was detected in
cellular fractions and appeared to accumulate in the
nucleus, representing
approximately 40% of this fraction after 72 h treatment.
These findings suggest
that MCF-7 cells do not vigorously metabolize I3C and that
the major route of
reaction is with cellular thiols such as glutathione and
proteins. The accumulation
of DIM in the nucleus suggests that this product may
have a role in the
cellular biological activities of I3C.3
PMID: 11849035
Carcinogenesis. 2001
Nov;22(11):1809-17.
Cytostatic effects of
3,3'-Diindolylmethane (DIM) in human endometrial cancer cells
result from an estrogen
receptor-mediated increase in transforming growth
factor-alpha expression.
Leong H, Firestone GL,
Bjeldanes LF.
Department of Nutritional
Sciences and Toxicology, University of
California-Berkeley,
Berkeley, CA 94720, USA.
3,3'-Diindolylmethane
(DIM), a major in vivo product of indole-3-carbinol (I3C),
is a promising anticancer
agent derived from vegetables of the Brassica genus
including broccoli,
Brussels sprouts and cabbage. We report here that DIM has a
potent cytostatic effect
in cultured human Ishikawa endometrial cancer cells. A
combination of northern
blot and quantitative PCR analyses revealed that DIM
induced the level of TGF-alpha
transcripts by approximately 4-fold within 24 h of
indole treatment. DIM also
induced a 4-fold increase in the activity of the
estrogen response marker,
alkaline phosphatase (AP). Co-treatment of cells with
the estrogen receptor (ER)
antagonist ICI, or with the inhibitor of PKA-mediated
activation of the ER, H89,
ablated the DIM induction of both TGF-alpha expression
and AP activity.
Furthermore, DIM increased the maximum stimulatory effect of
estrogen on TGF-alpha
expression. Co-treatment with the protein synthesis
inhibitor, cycloheximide,
abolished the inductive effects of DIM, indicating
differences in the
mechanistic requirements of DIM and estrogen. DIM treatment
also stimulated levels of
secreted TGF-alpha protein by >10-fold. The ectopic
addition of TGF-alpha
inhibited the growth of Ishikawa cells, whereas incubation
with a TGF-alpha antibody
partially reversed the growth inhibitory effects of
DIM. Taken together, these
results extend our previous findings of the ligand
independent estrogen
receptor agonist activity of DIM, and uncover an essential
role for the stimulation
in TGF-alpha expression and the TGF-alpha activated
signal transduction
pathway in the potent cytostatic effects of DIM in
endometrial cancer cells.
PMID: 11698343
Cancer Res. 2001 Aug
15;61(16):6120-30.
Dietary indoles and
isothiocyanates that are generated from cruciferous
vegetables can both
stimulate apoptosis and confer protection against DNA damage
in human colon cell lines.
Bonnesen C, Eggleston IM,
Hayes JD.
Biomedical Research
Centre, Ninewells Hospital and Medical School, University of
Dundee, Dundee DD1 9SY,
Scotland, United Kingdom.
The natural indoles
3,3'-diindolylmethane (DIM), ascorbigen (ASG),
indole-3-carbinol (I3C),
and indolo[3,2-b]carbazole (ICZ), as well as the natural
isothiocyanates
sulforaphane (SUL), benzyl isothiocyanate (BITC) and phenethyl
isothiocyanate (PEITC),
all possess cancer chemopreventive properties. It is now
shown that DIM, ICZ, SUL,
and BITC can each stimulate apoptosis in human colon
adenocarcinoma LS-174 and
Caco-2 cells. Treatment of LS-174 cells with nontoxic
doses of DIM, ASG, I3C, or
ICZ affected an increase of up to 21-fold in
cytochrome P450 1A1
(CYP1A1). None of these indoles caused an elevation in either
aldo-keto reductase 1C1
(AKR1C1) or the gamma-glutamylcysteine synthetase heavy
subunit (GCS(h)), but DIM,
I3C, and ICZ produced a very modest increase in
NAD(P)H:quinone
oxidoreductase 1 (NQO1). By contrast, nontoxic doses of SUL,
BITC, or PEITC failed to
induce expression of CYP1A1 in LS-174 cells, but caused
an increase of between 11-
and 17-fold in the protein levels of AKR1C1, NQO1, and
GCS(h). Treatment of the
colon cell line with ICZ or SUL caused increases in the
levels of mRNA for CYP1A1,
AKR1C1, and NQO1 that were consistent with the enzyme
data. Exposure of Caco-2
cells to media containing indoles or isothiocyanates
gave similar results to
those obtained using LS-174 cells. Evidence is presented
that the ability of
indoles and isothiocyanates to stimulate either xenobiotic
response element- or
antioxidant response element-driven gene expression accounts
for the two groups of
phytochemicals inducing different gene batteries.
Pretreatment of LS-174
cells for 24 h with ICZ and SUL before exposure for 24 h
to benzo(a)pyrene (BaP)
reduced to <20% the number of single-strand DNA breaks
produced by the
carcinogen. Neither ICZ alone nor SUL alone were able to confer
the same degree of
protection against DNA damage produced by BaP as they achieved
in combination. Similar
results were obtained with H(2)O(2) as the genotoxic
agent. Together, these
phytochemicals may prevent colon tumorigenesis by both
stimulating apoptosis and
enhancing intracellular defenses against genotoxic
agents.
PMID: 11507062
Breast Cancer Res Treat.
2001 Mar;66(2):147-57.
Methyl-substituted
diindolylmethanes as inhibitors of estrogen-induced growth of
T47D cells and mammary
tumors in rats.
McDougal A, Gupta MS,
Morrow D, Ramamoorthy K, Lee JE, Safe SH.
Department of Veterinary
Physiology & Pharmacology, Texas A &M University,
College Station
77843-4466, USA.
Diindolylmethane (DIM) is
formed by acid catalyzed dimerization of the
phytochemical
indole-3-carbinol, and both compounds inhibit formation and/or
growth of mammary tumors
in rodents. In this study, we have investigated the aryl
hydrocarbon receptor (AhR)
agonist activity and inhibitory AhR-estrogen receptor
crosstalk induced by the
following methyl-substituted DIMs: 1,1'-dimethyl-,
2,2'-dimethyl-, 5,5'-dimethyl-,
6,6'-dimethyl-, and 7,7'-dimethylDIM and
1,1',2,2'-tetramethylDIM.
The six compounds bound to the rat cytosolic AhR in a
transformation assay but,
at concentrations < or = 10 microM, exhibited minimal
to non-detectable AhR
agonist or antagonist activities associated with CYP1A1
induction. In contrast,
the methyl-substituted DIMs inhibited estrogen-induced
T47D human breast cancer
cell growth and the four most active compounds (1,1'-,
2,2'-, 5,5'-dimethylDIM
and 1,1',2,2'-tetramethylDIM) inhibited one or more
estrogen-induced responses
in the 21-day-old female B6C3F1 mice at a dose of 100
mg/kg/day (X3). Induction
of hepatic CYP1A1-dependent activity was not observed
at this high dose. The
antitumorigenic activity of these compounds was examined
in
7,12-dimethylbenz[a]anthracene-induced rat mammary tumor model in which the
DIM analogs were orally
administered (by gavage in corn oil) at a dose of 1
mg/kg/day (X10).
