Diindolylmethane (DIM) Information Resource Center

This section is updated on a monthly basis. Paper abstracts are presented in chronological order from most recent.

 

Food Chem Toxicol. 2008 Jul;46(7):2451-8. Epub 2008 Apr 6.

Inhibitory effects of a dietary phytochemical 3,3'-diindolylmethane (DIM) on the
phenobarbital-induced hepatic CYP mRNA expression and CYP-catalyzed reactions in
female rats.

Parkin DR, Lu Y, Bliss RL, Malejka-Giganti D.

Veterans Affairs Medical Center, Minneapolis, MN 55417, USA.

3,3'-diindolylmethane (DIM), derived from indole-3-carbinol (I3C), is used as a
dietary supplement for its putative anticancer effects that include suppression
of mammary tumor growth in female rats. The mechanism of action DIM may involve
its interaction(s) with hepatic cytochromes P450 (CYPs) catalyzing oxidations of
17beta-estradiol (E2). Our study showed that DIM added to hepatic microsomes of
female Sprague-Dawley rats was primarily a competitive inhibitor of
beta-naphthoflavone (beta-NF)- or I3C-induced CYP1A1 probe activity, and a potent
mixed or uncompetitive inhibitor of phenobarbital (PB)-induced CYP2B1 or CYP2B2
probe activity, respectively. Microsomal metabolites of DIM were tentatively
identified as two mono-hydroxy isomers of DIM, each formed preferentially by
CYP1A1- or CYP2B1/2-catalyzed reaction. Evaluation of the effects of co-treatment
of rats with PB and DIM by a full factorial ANOVA showed that DIM decreased the
PB-induced CYP2B1 and CYP2B2 mRNA expression levels, and the rates of 2- and
4-hydroxylation of E2, and total E2 metabolite formation. The results suggest
that interactions of DIM, and/or its mono-hydroxy metabolites, with CYP2B1 and
CYP2B2 found to occur in hepatic microsomes upon addition of DIM or co-treatment
of rats with DIM affect the rates of relevant oxidations of E2, and potentially
protect against estrogen-dependent tumorigenesis.

PMID: 18486294

 

Carcinogenesis. 2008 Jun;29(6):1139-47. Epub 2008 May 5.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell
and tumor growth through activation of c-jun N-terminal kinase.

Lei P, Abdelrahim M, Cho SD, Liu S, Chintharlapalli S, Safe S.

Institute of Biosciences and Technology, Texas A&M University Health Science
Center, Houston, TX 77030-3303, USA.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) activate the orphan
receptors peroxisome proliferator-activated receptor gamma (PPARgamma) and Nur77
and induce receptor-dependent and -independent apoptotic pathways in colon and
other cancer cells. Structure-activity studies show that the p-bromo
(DIM-C-pPhBr) and p-fluoro (DIM-C-pPhF) analogs, which exhibit minimal activation
of Nur77 and PPARgamma, induce expression of CCAAT/enhancer-binding protein
homologous protein (CHOP/GADD153) in colon cancer cells. Moreover, among a series
of bromo and fluoro C-DIM analogs, their induction of CHOP was dependent on the
position of the phenyl substituents (para >/= meta >/= ortho) and required a free
indole group. DIM-C-pPhBr and DIM-C-pPhF not only induced CHOP but also activated
death receptor 5 (CHOP dependent), cleavage of caspase 8 and poly (ADP ribose)
polymerase (PARP) that is consistent with activation of the extrinsic pathway of
apoptosis. These responses were associated with the activation of c-jun
N-terminal kinase (JNK) pathway since inhibition of JNK inhibited induction of
the extrinsic apoptotic pathway by these C-DIMs. However, in contrast to
classical inducers of endoplasmic reticulum (ER) stress such as tunicamycin and
thapsigargin, the C-DIM compounds did not induce glucose-related protein 78 that
is a marker of ER stress. Proapoptotic and anticarcinogenic effects were also
observed in athymic nude mice bearing RKO cell xenografts and treated with 30
mg/kg/day DIM-C-pPhBr and this was accompanied by increased JNK phosphorylation
in the tumors. Thus, the anticarcinogenic activity of DIM-C-pPhBr in colon cancer
cells and tumors is related to a novel ER stress-independent activation of JNK.

PMID: 18460448

 

Cancer Let. 2008 Jun 28;265(1):113-23. Epub 2008 Apr 2.

CXCR4 and CXCL12 down-regulation: a novel mechanism for the chemoprotection of
3,3'-diindolylmethane (DIM) for breast and ovarian cancers.

Hsu EL, Chen N, Westbrook A, Wang F, Zhang R, Taylor RT, Hankinson O.

Molecular Toxicology Interdepartmental Doctoral Program, University of
California-Los Angeles, Los Angeles, CA 90095-1732, USA.

Cruciferous vegetables are thought to protect against numerous types of cancer.
3,3'-Diindolylmethane (DIM) is an acid-catalyzed product generated during the
consumption of cruciferous vegetables and appears to be chemoprotective for
breast cancer. The interaction between the chemokine receptor, CXCR4, and its
unique ligand, CXCL12, is known to mediate the progression and metastasis of
breast and other cancers. Organs to which these cancers metastasize secrete
CXCL12, which binds to CXCR4 expressed on the surface of primary cancer cells.
This process subsequently stimulates the invasive properties of the cancer cells
and attracts them to the preferred organ sites of metastases. We have found that
DIM down-regulates both CXCR4 and CXCL12 in MCF-7 and MDA-MB-231 breast cancer
cells as well as in BG-1 ovarian cancer cells at the transcriptional level and in
an estrogen-independent manner. We demonstrate that the potential of MDA-MB-231
and BG-1 cells for chemotaxis and invasion towards CXCL12, but not towards IL-6
or fetal bovine serum, respectively, is inhibited by DIM. Furthermore, we show
that DIM down-regulates CXCR4 under hypoxia and CXCL12 under estradiol-inducing
conditions. Our data suggest that one mechanism whereby DIM protects against
breast, ovarian, and possibly other cancers is through the repression of CXCR4
and/or CXCL12, thereby lowering the invasive and metastatic potential of these
cells.

PMID: 18378071

 

Mol Cancer Ther. 2008 Jun;7(6):1708-19.

Apoptosis-inducing effect of erlotinib is potentiated by 3,3'-diindolylmethane (DIM) in
vitro and in vivo using an orthotopic model of pancreatic cancer.

Ali S, Banerjee S, Ahmad A, El-Rayes BF, Philip PA, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI, USA.

Blockade of epidermal growth factor receptor (EGFR) by EGFR tyrosine kinase
inhibitors is insufficient for effective antitumor activity because of
independently activated survival pathways. A multitargeted approach may therefore
improve the outcome of anti-EGFR therapies. In the present study, we determined
the effects of 3,3'-diindolylmethane on cell viability and apoptosis with
erlotinib in vitro and in vivo using an orthotopic animal tumor model. BxPC-3 and
MIAPaCa cells with varying levels of EGFR and nuclear factor-kappaB (NF-kappaB)
DNA-binding activity were treated with DIM (20 micromol/L), erlotinib (2
micromol/L), and the combination. Cell survival and apoptosis was assessed by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and histone-DNA
ELISA. Electrophoretic mobility shift assay was used to evaluate NF-kappaB
DNA-binding activity. We found significant reduction in cell viability by both
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic
assays, induction of apoptosis, down-regulation of EGFR phosphorylation,
NF-kappaB DNA-binding activity, and expression of antiapoptotic genes in BxPC-3
cells when treated with the combination of erlotinib and DIM compared with
either agent alone. In contrast, no such effect was observed in MIAPaCa cells by
similar treatment. Most importantly, these in vitro results were recapitulated in
animal model showing that DIM in combination with erlotinib was much more
effective as an antitumor agent compared with either agent alone. These results
suggest that the utilization of DIM could be a useful strategy for achieving
better treatment outcome in patients with activated status of EGFR and NF-kappaB
in their tumors.

PMID: 18566242 [PubMed - in process]

Biochem Pharmacol. 2008 May 1;75(9):1858-67.

3,3'-diindolylmethane (DIM) reduces levels of HIF-1alpha and HIF-1 activity in hypoxic 
cultured human cancer cells.

Riby JE, Firestone GL, Bjeldanes LF.

Department of Nutritional Sciences and Toxicology, 217 Morgan Hall, University of
California, Berkeley, CA 94720, USA.

3,3'-diindolylmethane (DIM) is a chemopreventive and chemotherapeutic
phytochemical derived from the metabolism of indoles found at high concentrations
in cruciferous vegetables. We have previously shown that DIM exhibits
anti-angiogenic properties in cultured vascular endothelial cells and in Matrigel
plug assays in rodents. In the present study, we demonstrate that DIM reduces the
level of hypoxia-inducible factor (HIF)-1alpha in hypoxic tumor cell lines, as
well as HIF-1 transcriptional activity as measured by a reporter assay. Moreover,
DIM inhibited the expression of HIF-1-responsive endogenous genes, resulting in
the reduced expression of key hypoxia responsive factors, VEGF, furin, enolase-1,
glucose transporter-1 and phosphofructokinase. DIM reduced the level of
HIF-1alpha in hypoxic cells by increasing the rate of the prolylhydroxylase- and 
proteasome-mediated degradation of HIF-1alpha, and by decreasing the rate of
HIF-1alpha transcription. Using enzyme kinetics studies, we established that DIM 
interacts with the oligomycin-binding site on the F0 transmembrane component of
mitochondrial F1F0-ATPase. The contributions of the resulting increases in levels
of ROS and O2 in hypoxic cells to the inhibitory effects of DIM on HIF-1alpha
expression are discussed. These studies are the first to show that DIM can
decrease the accumulation and activity of the key angiogenesis regulatory factor,
HIF-1alpha, in hypoxic tumor cells.

PMID: 18329003

Pharm Res. 2008 Apr 22

Chemoprevention of Pancreatic Cancer: Characterization of Par-4 and its
Modulation by 3,3' Diindolylmethane (DIM).

Azmi AS, Ahmad A, Banerjee S, Rangnekar VM, Mohammad RM, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, 9374 Scott Hall, 540 E Canfield, Detroit, Michigan, 48201, USA.

PURPOSE: Cancer chemoprevention is defined as the use of natural, synthetic, or
biological agents to suppress, reverse or prevent the carcinogenic process from
turning into aggressive cancer. Prostate apoptosis response-4 (Par-4) is a unique
pro-apoptotic protein that selectively induces apoptosis in prostate cancer
cells. However, its role in other malignancies has not been fully explored. This 
study tries to identify the functional significance of Par-4 in pancreatic
cancer. METHODS: Multiple molecular techniques such as Western blot analysis,
trypan blue assay for cell viability, MTT assay for cell growth inhibition and
Histone/DNA ELISA for apoptosis were used. RESULTS: Western blot analysis
revealed that 3,3'-diindolylmethane (DIM) a chemopreventive agent, specifically
its more bioavailable formulation, DIM, at low doses (20 mumol/L) induces
Par-4, in L3.6pl and Colo-357 pancreatic cancer cells. At similar doses, DIM
reduced cell viability and caused cell growth inhibition and apoptosis. Moreover,
DIM pre-treatment sensitized the cells to cytotoxic action of chemotherapeutic
drug gemcitabine through up-regulation of Par-4. CONCLUSION: The induction of
Par-4 is indirectly related to increased sensitivity and cell death through
apoptosis. To our knowledge the results reported here showed, for the first time,
the induction of Par-4 by chemopreventive agents, in general, and DIM, in
particular, in pancreatic cancer cells in vitro.

PMID: 18427961

Cancer Let. 2008 Mar 28 

CXCR4 and CXCL12 down-regulation: A novel mechanism for the chemoprotection of
3,3'-diindolylmethane (DIM) for breast and ovarian cancers.

Hsu EL, Chen N, Westbrook A, Wang F, Zhang R, Taylor RT, Hankinson O.

Molecular Toxicology Interdepartmental Doctoral Program, University of
California-Los Angeles, Los Angeles, CA 90095-1732, USA; Department of Pathology 
and Laboratory Medicine, University of California-Los Angeles, Los Angeles, CA
90095-1732, USA; Jonsson Comprehensive Cancer Center, University of
California-Los Angeles, Los Angeles, CA 90095-1732, USA.

Cruciferous vegetables are thought to protect against numerous types of cancer.
3,3'-Diindolylmethane (DIM) is an acid-catalyzed product generated during the
consumption of cruciferous vegetables and appears to be chemoprotective for
breast cancer. The interaction between the chemokine receptor, CXCR4, and its
unique ligand, CXCL12, is known to mediate the progression and metastasis of
breast and other cancers. Organs to which these cancers metastasize secrete
CXCL12, which binds to CXCR4 expressed on the surface of primary cancer cells.
This process subsequently stimulates the invasive properties of the cancer cells 
and attracts them to the preferred organ sites of metastases. We have found that 
DIM down-regulates both CXCR4 and CXCL12 in MCF-7 and MDA-MB-231 breast cancer
cells as well as in BG-1 ovarian cancer cells at the transcriptional level and in
an estrogen-independent manner. We demonstrate that the potential of MDA-MB-231
and BG-1 cells for chemotaxis and invasion towards CXCL12, but not towards IL-6
or fetal bovine serum, respectively, is inhibited by DIM. Furthermore, we show
that DIM down-regulates CXCR4 under hypoxia and CXCL12 under estradiol-inducing
conditions. Our data suggest that one mechanism whereby DIM protects against
breast, ovarian, and possibly other cancers is through the repression of CXCR4
and/or CXCL12, thereby lowering the invasive and metastatic potential of these
cells.

PMID: 18378071

Cancer Res. 2008 Mar 15;68(6):1927-34.

Mammalian target of rapamycin repression by 3,3'-diindolylmethane (DIM) inhibits
invasion and angiogenesis in platelet-derived growth factor-D-overexpressing PC3 
cells.

Kong D, Banerjee S, Huang W, Li Y, Wang Z, Kim HR, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI 48201, USA.

Platelet-derived growth factor-D (PDGF-D) is a newly recognized growth factor
known to regulate many cellular processes, including cell proliferation,
transformation, invasion, and angiogenesis. Recent studies have shown that PDGF-D
and its cognate receptor PDGFR-beta are expressed in prostate tumor tissues,
suggesting that PDGF-D might play an important role in the development and
progression of prostate cancer. However, the biological role of PDGF-D in
tumorigenesis remains elusive. In this study, we found that PDGF-D-overexpressing
PC3 cells (PC3 cells stably transfected with PDGF-D cDNA and referred to as PC3
PDGF-D) exhibited a rapid growth rate and enhanced cell invasion that was
associated with the activation of mammalian target of rapamycin (mTOR) and
reduced Akt activity. Rapamycin repressed mTOR activity and concomitantly
resulted in the activation of Akt, which could attenuate the therapeutic effects 
of mTOR inhibitors. In contrast, DIM significantly inhibited both mTOR and Akt in

PC3 PDGF-D cells, which were correlated with decreased cell proliferation and invasion.
Moreover, conditioned medium from PC3 PDGF-D cells significantly increased the
tube formation of human umbilical vein endothelial cells, which was inhibited by 
DIM treatment concomitant with reduced full-length and active form of PDGF-D.
Our results suggest that DIM could serve as a novel and efficient
chemopreventive and/or therapeutic agent by inactivation of both mTOR and Akt
activity in PDGF-D-overexpressing prostate cancer.