1,1'-DimethylDIM, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM
significantly inhibited
mammary tumor growth, and this was not accompanied by
changes in organ/body
weights or histopathology. These studies demonstrate that
methyl-substituted DIMs
are selective AhR modulators (SAhRMs) with potential for
clinical treatment of
breast cancer.
PMID: 11437101
Toxicol Sci. 2001
May;61(1):40-8.
2,3,7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce
cytochrome P450 1A1, 1B1,
and 19 in H295R human adrenocortical carcinoma cells.
Sanderson JT, Slobbe L,
Lansbergen GW, Safe S, van den Berg M.
Research Institute for
Toxicology, Utrecht University, P.O. Box 80176, 3508 TD
Utrecht, The Netherlands.
Diindolylmethane (DIM) is
an acid-catalyzed condensation product of
indole-3-carbinol, a
constituent of cruciferous vegetables, and is formed in the
stomach. DIM alters
estrogen metabolism and inhibits carcinogen-induced mammary
tumor growth in rodents.
DIM is a weak agonist for the aryl hydrocarbon (Ah)
receptor and blocks the
effects of estrogens via inhibitory Ah receptor-estrogen
receptor cross-talk. DIM
and various structural analogs were examined in H295R
cells for effects on 3
cytochrome P450 (CYP) enzymes involved in estrogen
synthesis and/or
metabolism: CYP1A1, CYP1B1, and CYP19 (aromatase). Aromatase
activity was measured by
conversion of 1 beta-(3)H-androstenedione to estrone and
(3)H(2)O. H295R cells were
exposed to the test chemicals dissolved in dimethyl
sulfoxide for 24 h prior
to analyses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
(0--30 nM) and DIM (0--10
microM) induced ethoxyresorufin-O-deethylase (EROD)
activity, as a measure of
CYP1A1 and possibly 1B1 activity, with EC(50) values of
about 0.3 nM and 3 microM,
respectively. DIM, but not TCDD, induced aromatase
activity with an
apparently maximal 2-fold increase at 10 microM; higher
concentrations of DIM and
many of its analogs were cytotoxic. TCDD (30 nM)
significantly increased
CYP1A1 and 1B1 mRNA levels, but had no effect on mRNA for
CYP19. DIM (3 microM)
significantly increased mRNA levels for all three CYPS: DIM
analogs with substitutions
on the 5 and 5' position (3 microM) induced aromatase
and EROD activity,
together with mRNA levels of CYP1A1, 1B1, and 19; analogs that
were substituted on the
central carbon of the methane group showed little or no
inductive activity toward
the CYPS: In conclusion, DIM and several of its analogs
appear to induce CYPs via
multiple yet distinct pathways in H295R human
adrenocortical carcinoma
cells.
PMID: 11294972
Gene. 2001 Jan
10;262(1-2):207-14.
Identification of
estrogen-induced genes downregulated by AhR agonists in MCF-7
breast cancer cells using
suppression subtractive hybridization.
Chen I, Hsieh T, Thomas T,
Safe S.
Department of Veterinary
Physiology and Pharmacology, A&M University, College
Station, TX 77843, USA.
Aryl hydrocarbon receptor
(AhR) agonists inhibit 17beta-estradiol (E2) induced
growth of MCF-7 human
breast cancer cells in vitro and rodent mammary tumor
growth in vivo. Genes
associated with inhibitory AhR-estrogen receptor (ER)
crosstalk were
investigated in MCF-7 human breast cancer cells using
poly(A)(+)RNA from cells
treated with either 1 nM E2 (target) or E2 plus 1 nM
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (reference) or 25 microM
diindolylmethane (DIM) as
AhR agonists in MCF-7 cells. Suppression subtractive
hybridization (SSH) was
subsequently used to identify 33 genes with sequence
homology to known human
genes that are induced by E2 and inhibited by AhR
agonists in MCF-7 cells;
two unknown genes were also identified. Many of these
genes are involved in cell
proliferation and these include cell cycle regulators
(cdc28/cdc2-associated
protein), nucleotide synthases (thymidylate synthase),
early intermediate genes
(early growth response alpha, EGRalpha) and other
proteins involved in
signaling pathways (calmodulin, ATP synthase alpha subunit).
Thus SSH has identified a
diverse spectrum of new genes that are affected by
inhibitory AhR-ER
crosstalk and among this group are a subset of genes that may
be critical for the in
vivo antitumorigenic effects of AhR agonists.
PMID: 11179685
Drug Metab Dispos. 2000
Aug;28(8):930-6.
Concurrent flavin-containing
monooxygenase down-regulation and cytochrome P-450
induction by dietary
indoles in rat: implications for drug-drug interaction.
Katchamart S, Stresser DM,
Dehal SS, Kupfer D, Williams DE.
Department of
Environmental and Molecular Toxicology, Oregon State University,
Corvallis, Oregon, USA.
Our laboratory has
previously shown that dietary administration of
indole-3-carbinol (I3C) to
male Fischer 344 rats has the very unusual property of
inducing hepatic levels of
a number of cytochrome P450s (CYPs), especially
CYP1A1, while markedly
inhibiting the levels of flavin-containing monooxygenase
(FMO) 1 protein and its
catalytic activity. We hypothesized that rats fed I3C or
3,3'-diindolylmethane
(DIM), one of its major acid condensation products formed
in vivo, should exhibit a
marked shift in the metabolic profiles of drugs or
xenobiotics that are
substrates for both monooxygenase systems. Male rats were
fed AIN-76A powdered diets
containing 0, 1000, or 2500 ppm I3C or DIM for 4
weeks. Dietary I3C and DIM
reduced FMO1 protein levels (8% reduction with I3C and
84% with DIM at 1000 ppm,
and 90% reduction with I3C and 97% with DIM at 2500
ppm) in hepatic microsomes.
The ratio of FMO (N-oxygenation)- to CYP
(N-demethylation)-mediated
metabolism of N,N-dimethylaniline decreased in liver
microsomes from I3C- or
DIM-fed rats from near unity to 0.02 at the highest
dietary doses. FMO-mediated
N-oxygenation (nicotine N-1'-oxide) was decreased,
whereas CYP-mediated (nornicotine
and nicotine delta (1,5)-iminium ion)
metabolism of nicotine was
unchanged in liver microsomes from rats fed I3C or
DIM. Similarly, the ratio
of FMO to CYP metabolites of tamoxifen decreased due to
a reduction in
N-oxygenation. This study demonstrates alteration of FMO- and
CYP-mediated drug
metabolism in vitro by dietary I3C or DIM and suggests the
potential for altered
toxicity of tamoxifen and nicotine in vivo.
PMID: 10901703
Biochem Pharmacol. 2000
Jul 15;60(2):167-77.
Ligand-independent
activation of estrogen receptor function by 3,
3'-diindolylmethane in
human breast cancer cells.
Riby JE, Chang GH,
Firestone GL, Bjeldanes LF.
Division of Nutritional
Sciences and Toxicology, University of California,
Berkeley, CA 94720, USA.
3,3'-Diindolylmethane
(DIM), a major in vivo product of acid-catalyzed
oligomerization of
indole-3-carbinol (I3C), is a promising anticancer agent
present in vegetables of
the Brassica genus. We investigated the effects of DIM
on estrogen-regulated
events in human breast cancer cells and found that DIM was
a promoter-specific
activator of estrogen receptor (ER) function in the absence
of 17beta-estradiol
(E(2)). DIM weakly inhibited the E(2)-induced proliferation
of ER-containing MCF-7
cells and induced proliferation of these cells in the
absence of steroid, by
approximately 60% of the E(2) response. DIM had little
effect on proliferation of
ER-deficient MDA-MB-231 cells, suggesting that it is
not generally toxic at
these concentrations. Although DIM did not bind to the ER
in this concentration
range, as shown by a competitive ER binding assay, it
activated the ER to a
DNA-binding species. DIM increased the level of transcripts
for the endogenous pS2
gene and activated the estrogen-responsive pERE-vit-CAT
and pS2-tk-CAT reporter
plasmids in transiently transfected MCF-7 cells. In
contrast, DIM failed to
activate transcription of the simple E(2)- and
diethylstilbesterol-responsive
reporter construct pATC2. The estrogen antagonist
ICI 182780
(7alpha-[9-[(4,4,5,5,
5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3, 17beta-diol) was
effective against
DIM-induced transcriptional activity of the pERE-vit-CAT
reporter, which further
supports the hypothesis that DIM is acting through the
ER. We demonstrated that
ligand-independent activation of the ER in MCF-7 cells
could be produced
following treatment with the D1 dopamine receptor agonist
SKF-82958
[(+/-)6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,
5-tetrahydro-1H-3-benzazepinehydrobromide]. We also demonstrated that the
agonist
effects of SKF-82958 and
DIM, but not of E(2), could be blocked by co-treatment
with the protein kinase A
(PKA) inhibitor H-89
(N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide). These results
have
uncovered a
promoter-specific, ligand-independent activation of ER signaling for
DIM that may require
activation by PKA, and suggest that this major I3C product
may be a selective
activator of ER function.
PMID: 10825461
Cancer Lett. 2000 Apr
14;151(2):169-79.
Inhibition of
carcinogen-induced rat mammary tumor growth and other
estrogen-dependent
responses by symmetrical dihalo-substituted analogs of
Diindolylmethane (DIM).
McDougal A, Sethi Gupta M,
Ramamoorthy K, Sun G, Safe SH.
Department of Veterinary
Physiology and Pharmacology, Texas A&M University,
College Station, TX
77843-4466, USA.
90%) by the haloDIMs at
concentrations of 5 or 10 microM, and only 4,
4'-dichloroDIM alone
increased cell proliferation. With the exception of
5,5'-difluoroDIM, the
remaining compounds also inhibited E2-induced growth of
MCF-7 human breast cancer
cells. DihaloDIMs (100 mg/kg/dayx3) were not estrogenic
in the immature female
B6C3F1 mouse uterus; however, in animals co-treated with
E2 (0.02 microg/mouse),
5,5'-dichloro- and 6,6'-dichloroDIM inhibited uterine
progesterone receptor (PR)
binding and uterine peroxidase activity, whereas
5,5'-dichloro- and
5,5'-dichloro-2,2'-dimethylDIM inhibited only the latter
response. The
antitumorigenic activities of the dihaloDIMs were determined by
their inhibition of
carcinogen-induced mammary tumor growth in female
Sprague-Dawley rats. 4,4'-Dichloro-,
5,5'-dibromo- and 6,6'-dichloroDIM,
significantly inhibited
mammary tumor growth at doses of 1 mg/kg every second
day, and no significant
changes in organ weights or liver and kidney
histopathology were
observed. These three compounds were more active than DIM in
the same in vivo assay.
PMID: 10738111
Toxicol Sci. 1999
Dec;52(2):178-88.
The anti-estrogenicity of
Ah receptor agonists in carp (Cyprinus carpio)
hepatocytes.
Smeets JM, van Holsteijn
I, Giesy JP, van den Berg M.
Research Institute of
Toxicology, Utrecht University, The Netherlands.
Cultured hepatocytes of
female carp (Cyprinus carpio) were coexposed for 4 days
to 200 nM 17beta-estradiol
(E2), and concentration ranges of nine known Ah
receptor (AhR) agonists:
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
3,3'4,4'5-pentachlorobiphenyl (PCB 126), 2,3'4,4'5-pentachlorobiphenyl (PCB
118),
beta-naphthoflavone (BNF),
benzo(a)pyrene (BaP), benzo(a)anthracene (BaA),
diindolylmethane (DIM),
6-methyl-1,3,8-trichlorodibenzofuran (MCDF) and
hexachlorobenzene (HCB).
TCDD caused a greater than 100-fold induction of
cytochrome P4501A (CYP1A)
activity, measured as ethoxyresorufin O-deethylase
(EROD), with an EC50 of 6
pM. Based on EC50 values, the order of potency as CYP1A
inducers was TCDD > PCB
126 > BNF > BaP > BaA > PCB 118. DIM and MCDF
caused a lower maximum CYP1A
induction (< 9-fold), whereas HCB caused no EROD
induction at concentrations up
to 6 microM. TCDD, PCB 126, BNF, BaP, and DIM also
caused a
concentration-dependent suppression of the secretion of the yolk protein
vitellogenin (Vtg),
relative to E2-treated hepatocytes. Suppression of Vtg
secretion was not directly
correlated with EROD activity, and the antiestrogenic
effects occurred at higher
concentrations than the induction of CYP1A. This
indicates that the anti-estrogenicity
was not caused by increased metabolism of
E2 due to induction of
CYP1A. Nevertheless, the order of potency of the tested
compounds for suppression
of Vtg secretion was comparable to the order of potency
for CYP1A induction. This
concurrence suggests that the anti-estrogenicity of
these compounds is AhR-mediated,
but does not involve CYP1A. This could be
relevant for feral fish
populations, as they are frequently exposed to AhR
agonists, to an extent
that AhR-mediated effects are observed.
PMID: 10630570
Toxicol Sci. 1999
Nov;52(1):1-8.
Symposium on mechanisms of
action of naturally occurring anticarcinogens.
Safe S, Wargovich MJ,
Lamartiniere CA, Mukhtar H.
Department of Veterinary
Physiology and Pharmacology, Texas A&M University,
College Station
77843-4466, USA.
PMID: 10568692
Food Chem Toxicol. 1999
Jun;37(6):609-18.
Effect of some indole
derivatives on xenobiotic metabolism and xenobiotic-induced
toxicity in cultured rat
liver slices.
Renwick AB, Mistry H,
Barton PT, Mallet F, Price RJ, Beamand JA, Lake BG.
BIBRA International,
Carshalton, Surrey, UK.
In this study the effect
of some indole derivatives on xenobiotic metabolizing
enzymes and xenobiotic-induced
toxicity has been examined in cultured
precision-cut liver slices
from male Sprague-Dawley rats. While treatment of rat
liver slices for 72 hours
with 2-200 microM of either indole-3-carbinol (I3C) or
indole-3-acetonitrile
(3-ICN) had little effect on cytochrome P-450
(CYP)-dependent enzyme
activities, enzyme induction was observed after in vivo
administration of I3C. The
treatment of rat liver slices with 50 microM
3,3'-diindolylmethane
(DIM; a dimer derived from I3C under acidic conditions) for
72 hours resulted in a
marked induction of CYP-dependent enzyme activities. DIM
appears to be a mixed
inducer of CYP in rat liver slices having effects on CYP1A,
CYP2B and CYP3A subfamily
isoforms. Small increases in liver slice reduced
glutathione levels and
glutathione S-transferase activity were also observed
after DIM treatment. While
aflatoxin B1 and monocrotaline produced a
concentration-dependent
inhibition of protein synthesis in 72-hour-cultured rat
liver slices, cytotoxicity
was markedly reduced in liver slices cultured with 50
microM DIM. These results
demonstrate that cultured rat liver slices may be
employed to evaluate the
effects of chemicals derived from cruciferous and other
vegetables on CYP isoforms.