PMID: 18339874

Zhonghua Yi Xue Za Zhi. 2008 Mar 11;88(10):661-4.

3,3-diindolylmethane (DIM) enhances the inhibitory effect of idarubicin on the growth 
of human prostate cancer cells (Article in Chinese)

Zhao YY, Zhou L, Pan YZ, Zhao LJ, Liu YN, Yu H, Li Y, Zhao XJ.

Prostate Diseases Prevention and Treatment Research Center, Jilin University,
Changchun 130021, China.

OBJECTIVE: To study the effects of idarubicin (IDA) combined with 3,
3-diindolylmethane (DIM) on the growth inhibition of human prostate cancer cells.
METHODS: Human prostate cancer cells of the line PC-3M were cultured and then
divided into the following groups: control group with solvent added into the
culture fluid; IDA groups, with IDA of the terminal concentrations of 0.5, 1 or 5
mg/L added into the culture fluid; DIM groups, with DIM of the terminal
concentrations of 30, 60 or 100 micromol/L added into the culture fluid; and DIM 
+ IDA groups, with 0. 5 mg/L IDA and DIM 30, 60 or 100 micromol/L added into the 
culture fluid. 48 h later the cell growth inhibition rate was detected by MTT
assay. Flow cytometry and acridine orange staining were used to detect the cell
cycle and apoptosis. RT-PCR and Western blotting were used to detect the mRNA and
protein expression of caspase 9, an apoptosis gene. RESULTS: Both IDA and DIM
dose-dependently inhibited the growth of the PC-3M cells. The growth inhibition
rate of the 60 micromol/L DIM + 0.5 mg/L IDA group was 69.9%, almost 10 times as 
that of the 0.5 mg/L IDA group. The apoptosis rate of the 60 micromol/L DIM + 0. 
5 mg/L IDA group was 47.0%, significantly higher than that of the 0.5 mg/L IDA
group (3.2%, P < 0.05). RT-PCR and Western blotting showed that the combination
of DIM and IDA significantly enhanced the mRNA and protein expression of caspase 
9. CONCLUSION: DIM enhances the growth inhibition effect of IDA on human prostate
cancer cells by the mechanism of induction of apoptosis.

PMID: 18642764

Mol Cancer Ther. 2008 Feb;7(2):341-9.

Induction of growth arrest and apoptosis in human breast cancer cells by
3,3-diindolylmethane (DIM) is associated with induction and nuclear localization of
p27kip.

Wang Z, Yu BW, Rahman KM, Ahmad F, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI 48201, USA.

3,3'-Diindolylmethane (DIM) is a stable condensation product of
indole-3-carbanol, a potential breast cancer chemoprevention agent. Human breast 
cancer cell lines were studied to better understand its mechanisms. In vitro
experiments were done in MCF-7, T47D, BT-20 and BT-474 cells using MTT, ELISA,
immunoblotting assays, reverse transcription-PCR, protein half-life, confocal
microscopy, cell fractionation, and immunoprecipitation assays. We found that DIM
inhibited the growth of all four breast cancer cell lines (IC(50)s, 25-56
micromol/L). Because BT-20 and BT-474 overexpressed Her-2 and activated Akt, and 
BT-20 lacks estrogen receptor, these were studied further. In both cell lines,
DIM appeared to induce expression of p27(kip) protein before the loss of cell
viability and apoptosis. In BT-20 cells, DIM also inhibited expression of
activated Akt, but this appeared after p27(kip) induction. In both cell lines,
DIM induced p27(kip) transcript expression within 6 h. DIM prolonged the p27(kip)
protein half-life in BT-20 but not BT-474 cells. We also showed, for the first
time, that DIM induced nuclear localization of p27(kip) in both cell lines.
Moreover, in BT-20 cells, DIM induced a decrease in p27(kip) phosphorylation at
Thr(187), and its association with the 14-3-3 protein, which helped to explain
the protein half-life increase and nuclear localization, respectively. DIM
modulates p27(kip) through transcription, prolongation of protein half-life, and 
nuclear localization. These effects appear to be independent of Her-2, Akt, or
estrogen receptor status and should support further study for its chemoprevention
potential in breast cancer.

PMID: 18281517

J Nutr. 2008 Jan;138(1):17-23.

 

3,3'-Diindolylmethane (DIM) suppresses the inflammatory response to lipopolysaccharide

in murine macrophages.

 

Cho HJ, Seon MR, Lee YM, Kim J, Kim JK, Kim SG, Park JH.

 

Center for Efficacy Assessment and Development of Functional Foods and Drugs,

Hallym University, Chuncheon 200-702, South Korea.

 

3,3'-Diindolylmethane (DIM), a major acid-condensation product of

indole-3-carbinol, has been shown to have multiple anticancer effects in

experimental models. Because recurrent or chronic inflammation has been

implicated in the development of a variety of human cancers, this study examined

the antiinflammatory effects of DIM and the underlying mechanisms using

lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages. DIM

significantly decreased the release of nitric oxide (NO), prostaglandin (PG)E2,

tumor necrosis factor alpha, interleukin (IL)-6, and IL-1beta by RAW264.7 cells

treated with LPS. DIM inhibited LPS-induced increases in protein levels of

inducible NO synthase (iNOS), which were accompanied by decreased iNOS mRNA

levels and transcriptional activity. The mRNA levels of phospholipase A2

decreased, whereas neither cyclooxygenases-2 protein nor transcript was altered

by DIM. In addition, DIM suppressed LPS-induced nuclear factor-kappaB (NF-kappaB)

transcriptional activity, NF-kappaB DNA-binding activity, translocation of p65

(RelA) to the nucleus, and degradation of inhibitor of kappaB alpha. Furthermore,

DIM decreased LPS-induced transcriptional activity of activator protein (AP)-1,

AP-1 DNA-binding activity, and phosphorylation of stress-activated protein

kinase/Jun-N-terminal kinase and c-Jun. We demonstrate that DIM inhibits

LPS-induced release of proinflammatory mediators in murine macrophages.

Downregulation of NF-kappaB and AP-1 signaling may be one of the mechanisms by

which DIM inhibits inflammatory responses.

 

PMID: 18156398 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

Carcinogenesis. 2007 Jul;28(7):1471-7. Epub 2007 Feb 28.

 

Quantitative combination effects between sulforaphane and 3,3'-diindolylmethane (DIM)

on proliferation of human colon cancer cells in vitro.

 

Pappa G, Strathmann J, Löwinger M, Bartsch H, Gerhäuser C.

 

Division of Toxicology and Cancer Risk Factors, German Cancer Research Center

(DKFZ), C010-2 Chemoprevention, Im Neuenheimer Feld 280, 69120 Heidelberg,

Germany.

 

Isothiocyanates (ITCs) and indoles derived from cruciferous vegetables possess

growth-inhibiting and apoptosis-inducing activities in cancer cell lines in

vitro. ITCs like sulforaphane (SFN) are cytotoxic, whereas indoles including

indole-3-carbinol or its condensation product 3,3'-diindolylmethane (DIM) are

acting by cytostatic mechanisms in human colon cancer cell lines. In the present

study, we have investigated the impact of defined combinations of SFN and DIM

(ratio 1:4, 1:2, 1:1, 2:1 and 4:1) on cell proliferation, cell-cycle progression

and apoptosis induction in cultured 40-16 colon carcinoma cells. Calculations of

combination effects were based on the method of Chou et al. (1984) Adv. Enzyme

Regul., 22, 27-55, and were expressed as a combination index (CI) with CI < 1, CI

= 1 or CI > 1 representing synergism, additivity or antagonism, respectively.