In addition, liver slices can also be utilized to
examine the ability of
such chemicals to modulate xenobiotic-induced toxicity.
PMID: 10478829
Biochem Pharmacol. 1999
Sep 1;58(5):825-34.
Cytostatic and
antiestrogenic effects of 2-(indol-3-ylmethyl)-3,3'-Diindolylmethane (DIM), a major in vivo product of dietary indole-3-carbinol.
Chang YC, Riby J, Chang GH,
Peng BC, Firestone G, Bjeldanes LF.
Division of Nutritional
Sciences and Toxicology, University of California,
Berkeley 94720, USA.
Under acidic conditions,
indole-3-carbinol (13C) is converted to a series of
oligomeric products
thought to be responsible for the biological effects of
dietary 13C.
Chromatographic separation of the crude acid mixture of 13C, guided
by cell proliferation
assay in human MCF-7 cells, resulted in the isolation of
2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr-1) as a major antiproliferative
component. LTr-1 inhibited
the growth of both estrogen-dependent (MCF-7) and
-independent (MDA-MB-231)
breast cancer cells by approximately 60% at a
non-lethal concentration
of 25 microM. LTr-1 had no apparent effect on the
proliferation of MCF-7
cells in the absence of estrogen. LTr-1 was a weak ligand
for the estrogen receptor
(ER) (IC50 70 microM) and efficiently inhibited the
estradiol (E2)-induced
binding of the ER to its cognate DNA responsive element.
The antagonist effects of
LTr-1 also were exhibited in assays of endogenous pS2
gene expression and in
cells transiently transfected with an estrogen-responsive
reporter construct (pERE-vit-CAT).
LTr-1 activated both binding of the aryl
hydrocarbon (Ah) receptor
to its cognate DNA responsive element and expression of
the Ah receptor-responsive
gene CYP1A1. LTr-1 was a competitive inhibitor of
CYP1A1-dependent
ethoxyresorufin-O-deethylase (EROD) activity. In summary, these
results demonstrated that
LTr-1, a major in vivo product of I3C, could inhibit
the proliferation of both
estrogen-dependent and -independent breast tumor cells
and that LTr-1 is an
antagonist of estrogen receptor function and a weak agonist
of Ah receptor function.
PMID: 10449193
Carcinogenesis. 1998
Sep;19(9):1631-9.
Aryl hydrocarbon
receptor-mediated antiestrogenic and antitumorigenic activity of
Diindolylmethane (DIM).
Chen I, McDougal A, Wang
F, Safe S.
Department of Veterinary
Physiology and Pharmacology, Texas A&M University,
College Station
77843-4466, USA.
Phytochemicals such as
indole-3-carbinol (I3C) and sulforaphane are components of
cruciferous vegetables
which exhibit antitumorigenic activity associated with
altered carcinogen
metabolism and detoxification. Diindolylmethane (DIM) is a
major acid-catalyzed
metabolite of I3C formed in the gut that binds to the aryl
hydrocarbon receptor (AhR)
and treatment of MCF-7 human breast cancer cells with
10-50 microM DIM resulted
in rapid formation of the nuclear AhR complex and
induction of CYP1A1 gene
expression was observed at concentrations >50 microM.
Previous studies have
demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD), a high affinity
AhR ligand, inhibits 17beta-estradiol (E2)-induced
responses in MCF-7 cells
and growth of E2-dependent 7,12-dimethylbenzanthracene
(DMBA)-induced mammary
tumors in female Sprague-Dawley rats. Results of this
study show that like TCDD,
DIM inhibits E2-induced proliferation of MCF-7 cells,
reporter gene activity in
cells transiently transfected with an E2-responsive
plasmid (containing a frog
vitellogenin A2 gene promoter insert) and
down-regulates the nuclear
estrogen receptor. Moreover, DIM (5 mg/kg every other
day) also inhibits DMBA-induced
mammary tumor growth in Sprague-Dawley rats and
this was not accompanied
by induction of hepatic CYP1A1-dependent activity. Thus,
DIM represents a new class
of relatively non-toxic AhR-based antiestrogens that
inhibit E2-dependent tumor
growth in rodents and current studies are focused on
development of analogs for
clinical treatment of breast cancer.
PMID: 9771935
Xenobiotica. 1998
Aug;28(8):803-11.
3,3'-Diindolylmethane (DIM)
induces CYP1A2 in cultured precision-cut human liver
slices.
Lake BG, Tredger JM,
Renwick AB, Barton PT, Price RJ.
BIBRA International,
Carshalton, UK.
1. The effect of
3,3'-diindolylmethane (DIM), an indole derivative derived from
cruciferous vegetables, on
cytochrome P450 (CYP) isoforms in the CYP1A and CYP3A
subfamilies has been
studied in 72-h cultured human liver slices. 2. In cultured
human liver slices 50
microM DIM induced 7-ethoxyresorufin O-deethylase and to a
lesser extent
7-methoxyresorufin O-demethylase activities. 3. Western
immunoblotting of liver
slice microsomes was performed with antibodies to rat
CYP1A2 and human CYP3A4.
Compared with control liver slice microsomes (dimethyl
sulphoxide-only treated),
DIM induced levels of CYP1A2 but had little effect on
levels of CYP3A4. The
treatment of human liver slices with 2 microg/ml of the
polycholorinated biphenyl
mixture Aroclor 1254 also resulted in an induction of
levels of CYP1A2, but had
no effect on CYP3A4. 4. These results demonstrate that
DIM induces CYP1A isoforms
in cultured human liver slices. Some variability in
the magnitude of induction
of enzyme activities by DIM was observed in four human
liver samples examined.
For 7-ethoxyresorufin O-deethylase, the magnitude of
induction by 50 microM DIM
ranged from 2.3- to 19.3-fold. 5. These results
demonstrate that cultured
human liver slices can be used to evaluate the effect
of chemicals derived from
cruciferous and other vegetables on CYP isoforms.
PMID: 9741959
Anticancer Drugs. 1998
Feb;9(2):141-8.
Selective cytostatic and
cytotoxic effects of glucosinolates hydrolysis products
on human colon cancer
cells in vitro.
Gamet-Payrastre L, Lumeau
S, Gasc N, Cassar G, Rollin P, Tulliez J.
INRA, Laboratoire des
Xιnobiotiques, Toulouse, France.
Glucosinolates hydrolysis
products are attracting increasing attention since many
studies have suggested
that they may be involved in the anticarcinogenic property
of cruciferous vegetables.
In this study, we show that diindolylmethane (DIM) and
sulforaphane, produced
during the hydrolysis of glucobrassicin and glucoraphanin,
respectively, exert a
dose-dependent cytotoxicity on human colon adenocarcinoma
HT29 cells. Moreover,
these products are able to inhibit quiescent cells to
re-enter the cell cycle.