Interestingly, at a total drug concentration of 2.5 microM, all combinations of

SFN and DIM were antagonistic. With increasing concentrations, the antagonistic

effect gradually turned into a synergistic interaction at the highest combined

cytotoxic concentration of 40 microM. Cell-cycle analyses with SFN:DIM ratios of

1:1, 1:2 and 1:4 and total concentrations between 10 and 25 microM confirmed

antagonism at low and additive effects at higher doses. SFN (10 microM) in

combination with DIM (10 microM) resulted in strong G(2)/M cell-cycle arrest,

which was not observed with either compound alone. Our results indicate that

cytotoxic concentrations of SFN:DIM combinations affect cell proliferation

synergistically. At low total concentrations (below 20 microM), which are

physiologically more relevant, the combined broccoli compounds showed

antagonistic interactions in terms of cell growth inhibition. These data stress

the need for elucidating mechanistic interactions for better predicting

beneficial health effects of bioactive food components.

 

PMID: 17331956 [PubMed - indexed for MEDLINE]

   

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 [PubMed - indexed for MEDLINE]

  

Mol Pharmacol. 2007 Feb;71(2):558-69. Epub 2006 Nov 8.

 

1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes inhibit growth, induce

apoptosis, and decrease the androgen receptor in LNCaP prostate cancer cells

through peroxisome proliferator-activated receptor gamma-independent pathways.

 

Chintharlapalli S, Papineni S, Safe S.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466

TAMU, Vet. Res. Bldg. 409, College Station, TX 77843-4466, USA.

 

1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes (C-DIMs) containing

para-trifluoromethyl, t-butyl, and phenyl groups are a novel class of peroxisome

proliferator-activated receptor (PPAR)gamma agonists. In LNCaP prostate cancer

cells, these compounds induce PPARgamma-dependent transactivation, inhibit cell

proliferation, and induce apoptosis. In addition, these PPARgamma agonists

modulate a number of antiproliferative and proapoptotic responses, including

induction of p27, activating transcription factor 3, and nonsteroidal

anti-inflammatory drug-activated gene-1 and down-regulation of cyclin D1 and

caveolin-1. Moreover, the PPARgamma antagonist 2-chloro-5-nitrobenzanilide

(GW9662) does not inhibit these effects. The C-DIM compounds also abrogate

androgen receptor (AR)-mediated signaling and decrease prostate-specific antigen

(PSA) and AR protein expression, and these responses were PPARgamma-independent.

The effects of C-DIMs on AR and PSA were due to decreased AR and PSA mRNA

expression in LNCaP cells. Thus, this series of methylene-substituted

diindolylmethane derivatives simultaneously activate multiple pathways in LNCaP

cells, including ablation of androgen-responsiveness and down-regulation of

caveolin-1. Both of these responses are associated with activation of

proapoptotic pathways in this cell line.

 

PMID: 17093136 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

   

Cancer Res. 2006 May 1;66(9):4952-60.

 

Gene expression profiling revealed survivin as a target of

3,3'-diindolylmethane-induced cell growth inhibition and apoptosis in breast

cancer cells.

 

Rahman KW, Li Y, Wang Z, Sarkar SH, Sarkar FH.

 

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

of Medicine, Detroit, Michigan 48201, USA.

 

The phytochemical indole-3-carbinol (I3C), found in cruciferous vegetables, and

its major acid-catalyzed reaction product 3,3'-diindolylmethane (DIM) showed

anticancer activity mediated by its pleiotropic effects on cell cycle

progression, apoptosis, carcinogen bioactivation, and DNA repair. To further

elucidate the molecular mechanism(s) by which 3,3'-diindolylmethane exerts its

effects on breast cancer cells, we have used microarray gene expression profiling

analysis. We found a total of 1,238 genes altered in

3,3'-diindolylmethane-treated cells, among which 550 genes were down-regulated

and 688 genes were up-regulated. Clustering analysis showed significant

alterations in some genes that are critically involved in the regulation of cell

growth, cell cycle, apoptosis, and signal transduction, including down-regulation

of survivin. Previous studies have shown that antiapoptotic protein survivin is

overexpressed in many human cancers, including breast cancer. However, very

little or no information is available regarding the consequence of

down-regulation of survivin for cancer therapy. We, therefore, hypothesized that

down-regulation of survivin as observed by 3,3'-diindolylmethane could be an

important approach for the treatment of breast cancer. We have tested our

hypothesis using multiple molecular approaches and found that

3,3'-diindolylmethane inhibited cell growth and induced apoptosis in MDA-MB-231

breast cancer cells by down-regulating survivin, Bcl-2, and cdc25A expression and

also caused up-regulation of p21(WAF1) expression, which could be responsible for

cell cycle arrest. Down-regulation of survivin by small interfering RNA before

3,3'-diindolylmethane treatment resulted in enhanced cell growth inhibition and

apoptosis, whereas overexpression of survivin by cDNA transfection abrogated

3,3'-diindolylmethane-induced cell growth inhibition and apoptosis. These results

suggest that targeting survivin by 3,3'-diindolylmethane could be a new and novel

approach for the prevention and/or treatment of breast cancer.

 

PMID: 16651453 [PubMed - indexed for MEDLINE]

 

Cancer Res. 2006 May 1;66(9):4880-7.

 

3,3'-Diindolylmethane (DIM) is a novel mitochondrial H(+)-ATP synthase inhibitor that

can induce p21(Cip1/Waf1) expression by induction of oxidative stress in human

breast cancer cells.

 

Gong Y, Sohn H, Xue L, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, California 94720, USA.

 

Epidemiologic evidence suggests that high dietary intake of Brassica vegetables,

such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis

in multiple organs. 3,3'-Diindolylmethane, one of the active products derived

from Brassica vegetables, is a promising antitumor agent. Previous studies in our

laboratory showed that 3,3'-diindolylmethane induced a G(1) cell cycle arrest in

human breast cancer MCF-7 cells by a mechanism that included increased expression

of p21. In the present study, the upstream events leading to p21 overexpression

were further investigated. We show for the first time that 3,3'-diindolylmethane

is a strong mitochondrial H(+)-ATPase inhibitor (IC(50) approximately 20

micromol/L). 3,3'-Diindolylmethane treatment induced hyperpolarization of

mitochondrial inner membrane, decreased cellular ATP level, and significantly

stimulated mitochondrial reactive oxygen species (ROS) production. ROS

production, in turn, led to the activation of stress-activated pathways involving

p38 and c-Jun NH(2)-terminal kinase. Using specific kinase inhibitors (SB203580

and SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal kinase

(JNK) pathways in 3,3'-diindolylmethane-induced p21 mRNA transcription. In

addition, antioxidants significantly attenuated 3,3'-diindolylmethane-induced

activation of p38 and JNK and induction of p21, indicating that oxidative stress

is the major trigger of these events. To further support the role of ROS in

3,3'-diindolylmethane-induced p21 overexpression, we showed that

3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial

respiratory chain deficient rho(0) MCF-7 cells, in which 3,3'-diindolylmethane

did not stimulate ROS production. Thus, we have established the critical role of

enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21

up-regulation in human breast cancer cells.

 

PMID: 16651444 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

  

Mutat Res. 2006 Jul 25;599(1-2):76-87. Epub 2006 Feb 24.

 

Comparison of growth inhibition profiles and mechanisms of apoptosis induction in

human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae.

 

Pappa G, Lichtenberg M, Iori R, Barillari J, Bartsch H, Gerhäuser C.

 

Division of Toxicology and Cancer Risk Factors, German Cancer Research Center,

DKFZ, 69120 Heidelberg, Germany.

 

The isothiocyanates sulforaphane and PEITC (beta-phenethyl isothiocyanate) as

well as the indoles indole-3-carbinol and its condensation product

3,3'-diindolylmethane are known to inhibit cancer cell proliferation and induce

apoptosis. In this study, we compared the cell growth inhibitory potential of the

four compounds on the p53 wild type human colon cancer cell line 40-16 (p53(+/+))

and its p53 knockout derivative 379.2 (p53(-/-)) (both derived from HCT116).