Interestingly, our results clearly show that low doses
of DIM and sulforaphane,
although very effective on undifferentiated intestinal
HT29 cells, do not affect
the viability of the differentiated CaCo2 cells. The
reversibility of their
effects has also been tested and is discussed.
PMID: 9510500
Biochem Biophys Res Commun.
1996 Nov 1;228(1):153-8.
3,3'-Diindolylmethane (DIM)
induces apoptosis in human cancer cells.
Ge X, Yannai S, Rennert G,
Gruener N, Fares FA.
Department of Food
Engineering and Biotechnology, Technion-Israel Institute of
Technology, Haifa, Israel.
3,3'-Diindolylmethane (DIM) is a dimer of indole-3-carbinol formed both in vivo and in
vitro. In this study,
human cancer cells MCF-7 (with wild-type p53), T47-D
(mutant p53), and Saos-2
(deficient in p53 gene), were used to examine the
anticancer activities of
3,3'-Diindolylmethane (DIM). The dose-dependent growth
inhibitory effect was
found in all these cell lines. Exposure of the cells to 50
microM solution of
3,3'-Diindolylmethane (DIM) for 48 h, apoptosis (programmed cell
death) was evidenced by
the characteristic morphology of cell nuclei under
fluorescence microscope
and the DNA "ladder" in agarose gel electrophoresis. The
percentage of apoptotic
cells in each cell line was found to be 12% for MCF-7,
14% for T47D and 13% for
Saos2 cells. Exposure of MCF-7 cells to 100 microM
3,3'-Diindolylmethane (DIM) for
24 h, 19% of apoptotic cells were detected by flow
cytometry analysis. The
lowest dose required for induction of apoptosis in MCF-7
cells was found to be 10
microM after 72 h incubation. Western blot showed that
wild-type p53 protein was
unchanged after MCF-7 cells had been exposed to 50
microM
3,3'-Diindolylmethane (DIM) for 8 h. This study provides evidences that
3,3'-Diindolylmethane (DIM)
induces apoptosis in human cancer cells and that the
induction of apoptosis is
independent of p53 pathway.
PMID: 8912651
Biochem Pharmacol. 1996
Apr 26;51(8):1069-76.
Indole-3-carbinol and
Diindolylmethane (DIM) as aryl hydrocarbon (Ah) receptor agonists
and antagonists in T47D
human breast cancer cells.
Chen I, Safe S, Bjeldanes
L.
Veterinary Physiology and
Pharmacology, Texas A&M University, College Station
77843-4466, USA.
Indole-3-carbinol (I3C) is
a major component of Brassica vegetables, and
diindolylmethane (DIM) is
the major acid-catalyzed condensation product derived
from I3C. Both compounds
competitively bind to the aryl hydrocarbon (Ah) receptor
with relatively low
affinity. In Ah-responsive T47D human breast cancer cells,
I3C and DIM did not induce
significantly CYP1A1-dependent ethoxyresorufin
O-deethylase (EROD)
activity or CYP1A1 mRNA levels at concentrations as high as
125 or 31 microM,
respectively. A 1 nM concentration of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced EROD activity in these cells,
and cotreatment with TCDD
plus different concentrations of I3C (1-125 microM) or
DIM (1-31 microM) resulted
in a > 90% decrease in the induced response at the
highest concentration of
I3C or DIM. I3C or DIM also partially inhibited (< 50%)
induction of CYP1A1 mRNA
levels by TCDD and reporter gene activity, using an
Ah-responsive plasmid
construct in transient transfection assays. In T47D cells
cotreated with 5 nM
[3H]TCDD alone or in combination with 250 microM I3C or 31
microM DIM, there was a 37
and 73% decrease, respectively, in formation of the
nuclear Ah receptor. The
more effective inhibition of induced EROD activity by
I3C and DIM was due to in
vitro inhibition of enzyme activity. Thus, both I3C and
DIM are partial Ah
receptor antagonists in the T47D human breast cancer cell
line.
PMID: 8866829
Food Chem Toxicol. 1995
Oct;33(10):851-7.
Mechanisms of
indole-3-carbinol (I3C) anticarcinogenesis: inhibition of aflatoxin
B1-DNA adduction and
mutagenesis by I3C acid condensation products.
Takahashi N, Dashwood RH,
Bjeldanes LF, Williams DE, Bailey GS.
Department of Food Science
& Technology, Oregon State University, Corvallis
97331, USA.
Possible inhibitory
mechanisms of indole-3-carbinol (I3C) against aflatoxin B1
(AFB1), a potent
hepatocarcinogen, were examined in rainbow trout. In the
Salmonella assay using a
trout post-mitochondrial activation system, I3C itself
was not an antimutagen
against AFB1. The study also evaluated: the antimutagenic
ability of I3C oligomers;
an acid reaction mixture (RXM) of I3C, generated at low
pH to simulate I3C
products formed under acidic conditions of the stomach;
3,3-diindolylmethane
(DIM), the major derivative of I3C found in trout liver;
and 5,6,11,12,17,18-
hexahydrocyclononal [1,2-b:4,5-b':7,8-b"]triindole , the
cyclic trimer of I3C (CT),
a derivative of I3C in liver and one of the major
components of RXM.
Concentrations of 3.5 microM and greater of I33', CT or RXM
showed about 80%
inhibition compared with the control. Higher concentrations (70
microM) of the various I3C
oligomers also inhibited (to a maximum of 55%)
mutagenesis of synthetic
AFB1-8,9-epoxide added to the Salmonella assay, in the
absence of activating
enzymes. I33' inhibited total microsome catalysed AFB1-DNA
binding in vitro in an
apparently non-competitive manner (Kis = 27.6 +/- 9.4
microM, Kii = 37.5 +/-
32.2 microM). These results suggest that the
anticarcinogenic effect of
I3C against AFB1 in rainbow trout, and perhaps other
species, is due in part to
inhibition of AFB1 bioactivation enzymes and to
scavenging of the
activated AFB1-8,9-epoxide.
PMID: 7590529
J Biochem Toxicol. 1995
Aug;10(4):191-201.
The anticarcinogen
3,3'-Diindolylmethane (DIM) is an inhibitor of cytochrome P-450.
Stresser DM, Bjeldanes LF,
Bailey GS, Williams DE.
Department of Food Science
and Technology, Oregon State University, Corvallis
97331-6602, USA.
Dietary indole-3-carbinol
inhibits carcinogenesis in rodents and trout. Several
mechanisms of inhibition
may exist. We reported previously that
3,3'-Diindolylmethane (DIM), an
in vivo derivative of indole-3-carbinol, is a potent
noncompetitive inhibitor
of trout cytochrome P450 (CYP) 1A-dependent
ethoxyresorufin O-deethylase
with Ki values in the low micromolar range. We now
report a similar potent
inhibition by 3,3'-Diindolylmethane (DIM) of rat and human
CYP1A1, human CYP1A2, and
rat CYP2B1 using various CYP-specific or preferential
activity assays.