Using sulforhodamin B staining to assess cell proliferation, we found that the

isothiocyanates were strongly cytotoxic, whereas the indoles inhibited cell

growth in a cytostatic manner. Half-maximal inhibitory concentrations of all four

compounds in both cell lines ranged from 5-15 microM after 24, 48 and 72 h of

treatment. Apoptosis induction was analyzed by immunoblotting of

poly(ADP-ribose)polymerase (PARP). Treatment with sulforaphane (15 microM), PEITC

(10 microM), indole-3-carbinol (10 microM) and 3,3'-diindolylmethane (10 microM)

induced PARP cleavage after 24 and 48 h in both 40-16 and the 379.2 cell lines,

suggestive of a p53-independent mechanism of apoptosis induction. In cultured

40-16 cells, activation of caspase-9 and -7 detected by Western blotting

indicated involvement of the mitochondrial pathway. We detected time- and

concentration-dependent changes in protein expression of anti-apoptotic Bcl-x(L)

as well as pro-apoptotic Bax and Bak proteins. Of note is that for sulforaphane

only, ratios of pro- to anti-apoptotic Bcl-2 family protein levels directly

correlated with apoptosis induction measured by PARP cleavage. Taken together, we

demonstrated that the glucosinolate breakdown products investigated in this study

have distinct profiles of cell growth inhibition, potential to induce

p53-independent apoptosis and to modulate Bcl-2 family protein expression in

human colon cancer cell lines.

 

PMID: 16500682 [PubMed - indexed for MEDLINE]

   

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

Mol Cancer Ther. 2005 Dec;4(12):1972-81.

 

Down-regulation of c-FLIP contributes to the sensitization effect of

3,3'-diindolylmethane (DIM) on TRAIL-induced apoptosis in cancer cells.

 

Zhang S, Shen HM, Ong CN.

 

Department of Community, Occupational and Family Medicine, Faculty of Medicine,

National University of Singapore, 16 Medical Drive, Singapore 117597, Singapore.

 

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of

the tumor necrosis factor superfamily, which has been shown to preferentially

induce apoptosis in cancer cells without adverse effects on normal cells.

However, there are still some cancer cells, especially those with high

malignancy, resistant to TRAIL-induced apoptosis, impeding the clinical

anticancer efficiency of TRAIL. In this report, we showed that

3,3'-diindolylmethane, an indole compound derived from cruciferous vegetables, is

capable of overcoming TRAIL resistance by sensitizing TRAIL-induced apoptosis in

human cancer cells. Noncytotoxic concentrations of 3,3'-diindolylmethane

significantly enhanced TRAIL-resistant cancer cells to TRAIL-induced apoptosis

via promoting the caspase cascade, a process independent of nuclear factor-kappaB

activation and cell surface TRAIL receptor expression. In the search of the

molecular mechanisms involved in the sensitization activity of

3,3'-diindolylmethane, we found that combined treatment of 3,3'-diindolylmethane

and TRAIL led to significant down-regulation of the cellular FLICE inhibitory

protein expression (c-FLIP). Furthermore, we provided evidence showing that the

reduced c-FLIP level is predominately mediated by the ubiquitin-proteasome

degradation system. These findings reveal a novel anticancer property of

3,3'-diindolylmethane and suggest that this compound could have potential use in

cancer therapy to overcome TRAIL resistance.

 

PMID: 16373712 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

  

Mol Pharmacol. 2006 Feb;69(2):430-9. Epub 2005 Nov 2.

 

Activation and potentiation of interferon-gamma signaling by

3,3'-diindolylmethane in MCF-7 breast cancer cells.

 

Riby JE, Xue L, Chatterji U, Bjeldanes EL, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, 94720-3104, USA.

 

3,3'-Diindolylmethane (DIM), a natural autolytic product in plants of the

Brassica genus, including broccoli, cauliflower, and Brussels sprouts, exhibits

promising cancer protective activities, especially against mammary neoplasia in

animal models. We observed previously that DIM induced a G(1) cell-cycle arrest

and strong induction of cell-cycle inhibitor p21 expression and promoter activity

in both estrogen-responsive and -independent breast cancer cell lines. We showed

recently that DIM up-regulates the expression of interferon gamma (IFNgamma) in

human MCF-7 breast cancer cells. This novel effect may contribute to the

anticancer effects of DIM because IFNgamma plays an important role in preventing

the development of primary and transplanted tumors. In this study, we observed

that DIM activated the IFNgamma signaling pathway in human breast cancer cells.

DIM activated the expression of the IFNgamma receptor (IFNGR1) and

IFNgamma-responsive genes p56- and p69-oligoadenylate synthase (OAS). In

cotreatments with IFNgamma, DIM produced an additive activation of endogenous

p69-OAS and of an OAS-Luc reporter and a synergistic activation of a GAS-Luc

reporter. DIM synergistically augmented the IFNgamma induced phosphorylation of

signal transducer and activator of transcription factor 1, further evidence of

DIM activation of the IFNgamma pathway. DIM and IFNgamma produced an additive

inhibition of cell proliferation and a synergistic increase in levels of major

histocompatibility complex class-1 (MHC-1) expression, accompanied by increased

levels of mRNAs of MHC-1-associated proteins and transporters. These results

reveal novel immune activating and potentiating activities of DIM in human tumor

cells that may contribute to the established effectiveness of this dietary indole

against various tumors types.

 

PMID: 16267208 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE] 

  

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 [PubMed - indexed for MEDLINE]

  

J Agric Food Chem. 2005 May 18;53(10):3895-901.

 

Effects of Brussels sprout juice on the cell cycle and adhesion of human

colorectal carcinoma cells (HT29) in vitro.

 

Smith TK, Lund EK, Clarke RG, Bennett RN, Johnson IT.

 

Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA,

United Kingdom.

 

Consumption of Brassica vegetables is associated with a reduced risk of cancer of

the alimentary tract in animal models and human populations. We used raw juice

extracted from Brussels sprouts rich in the glucosinolate sinigrin to explore the

effect of naturally occurring glucosinolate breakdown products on cell cycle

progression and apoptosis in human colorectal carcinoma cells (HT29). Juice was

prepared from sprout tissue immediately before use, and the glucosinolate

breakdown products were determined by gas chromatography mass spectrometry and

liquid chromatography mass spectrometry. The cell cycle was analyzed by flow

cytometry on detached and adherent cells, and apoptosis was measured in the

detached population by annexin V staining. Twenty-four hours after challenge with

juice (10 microL/mL), 7-13% of adherent cells had detached from the substratum

but the majority (82%) of these cells had not entered apoptosis, whereas only 33%

of detached control cells were not apoptotic (p < 0.05). The main glucosinolate

breakdown products were as follows: the sinigrin breakdown product,

1-cyano-2,3-epithiopropane (ca. 38 mM); the gluconapin hydrolysis product,

3-butenyl isothiocyanate (ca. 2.2.mM); the glucobrassicin metabolite, ascorbigen

(ca. 8 mM); and low concentrations of other indole glucosinolate-derived

hydrolysis products such as neoascorbigen and 3,3'-diindolylmethane. A variety of

biologically active glucosinolate breakdown products are released by mechanical

disruption of raw Brussels sprout tissue, but contrary to previous assumptions,

allyl isothiocyanate is not the main compound responsible for the inhibition of

cell proliferation.

 

PMID: 15884814 [PubMed - indexed for MEDLINE]

   

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 [PubMed - indexed for MEDLINE]

  

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 [PubMed - indexed for MEDLINE]

 

Biochem Biophys Res Commun. 2005 Mar 4;328(1):63-9.

 

Indole-3-carbinol and 3,3'-diindolylmethane induce expression of NAG-1 in a

p53-independent manner.

 

Lee SH, Kim JS, Yamaguchi K, Eling TE, Baek SJ.

 

Laboratory of Environmental Carcinogenesis, Department of Pathobiology, College

of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.

 

Indole-3-carbinol (I3C), present in cruciferous vegetables, and its major in vivo

product 3,3'-diindolylmethane (DIM), have been reported to suppress cancer

development. However, the responsible molecular mechanisms are not fully

understood. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a

TGF-beta superfamily gene associated with pro-apoptotic and anti-tumorigenic

activities. The present study was performed to investigate whether I3C and DIM

influence NAG-1 expression and to provide the potential molecular mechanism of

their effects on anti-tumorigenesis. The I3C repressed cell proliferation and

induced NAG-1 expression in a concentration-dependent manner. In addition, DIM

increased the expression of NAG-1 as well as activating transcription factor 3

(ATF3), and the induction of ATF3 was earlier than that of NAG-1. The DIM

treatment increased luciferase activity of NAG-1 in HCT-116 cells transfected

with NAG-1 promoter construct. The results suggest that I3C represses cell

proliferation through up-regulation of NAG-1 and that ATF3 may play a pivotal

role in DIM-induced NAG-1 expression in human colorectal cancer cells.

Furthermore, the mixture of I3C with resveratrol enhances NAG-1 expression,

suggesting the synergistic effect of these two unrelated compounds on NAG-1

expression.

 

PMID: 15670751 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

Carcinogenesis. 2005 Apr;26(4):771-8. Epub 2005 Jan 20.

 

3,3'-Diindolylmethane inhibits angiogenesis and the growth of transplantable

human breast carcinoma in athymic mice.

 

Chang X, Tou JC, Hong C, Kim HA, Riby JE, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720, USA.

 

Studies have linked the consumption of broccoli and other cruciferous vegetables

to a reduced risk of breast cancer. The phytochemical indole-3-carbinol (I3C),

present in cruciferous vegetables, and its major acid-catalyzed reaction product

3,3'-diindolylmethane (DIM) have bioactivities relevant to the inhibition of

carcinogenesis. In this study, the effect of DIM on angiogenesis and

tumorigenesis in a rodent model was investigated. We found that DIM produced a

concentration-dependent decrease in proliferation, migration, invasion and

capillary tube formation of cultured human umbilical vein endothelial cells

(HUVECs). Consistent with its antiproliferative effect, which was significant at

only 5 microM DIM, this indole caused a G1 cell cycle arrest in actively

proliferating HUVECs. Furthermore, DIM downregulated the expression of

cyclin-dependent kinases 2 and 6 (CDK2, CDK6), and upregulated the expression of

CDK inhibitor, p27(Kip1), in HUVECs. We observed further in a complementary in

vivo Matrigel plug angiogenesis assay that, compared with vehicle control,

neovascularization was inhibited up to 76% following the administration of 5

mg/kg DIM to female C57BL/6 mice. Finally, this dose of DIM also inhibited the

growth of human MCF-7 cell tumor xenografts by up to 64% in female athymic

(nu/nu) mice, compared with the vehicle control. This is the first study to show

that DIM can strongly inhibit the development of human breast tumor in a

xenograft model and to provide evidence for the antiangiogenic properties of this

dietary indole.

 

PMID: 15661811 [PubMed - indexed for MEDLINE]

 

Nutr Cancer. 2004;50(2):161-7.

 

Pilot study: effect of 3,3'-diindolylmethane supplements on urinary hormone

metabolites in postmenopausal women with a history of early-stage breast cancer.

 

Dalessandri KM, Firestone GL, Fitch MD, Bradlow HL, Bjeldanes LF.

 

Department of Molecular and Cell Biology, University of California, Berkeley,

94720-3200, USA.

 

Dietary indoles, present in Brassica plants such as cabbage, broccoli, and

Brussels sprouts, have been shown to provide potential protection against

hormone-dependent cancers. 3,3'-Diindolylmethane (DIM) is under study as one of

the main protective indole metabolites. Postmenopausal women aged 50-70 yr from

Marin County, California, with a history of early-stage breast cancer, were

screened for interest and eligibility in this pilot study on the effect of DIM supplements

on urinary hormone metabolites. The treatment group received daily DIM (108 mg

DIM/day) supplements for 30 days, and the control group received a placebo capsule

daily for 30 days. Urinary metabolite analysis included 2-hydroxyestrone (2-OHE1),

16-alpha hydroxyestrone (16alpha-OHE1), DIM, estrone (El), estradiol(E2), estriol (E3),

6beta-hydroxycortisol (6beta-OHC), and cortisol in the first morning urine sample

before intervention and 31 days after intervention. Nineteen women completed the

study,for a total of 10 in the treatment group and 9 in the placebo group.

DIM-treated subjects, relative to placebo, showed a significant increase in

levels of2-OHE1 (P=0. 020), DIM (P =0. 045), and cortisol (P = 0.039), and a

nonsignificant increase of 47% in the 2-OHE1/16alpha-OHE1 ratio from 1.46 to 2.14

(P=0.059). In this pilot study, DIM increased the 2-hydroxylation of estrogen

urinary metabolites.

 

PMID: 15623462 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

  

Br J Cancer. 2004 Oct 4;91(7):1358-63.

 

Induction of apoptosis in human prostate cancer cell line, PC3, by

3,3'-diindolylmethane through the mitochondrial pathway.

 

Nachshon-Kedmi M, Yannai S, Fares FA.

 

Faculty of Food Engineering and Biotechnology, Technion-Israel Institute of

Technology, Haifa 32000, Israel.

 

Prostate cancer is the most common malignancy and the second leading cause of

male death in Western countries. Prostate cancer mortality results from

metastases to the bones and lymph nodes and progression from androgen-dependent

to androgen-independent disease. Although androgen ablation was found to be

effective in treating androgen-dependent prostate cancer, no effective

life-prolonging therapy is available for androgen-independent cancer.

Epidemiological studies have shown a strong correlation between consumption of

cruciferous vegetables and a lower risk of prostate cancer. These vegetables

contain glucosinolates, which during metabolism give rise to several breakdown

products, mainly indole-3-carbinol (I3C), which may be condensed to polymeric

products, especially 3,3'-diindolylmethane (DIM). It was previously shown that

these indole derivatives have significant inhibitory effects in several human

cancer cell lines, which are exerted through induction of apoptosis. We have

previously reported that I3C and DIM induce apoptosis in prostate cancer cell

lines through p53-, bax-, bcl-2- and fasL-independent pathways. The objective of

this study was examination of the apoptotic pathways that may be involved in the

effect of DIM in the androgen-independent prostate cancer cell line, PC3, in

vitro. Our results suggest that DIM induces apoptosis in PC3 cells, through the

mitochondrial pathway, which involves the translocation of cytochrome c from the

mitochondria to the cytosol and the activation of initiator caspase, 9, and

effector caspases, 3 and 6, leading to poly ADP-ribose polymerase (PARP) cleavage

and induction of apoptosis. Our findings may lead to the development of new

therapeutic strategies for the treatment of androgen-independent prostate cancer.

 

PMID: 15328526 [PubMed - indexed for MEDLINE]

 

Prostate. 2004 Oct 1;61(2):153-60.

 

Therapeutic activity of 3,3'-diindolylmethane on prostate cancer in an in vivo

model.

 

Nachshon-Kedmi M, Fares FA, Yannai S.

 

Faculty of Food Engineering and Biotechnology, Technion-Israel Institute of

Technology, Haifa, Israel.