3,3'-Diindolylmethane (DIM) also inhibited in vitro CYP-mediated
metabolism of the
ubiquitous food contaminant and potent hepatocarcinogen,
aflatoxin B1. There was no
inhibition of cytochrome c reductase. In addition, we
found
3,3'-Diindolylmethane (DIM) to be a substrate for rat hepatic microsomal
monooxygenase(s) and
tentatively identified a monohydroxylated metabolite. These
observations indicate that
3,3'-Diindolylmethane (DIM) can inhibit the catalytic
activities of a range of
CYP isoforms from lower and higher vertebrates in vitro.
This broadly based
inhibition of CYP-mediated activation of procarcinogens may be
an indole-3-carbinol
anticarcinogenic mechanism applicable to all species,
including humans.
PMID: 8568833
Food Chem Toxicol. 1995
Feb;33(2):111-20.
Regulation of hepatic
cytochrome P4501A by indole-3-carbinol: transient induction
with continuous feeding in
rainbow trout.
Takahashi N, Dashwood RH,
Bjeldanes LF, Bailey GS, Williams DE.
Department of Food Science
and Technology, Oregon State University, Corvallis
97331.
This study investigated
the kinetics of hepatic cytochrome P-4501A (CYP1A)
induction in rainbow trout
by indole-3-carbinol (I3C), a natural tumour modulator
from cruciferous
vegetables, and its low pH reaction products
3,3'-diindolylmethane
(DIM),
5,6,11,12,17,18-hexahydrocyclononal[1,2-b:4,5-b':7,8-b"]triindo le cyclic trimer
(CT), and the unresolved
I3C acid reaction mixture (RXM). RXM, CT and I33' were
potent inducers of total
embryonic CYP1A following direct microinjection, and of
fingerling hepatic CYP1A
following ip exposure, whereas I3C itself produced only
a transient and relatively
weak induction. It is also reported for the first time
that dietary I3C induced
hepatic CYP1A and its associated ethoxyresorufin
O-deethylase (EROD)
activity in trout but, again, the induction was weak and
transient even with
continuous I3C feeding. Mechanism studies and mixed exposures
with the Ah agonist beta-naphthoflavone
indicated that transient induction by I3C
was not due to diet
ageing, but appears to involve inactivation of the Ah
inductive pathway and
irreversible inactivation of CYP1A-mediated EROD activity
by I3C-derived
metabolites. Thus, I3C derivatives exhibit dual capacities for
CYP1A induction and
inhibition in trout.
PMID: 7867998
J Steroid Biochem Mol
Biol. 1993 Dec;46(6):791-8.
Influence of indole
carbinols and growth hormone on the metabolism of
4-androstenedione by rat
liver microsomes.
Jellinck PH, Makin HL,
Sepkovic DW, Bradlow HL.
Department of
Biochemistry, Queen's University, Kingston, Ontario, Canada.
The effect of
indole-3-carbinol (IC), an anticarcinogen present in cruciferous
vegetables, to alter the
metabolism of 4-androstenedione (AD) by female rat liver
microsomes was
investigated and compared to that of its main gastric conversion
product, diindolylmethane
(DIM) as well as other specific cytochrome P450
inducers. DIM was a more
potent inducer of the hydroxylase which converts
androsterone to its 6
beta-hydroxylated derivative 3 alpha, 6 beta-dihydroxy-5
alpha-androstan-17-one (A)
than IC after either oral or intraperitoneal
administration and was
also a better in vitro inhibitor. Isosafrole (ISF), which
like IC and DIM, induces
CYP1A2 as well as gestodene, were powerful inhibitors of
the in vitro reaction.
Naringenin produced only a weak inhibitory effect while
3-methylcholanthrene was
inactive. SKF-525A, a prototypic hydroxylase inhibitor,
or 17
beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androst-1-ene-3-one which
inhibits steroid 5 alpha-reductase,
also decreased the formation of A from AD by
liver microsomes. The
infusion of human growth hormone by osmotic minipump, which
feminizes hepatic steroid
metabolism, increased the ability of male rat liver
microsomes to convert AD
to A and to respond to induction by IC. The identity of
A, the main polar
derivative of AD, induced by IC, DIM and ISF, was tentatively
assigned by a combination
of GC-MS and results from metabolic studies with
intermediates in the
pathway leading to its formation. It is proposed that the
protective role of indole
carbinols against mammary carcinoma due to decreased
formation of 16 alpha-hydroxyestrone
from estrone may be further enhanced by the
diminished availability of
AD for aromatization to estrone.
PMID: 8274413
Biochem Pharmacol. 1993
Mar 9;45(5):1129-36.
Ah receptor binding
properties of indole carbinols and induction of hepatic
estradiol hydroxylation.
Jellinck PH, Forkert PG,
Riddick DS, Okey AB, Michnovicz JJ, Bradlow HL.
Department of
Biochemistry, Queen's University, Kingston, Ontario, Canada.
The effect of route of
administration on the ability of indole-3-carbinol (13C),
an anticarcinogen present
in cruciferous vegetables, to induce estradiol
2-hydroxylase (EH) in
female rat liver microsomes was investigated and compared
to that of its main
gastric conversion product, 3,3'-diindolylmethane (DIM). This
dimer was more potent than
13C after either oral or intraperitoneal
administration and was
also a better in vitro inhibitor of EH in control and
13C-induced hepatic
microsomes. The induction of both CYP1A1 and 1A2 in about
equal amounts by 13C and
DIM as well as of CYP2B1/2 was demonstrated using
monoclonal antibodies.
DIM, isosafrole, beta-naphthoflavone, 3-methylcholanthrene
and naringenin added in
vitro inhibited EH strongly in induced microsomes but
gestodene was a better
inhibitor of estrogen 2-hydroxylation in liver microsomes
from untreated female
rats. The binding affinities of 13C and DIM to the Ah
receptor were compared to
that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by
competition studies, and
the IC50 values were shown to be 2.0 x 10(-9) M, 5.0 x
10(-5) M and 2.3 x 10(-3)
M for TCDD, DIM and 13C, respectively. The ability of
13C or DIM to cause in
vitro transformation of the Ah receptor to a form able to
bind to the
dioxin-responsive element-3 (DRE3) was compared to that of TCDD and
shown to parallel their
abilities to compete for binding of [3H]TCDD to the Ah
receptor. These
experiments confirm and extend the proposals that dietary indoles
induce specific cytochrome
P450s in rat liver by a mechanism possibly involving
the Ah receptor. The
induced monooxygenases, in turn, increase the synthesis of
2-hydroxylated estrogens
in the competing pathways of 2- and 16
alpha-hydroxylation which
decreases the levels of 16 alpha-hydroxyestrone able to
form stable covalent
adducts with proteins including the estrogen receptor. Such
steroid-protein
interaction has been correlated with mammary carcinogenesis.
PMID: 8384853
Food Chem Toxicol. 1992
Jul;30(7):589-99.
Effects of
indole-3-carbinol on biotransformation enzymes in the rat: in vivo
changes in liver and small
intestinal mucosa in comparison with primary
hepatocyte cultures.
Wortelboer HM, van der
Linden EC, de Kruif CA, Noordhoek J, Blaauboer BJ, van
Bladeren PJ, Falke HE.
UTOX, c/o Research
Institute of Toxicology (RITOX), University of Utrecht, The
Netherlands.