 

BACKGROUND: Prostate cancer (PC) is the second leading cancer-related death in

men in Western countries. Hence, efficient anti-carcinogenic and therapeutic

compounds against PC are badly needed. We have previously shown that

3,3'-diindolylmethane (DIM) has a suppressive effect on the growth of human

breast and PC cell lines. The objective of this study was examination of the

potential therapeutic effects of DIM in an in vivo model. METHODS: TRAMP-C2, a

mouse PC cell line, was injected into the flank of male C57BL/6 mice. When tumors

appeared, mice were injected intraperitoneally with either corn oil (vehicle) or

DIM (2.5, 5, or 10 mg per kg body weight) 3-times a week, for 3 weeks, and tumor

volumes were measured bi-weekly with calibermeters. Later, the tumors were

removed, their final weights and volumes were measured, and tumor sections were

tested for histological studies. RESULTS: DIM had a significant inhibitory

effect, caused by diminished tumor growth. Histological examination of tumors

from treated groups revealed apoptosis and decreased cell proliferation, compared

with the controls. DIM didn't affect body weights or kidney and liver

functioning. CONCLUSIONS: The inhibitory action of DIM on tumor growth was

demonstrated in vivo. Hence, this compound at the concentrations tested may offer

an effective and non toxic therapeutic means against tumor growth in rodents, and

may serve as a potential natural anti-carconigenic compound in humans. Copyright

2004 Wiley-Liss, Inc

 

PMID: 15305338 [PubMed - indexed for MEDLINE]

 

Cell Stress Chaperones. 2004 Mar;9(1):76-87.

 

Endoplasmic reticulum stress as a correlate of cytotoxicity in human tumor cells

exposed to diindolylmethane in vitro.

 

Sun S, Han J, Ralph WM Jr, Chandrasekaran A, Liu K, Auborn KJ, Carter TH.

 

North Shore-Long Island Jewish Research Institute, Manhasset, NY 11030, USA.

 

The dietary phytochemical indole-3-carbinol (I3C) protects against cervical

cancer in animal model studies and in human clinical trials. I3C and its

physiologic condensation product diindolylmethane (DIM) also induce apoptosis of

tumor cells in vitro and in vivo, suggesting that these phytochemicals might be

useful as therapeutic agents as well as for cancer prevention. Deoxyribonucleic

acid microarray studies on transformed keratinocytes and tumor cell lines exposed

to pharmacologic concentrations of DIM in vitro are consistent with a cellular

response to nutritional deprivation or disruptions in protein homeostasis such as

endoplasmic reticulum (ER) stress. In this report we investigate whether specific

stress response pathways are activated in tumor cells exposed to DIM and whether

the ER stress response might contribute to DIM's cytotoxicity. Induction of the

stress response genes GADD153, GADD34 and GADD45A, XBP-1, GRP78,

GRP94, and asparagine synthase was documented by Western blot and real-time reverse

transcriptase-polymerase chain reaction in C33A cervical cancer cells, and

induction of a subset of these was also observed in cancer cell lines from breast

(MCF-7) and prostate (DU145). The results are consistent with activation of more

than 1 stress response pathway in C33A cells exposed to 75 microM DIM.

Phosphorylation elF2alpha was rapidly and transiently increased, followed by

elevated levels of ATF4 protein. Activation of IRE1alpha was indicated by a rapid

increase in the stress-specific spliced form of XBP-1 messenger ribonucleic acid

and a rapid and persistent phosphorylation of JNK1 and JNK2. Transcriptional

activation dependent on an ATF6-XBP-1 binding site was detected by transient

expression in MCF-7, C33A, and a transformed epithelial cell line (HaCaT);

induction of the GADD153 (CHOP) promoter was also confirmed by transient

expression. Cleavage of caspase 12 was observed in both DIM-treated and untreated

C33A cells but did not correlate with cytotoxicity, whereas caspase 7 was cleaved

at later times, coinciding with the onset of apoptosis. The results support the

hypothesis that cytotoxic concentrations of DIM can activate cellular stress

response pathways in vitro, including the ER stress response. Conversely, DIM was

especially cytotoxic to stressed cells. Thapsigargin and tunicamycin, agents that

induce ER stress, sensitized cells to the cytotoxic effects of DIM to differing

degrees; nutrient limitation had a similar, but even more pronounced, effect.

Because DIM toxicity in vitro is enhanced in cells undergoing nutritional

deprivation and ER stress, it is possible that stressed cells in vivo, such as

those within developing solid tumors, also have increased sensitivity to killing

by DIM.

 

PMID: 15270080 [PubMed - indexed for MEDLINE]

 

Cancer Detect Prev. 2004;28(1):72-9.

 

Differences in the hepatic P450-dependent metabolism of estrogen and tamoxifen in

response to treatment of rats with 3,3'-diindolylmethane and its parent compound

indole-3-carbinol.

 

Parkin DR, Malejka-Giganti D.

 

Veterans Affairs Medical Center, Minneapolis, MN 55417, USA.

 

Indole-3-carbinol (I3C), present in cruciferous vegetables, and its major in vivo

product 3,3'-diindolylmethane (DIM), have been reported to suppress

estrogen-responsive cancers. This effect may be mediated through the modification

of cytochrome P450 (CYP) complement and activities leading to estrogen

detoxification. We examined the effects of a 4-day treatment of female

Sprague-Dawley rats with DIM at 8.4 and 42 mg/kg body weight (bwt), on the

hepatic CYP protein level, CYP1A1, 1A2, 2B1/2 and 3A1/2 probe activities and

CYP-dependent metabolism of 17beta-estradiol (E2) and estrone (E1). At 42 mg/kg

bwt, DIM effected a small increase (2.8-fold) in CYP1A1 activity, and at both

dose levels it reduced CYP3A1/2 activity by approximately 40%. At the higher dose

level, DIM decreased the rates of oxidation of E2 to 4-OH-E2, 4-OH-E1,

6alpha-OH-E2 and 6(alpha+beta)-OH-E1 by 39, 44, 71 and 60%, respectively, and E1

to 6(alpha+beta)-OH-E1 by 39%. These effects were considerably different from

those of I3C reported by us previously. We also examined the effects of DIM and

I3C on the hepatic microsomal metabolism of tamoxifen (TAM). Whereas metabolism

of TAM was unaffected by DIM, formation of N-desmethyl-TAM (and its presumed

derivative) was increased approximately 3-fold by I3C at 250 mg/kg bwt. Since

N-desmethyl-TAM is transformed to a genotoxic metabolite, dietary exposure to I3C

may enhance hepatic carcinogenicity of TAM in the rat. The differences between

I3C and DIM in CYP-mediated activities and metabolism indicate that DIM is not a

proximate intermediate in the mechanism of action of I3C.

 

PMID: 15041081 [PubMed - indexed for MEDLINE]

  

Mol Endocrinol. 2004 Feb;18(2):291-302. Epub 2003 Nov 26.

 

Potent ligand-independent estrogen receptor activation by 3,3'-diindolylmethane

is mediated by cross talk between the protein kinase A and mitogen-activated

protein kinase signaling pathways.

 

Leong H, Riby JE, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, California 94720, USA.

 

We investigated the mechanism of ligand-independent activation of the estrogen

receptor (ER) by 3,3'-diindolylmethane (DIM), a promising anticancer agent

derived from vegetables of the Brassica genus, in Ishikawa and HEC-1B human

endometrial cancer cells. DIM stimulated the activity of an ER-responsive

reporter by over 40-fold, equivalent to the maximum induction produced by

estradiol (E2), whereas cotreatment of cells with the ER antagonist, ICI-182,780

(ICI), abolished the stimulatory effect of DIM. DIM also induced the expressions

of the endogenous genes, TGF-alpha, alkaline phosphatase, and progesterone

receptor similar to levels induced by E2. Induction of gene expression by DIM was

inhibited by the protein synthesis inhibitor, cycloheximide. In addition,

cotreatment of cells with the protein kinase A (PKA) inhibitor, H89, or the MAPK

inhibitor, PD98059, reduced DIM activation of the ER by 75% and 50%,

respectively. Simultaneous treatment of cells with both inhibitors completely

abolished the effect of DIM. DIM stimulated MAPK activity and induced

phosphorylation of the endogenous PKA target, cAMP response element binding

protein (CREB), in a PKA-dependent manner. Expression of MCREB, a

nonphosphorylatable CREB mutant, partially abolished activation of the ER by DIM.

These results demonstrate that DIM is a mechanistically novel activator of the ER

that requires PKA-dependent phosphorylation of CREB.

 

PMID: 14645498 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

Mol Cell Biol. 2003 Nov;23(21):7920-5.

 

Agonist and chemopreventative ligands induce differential transcriptional

cofactor recruitment by aryl hydrocarbon receptor.

 

Hestermann EV, Brown M.

 

Department of Molecular Oncology, Dana-Farber Cancer Institute, Brigham and

Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

 

Aryl hydrocarbon receptor (AHR) is a transcription factor whose activity is

regulated by environmental agents, including several carcinogenic agonists. We

measured recruitment of AHR and associated proteins to the human cytochrome

P4501A1 gene promoter in vivo. Upon treatment with the agonist

beta-naphthoflavone, AHR is rapidly associated with the promoter and recruits the

three members of the p160 family of coactivators as well as the p300 histone

acetyltransferase, leading to recruitment of RNA polymerase II (Pol II) and

induction of gene transcription. AHR, coactivators, and Pol II cycle on and off

the promoter, with a period of approximately 60 min. In contrast, the

chemopreventative AHR ligand 3,3'-diindolylmethane promotes AHR nuclear

translocation and p160 coactivator recruitment but, remarkably, fails to recruit

Pol II or cause histone acetylation. This novel mechanism of receptor antagonism

may account for the antitumor properties of chemopreventative compounds targeting

the AHR.

 

PMID: 14560034 [PubMed - indexed for MEDLINE]

  

J Nutr. 2003 Jul;133(7 Suppl):2470S-2475S.

 

Indole-3-carbinol and 3-3'-diindolylmethane antiproliferative signaling pathways

control cell-cycle gene transcription in human breast cancer cells by regulating

promoter-Sp1 transcription factor interactions.

 

Firestone GL, Bjeldanes LF.

 

Department of Molecular and Cell Biology and Cancer Research Laboratory,

University of California at Berkeley, Berkeley, CA 94720-3200, USA.

 

Indole-3-carbinol (I3C), a compound that occurs naturally in Brassica vegetables

such as cabbage and broccoli, can induce a G1 cell-cycle arrest of human MCF-7

breast cancer cells that is accompanied by the selective inhibition of

cyclin-dependent kinase 6 (Cdk6) expression and stimulation of p21(Waf1/Cip1)

gene expression. Construction and transfection of a series of promoter-reporter

plasmids demonstrate that the indole-regulated changes in Cdk6 and p21(Waf1/Cip1)

levels are due to specific effects on their corresponding promoters. Mutagenic

analysis reveals that I3C signaling targets a composite transcriptional element

in the Cdk6 promoter that requires both Sp1 and Ets transcription factors for

transactivation function. Analysis of protein-DNA complexes formed with nuclear

proteins isolated from I3C-treated and -untreated cells demonstrates that the Sp1

DNA element in the Cdk6 promoter interacts with an I3C-inhibited protein-protein

complex that contains the Sp1 transcription factor. In indole-treated cells, a

fraction of [(3)H]I3C was converted into its natural diindole product

(3)H-labeled 3-3'-diindolylmethane ([(3)H]DIM), which accumulates in the nucleus;

this suggests that DIM may have a role in the transcriptional activities of I3C.

Mutagenic analysis of the p21(Waf1/Cip1) promoter reveals that in transfected

breast cancer cells, DIM (as well as I3C) stimulates p21(Waf1/Cip1) transcription

through an indole-responsive region of the promoter that contains multiple Sp1

consensus sequences. Furthermore, DIM treatment regulates the presence of a

nuclear Sp1 DNA-binding activity. Our results demonstrate that both the Cdk6 and

p21(Waf1/Cip1) promoters are newly defined downstream targets of the

indole-signaling pathway, and that the observed transcriptional effects are due

to a combination of the cellular activities of I3C and DIM.

 

PMID: 12840223 [PubMed - indexed for MEDLINE]

 

Food Chem Toxicol. 2003 Jun;41(6):745-52.

 

Indole-3-carbinol and 3,3'-diindolylmethane induce apoptosis in human prostate

cancer cells.

 

Nachshon-Kedmi M, Yannai S, Haj A, Fares FA.

 

Faculty of Food Engineering and Biotechnology, Technion- Israel Institute of

Technology, 32000, Haifa, Israel.

 

Cruciferous vegetables contain glucobrassicin which, during metabolism, yields

indole-3-carbinol (I3C). In a low pH environment I3C is converted into polymeric

products, among which 3,3'-diindolylmethane (DIM) is the main one. The apoptotic

effects of I3C and DIM were exhibited in human breast cancer cells. The

objectives of this study were: (a) examination of the potential effects of I3C

and DIM on the proliferation and induction of apoptosis in human prostate cancer

cell lines with different p53 status; (b) to try to characterise the mechanism(s)

involved in these effects. Our results indicate that both indole derivatives

suppress the growth of these cells in a dose- and time-dependent manner, by

inducing apoptosis. It appears that these indolic compounds may offer effective

means against prostate cancer. Induction of apoptosis was p53-independent.

Moreover, the indole derivatives employed did not affect the levels of bcl-2, bax

and fasL.

 

PMID: 12738179 [PubMed - indexed for MEDLINE]

  

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 [PubMed - indexed for MEDLINE]

 

J Biol Chem. 2003 Jun 6;278(23):21136-45. Epub 2003 Mar 27.

 

Plant-derived 3,3'-Diindolylmethane is a strong androgen antagonist in human

prostate cancer cells.

 

Le HT, Schaldach CM, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, The University of California,

Berkeley, California 94720-3104, USA.

 

3,3'-Diindolylmethane (DIM) is a major digestive product of indole-3-carbinol, a

potential anticancer component of cruciferous vegetables. Our results indicate

that DIM exhibits potent antiproliferative and antiandrogenic properties in

androgen-dependent human prostate cancer cells. DIM suppresses cell proliferation

of LNCaP cells and inhibits dihydrotestosterone (DHT) stimulation of DNA

synthesis. These activities were not produced in androgen-independent PC-3 cells.

Moreover, DIM inhibited endogenous PSA transcription and reduced intracellular

and secreted PSA protein levels induced by DHT in LNCaP cells. Also, DIM

inhibited, in a concentration-dependent manner, the DHT-induced expression of a

prostate-specific antigen promoter-regulated reporter gene construct in

transiently transfected LNCaP cells. Similar effects of DIM were observed in PC-3

cells only when these cells were co-transfected with a wild-type androgen

receptor expression plasmid. Using fluorescence imaging with green fluorescent

protein androgen receptor and Western blot analysis, we demonstrated that DIM

inhibited androgen-induced androgen receptor (AR) translocation into the nucleus.

Results of receptor binding assays indicated further that DIM is a strong

competitive inhibitor of DHT binding to the AR. Results of structural modeling

studies showed that DIM is remarkably similar in conformational geometry and

surface charge distribution to an established synthetic AR antagonist, although

the atomic compositions of the two substances are quite different. Taken together

with our published reports of the estrogen agonist activities of DIM, the present

results establish DIM as a unique bifunctional hormone disrupter. To our

knowledge, DIM is the first example of a pure androgen receptor antagonist from

plants.

 

PMID: 12665522 [PubMed - indexed for MEDLINE]

   

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 [PubMed - indexed for MEDLINE]

 

Nutr