Groups of male Wistar rats
were fed semi-synthetic diets containing 0, 200 or 500
mg indole-3-carbinol
(13C)/kg for 2, 7, 14 or 28 days. After 2 days, P-450
activities were already
induced, but the isoenzyme pattern induced was different
in the liver and the small
intestine. Hepatic P4501A1, P4501A2 and P4502B1
apoprotein levels were
dose-relatedly enhanced, whereas in the small intestine
induced levels of P4502B1
and P4501A1 were detected but P4501A2 was not induced.
Pentoxy- and
ethoxyresorufin dealkylation (PROD and EROD) were dose-relatedly
enhanced in the liver (5-
and 7-fold, respectively, in the higher dose group) as
well as in the small
intestine (8- and 13-fold, respectively, at 500 mg 13C/kg
diet). Testosterone 16
alpha- and 16 beta-hydroxylation in the small intestine
were enhanced (6-9-fold)
from day 2 onwards, but in the liver these activities
were only slightly
enhanced from day 7 onwards. Thus, the major forms induced in
the liver appear to be
P4501A1, P4501A2, P4502B1 and, to a lesser extent, P4503A,
whereas in the small
intestine all of the effects that were found are associated
with only one cytochrome
P-450, P4502B1. After 2 days I3C (500 mg/kg) induced
glutathione S-transferase
in the liver (1.3-fold) and small intestine (1.5-fold).
Hepatic glucuronyl
transferase (GT1) was induced (about 1.6-fold) after 7, 14 and
28 days. DT-diaphorase was
induced in the liver (2.7-fold) and small intestine
(1.5-fold) after 14 days
of exposure to 500 mg I3C/kg diet. Treatment of rat
hepatocytes with
indole-3-acetonitrile and 3,3'-Diindolylmethane (DIM), but not I3C and
indole-3-carboxaldehyde,
enhanced EROD activity and halved testosterone 16 alpha-
and 2 alpha-hydroxylation.
All four indoles slightly induced glutathione
S-transferase in cultured
hepatocytes. Thus, the in vitro studies suggest that
the in vivo effects of I3C
have to be attributed to indole-condensation products,
such as
3,3'-Diindolylmethane (DIM), but not to I3C itself.
PMID: 1521833
Chem Biol Interact.
1991;80(3):303-15.
Structure elucidation of
acid reaction products of indole-3-carbinol: detection
in vivo and enzyme
induction in vitro.
De Kruif CA, Marsman JW,
Venekamp JC, Falke HE, Noordhoek J, Blaauboer BJ,
Wortelboer HM.
UTOX, University of
Utrecht, The Netherlands.
The potency of
indole-3-carbinol (I3C) to form condensation products under acidic
aqueous conditions was
studied. After identifying a known dimer,
3,3'-diindolylmethane
(DIM), we elucidated the structures of two trimers also
found in acid reaction
mixtures:
5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b':7,8-b"]tri-indole (CTI), and
2,3-bis[3-indolylmethyl]
indole (BII). The formation of these indole oligomers
was shown to be pH
dependent. The highest amounts of DIM and BII were formed in
aqueous solutions having a
pH value ranging from 4 to 5. No CTI could be detected
at pH values above 4.5. In
rats that received an oral dose of I3C we could detect
DIM and BII in gastric
contents, stomach tissue, small intestine and liver. No
CTI could be detected in
vivo after oral exposure to I3C. In in vitro
experiments, using rat
hepatocytes, the cytochrome P-450IA1 apoprotein level,
7-ethoxyresorufin O-deethylation
activity (EROD) and DT-diaphorase activity (DTD)
were markedly enhanced by
DIM and CTI as well as BII.
PMID: 1954658
Food Chem Toxicol. 1989
Jun;27(6):385-92.
In vivo disposition of the
natural anti-carcinogen indole-3-carbinol after po
administration to rainbow
trout.
Dashwood RH, Uyetake L,
Fong AT, Hendricks JD, Bailey GS.
Department of Food Science
and Technology, Oregon State University, Corvallis
97331.
Indole-3-carbinol (I3C), a
compound found naturally as a glucosinolate in
cruciferous vegetables
such as broccoli and cabbage, has been shown to modulate
the carcinogenic process
in a number of animal species. The lack of detailed
information on the
disposition of I3C in vivo provided the main impetus for the
study reported here, in
which the distribution and metabolic fate of I3C was
assessed in selected
tissues and excreta after po administration to rainbow trout
(Salmo gairdneri). Animals
were fasted for 3 days and given [5-3H]I3C either in
the diet or by single oral
gavage (40 mg/kg body weight; 15 muCi/kg body weight).
Following administration,
75% of the initial 3H-dose was detected within the
stomach between 0.5 and 12
hr, after which it was released to distal regions of
the gut for subsequent
uptake, distribution and elimination. At the end of the
study (72 hr) 25% of the
administered dose was recovered from the water which
reflected excretion
through the gills and urinary tract. Significant excretion
also occurred in the bile,
with approximately 5% of the initial 3H-dose recovered
from the bile sacs at 72
hr. Further analyses of the radioactive components in
the bile indicated that
one or more derivatives of I3C, but not the parent
compound itself, are
excreted as glucuronide conjugates using this route.
Radioactivity accumulated
in the liver throughout most of the study, reaching
levels of 1-1.5% between
48 and 72 hr of the administered dose. High-performance
liquid chromatography
analyses indicated the presence of four main radiolabelled
species in these livers,
one of which co-eluted with the parent compound, I3C.
The major radiolabelled
species recovered from the liver was tentatively
identified as the dimer,
3,3'-diindolylmethane (DIM), which comprised 40% of the
total hepatic radiolabel.
This dimer, DIM, was also found to accumulate in the
diet containing I3C, which
reflected a time-dependent dimerization of the parent
compound in vitro. These
findings are discussed in view of recent postulates of a
role for I3C condensation products such as DIM in the mechanism of I3C
anti-carcinogenesis.
PMID: 2792968
Food Chem Toxicol. 1987
May;25(5):363-8.
Differential induction of
mixed-function oxidase (MFO) activity in rat liver and
intestine by diets
containing processed cabbage: correlation with cabbage levels
of glucosinolates and
glucosinolate hydrolysis products.
McDanell R, McLean AE,
Hanley AB, Heaney RK, Fenwick GR.
Both white and Savoy-type
cabbage added to a semi-purified diet at 25% dry weight
and fed to rats ad lib.
for 5 days significantly induced ethoxyresorufin (ERR)
deethylation in the small
and large intestine. Savoy cabbage also induced hepatic
activity and, in general,
exhibited a greater inducing effect than white cabbage.
These enzyme-inducing
effects were altered when the cabbage had been processed.
The content of intact
glucosinolate was greater in Savoy than in white cabbage.
The indole glucosinolate (glucobrassicin)
content of both types of cabbage was
approximately halved by
cooking but was unaffected by fermentation, whilst
homogenization of Savoy
cabbage led to the total disappearance of intact
glucosinolates. Levels of
the indole glucosinolate breakdown products ascorbigen
and indole-3-carbinol were
highest in homogenized cabbage, and ascorbigen levels
were also higher in cooked
than in fresh cabbage of either type. When added to
the semi-purified diet and
fed ad lib. to rats for 5 days, indole-3-carbinol was
a potent inducer of
hepatic ERR deethylation and cytochrome P-450 activity, but
had much less effect in
the intestine. Other glucobrassicin metabolites,
Diindolylmethane (DIM) and
indole-3-acetonitrile, also had some inducing effect in the
liver but no effect in the
intestine, while ascorbigen significantly induced ERR
deethylation in the small
and large intestine but had no effect on hepatic MFO
activity.
PMID: 3609976
J Toxicol Environ Health.
1987;21(3):311-23.
Structure-activity
relationships of dietary indoles: a proposed mechanism of
action as modifiers of
xenobiotic metabolism.
Bradfield CA, Bjeldanes
LF.
In an effort to understand
the mechanism by which dietary indoles inhibit
chemically initiated
tumorigenesis in experimental animals, we have investigated
the potency of
3-substituted and 1,3-disubstituted indoles on the induction of
intestinal and hepatic
cytochrome P-448-dependent monooxygenases in the rat. Oral
intubation with
indole-3-carbinol (13C), 1-methoxyindole-3-carbinol (N13C),
1-methoxyindole-3-carboxaldehyde (NCHO), and 3,3'-diindolylmethane (DIM) at 31
mumol/animal led to
significant increases in hepatic ethoxyresorufin O-deethylase
activity (EROD; 15, 7, 6,
and 5-fold over control, respectively), while
intubation with indole (IND),
3-methylindole (3MI), indole-3-carboxaldehyde
(13CHO), and
indole-3-acetonitrile (IAN) did not increase this monooxygenase
activity over control
levels. For the eight indoles tested, there was a strong
relationship between
instability in acidic solution, as indicated by the
generation of insoluble
products, and capacity to induce hepatic EROD. Further
experiments indicated that
13C did not induce hepatic EROD when dosed ip (thus
bypassing the acidity of
the stomach). Acid treatment of 13C generated a reaction
mixture (RXM) that induced
EROD after ip or po dosing. Chromatographic
fractionation of the RXM
indicated that there exist at least four different 13C
acid-condensation products
in the RXM with the ability to induce EROD. The
results presented strongly
support the hypothesis that dietary indoles influence
the levels of
monooxygenase activities via a series of acid-condensation products
generated upon
introduction of the indole into the acidic environment of the
stomach.
PMID: 3495667
Cancer Res. 1978
May;38(5):1410-3.
Inhibition of polycyclic
aromatic hydrocarbon-induced neoplasia by naturally
occurring indoles.
Wattenberg LW, Loub WD.
Indole-3-carbinol,
3,3'-Diindolylmethane (DIM), and indole-3-acetonitrile, three
indoles occurring in
edible cruciferous vegetables, have been studied for their
effects on
7,12-dimethylbenz(a)anthracene-induced mammary tumor formation in
female Sprague-Dawley rats
and on benzo(a)pyrene-induced neoplasia of the
forestomach in female ICR/Ha
mice. When given by p.o. intubation 20 hr prior to
7,12-dimethylbenz(a)anthracene administration, indole-3-carbinol and
3,3'-Diindolylmethane (DIM) had
an inhibitory effect on mammary tumor formation, but
indole-3-acetonitrile was
inactive. Indole-3-carbinol when added to the diet for
8 days prior to challenge
with 7,12-dimethylbenz(a)anthracene inhibited mammary
tumor formation, whereas
indole-3-acetonitrile did not. Dietary administration of
all three indoles
inhibited benzo(a)pyrene-induced neoplasia of the forestomach
in ICR/Ha mice. The
identification of dietary constituents that can inhibit
chemical carcinogens
ultimately may be of value in understanding the balance of
factors that determines
the neoplastic response to these cancer-producing agents
in the environment.
PMID: 416908
Fed Proc. 1976 May
1;35(6):1327-31.
Dietary constituents
altering the responses to chemical carcinogens.
Wattenberg LW, Loub WD,
Lam LK, Speier JL.
This paper deals with two
categories of compounds having the capacity to inhibit
the neoplastic effects of
chemical carcinogens on the host. The first are
inducers of increased
microsomal mixed function oxidase activity. An increasing
number of these inducers
are being found in natural products. Cruciferous
vegetables including
brussels sprouts, cabbage, and cauliflower contain such
compounds. Recently
indole-3-acetonitrile, indole-3-carbinol and
3,3'-Diindolylmethane (DIM) have
been identified as inducers in these three plants.
Other naturally occurring
inducers include flavones, safrole, isosafrole,
beta-ionone, and oxidized
sterols. Since previous work has shown that synthetic
inducers may protect
against chemical carcinogens, the composition of the diet
could play a role in
inhibiting the neoplastic response to these carcinogenic
agents. The second
category of inhibitors comprises the antioxidants. Several of
these compounds have been
found to inhibit the carcinogenic effects of a variety
of chemical carcinogens.
Considerable work of this nature has been done with
butylated hydroxyanisole
and butylated hydroxytoluene two antioxidants
extensively used as food
additives. Other antioxidants having carcinogen
inhibiting capacities
include ethoxyquin, disulfiram, and
dimethyldithiocarbamate.
PMID: 1261715
Cancer Res. 1975 Nov;35(11
Pt. 2):3326-31.
Effects of dietary
constituents on the metabolism of chemical carcinogens.
Wattenberg LW.
Dietary constituents of 2
types have been shown to affect the metabolism of
chemical carcinogens by
the microsomal mixed-function oxidase system. Naturally
occurring inducers of
increased activity of this system are present in plants.
Cruciferous vegetables
including Brussels sprouts, cabbage, and cauliflower are
relatively potent in this
regard. From these vegetables, three indoles with
inducing activity have
been identified. These are indole-3-acetonitrile,
indole-3-carbinol, and
3,3'-Diindolylmethane (DIM). A 2nd type of dietary constituent
affecting the microsomal
mixed-function oxidase system is added phenolic
antioxidant, i.e.,
butylated hydroxyanisole (BHA) and butylated hydroxytoluene.
Studies of the effect of
BHA on metabolism of bezo(a)-pyrene by liver microsomes
have been carried out. BHA
feeding results in microsomal changes. The cytochrome
P-450 shows altered
spectral characteristics, and the aryl hydrocarbon
hydroxylase system of
these microsomes has an increased sensitivity to inhibition
by alpha-naphthoflavone.
In addition, there is a decrease in binding of
metabolites of
benzo(a)pyrene to DNA upon incubation of these microsomes of
induction of increased
mixed function oxidase activity have shown that increased
levels of activity protect
against administration of chemical carcinogens. BHA
and butylated
hydroxytoluene also have been found to exert a protective effect
against chemical
carcinogens. Thus the constituents of the diet could be of
consequence in the
neoplastic response to exposure to carcinogens in the
environment.
PMID: 1104144
J Natl Cancer Inst. 1975
Apr;54(4):985-8.
Aryl hydrocarbon
hydroxylase induction in rat tissues by naturally occurring
indoles of cruciferous
plants.
Loub WD, Wattenberg LW,
Davis DW.
A phytochemical
investigation to identify inducers of increased aryl hydrocarbon
hydroxylase (AHH) activity
from three cruciferous vegetables, Brussels sprouts,
cabbage, and cauliflower,
resulted in the identification of
indole-3-acetonitrile,
indole-3-carbinol, and 3,3'-diindolylmethane (DIM) as naturally
occurring inducers. These
compounds are produced during the hydrolysis of
indolyl-methyl
glucosinolate by the plant enzyme myrosinase.
PMID: 1127728
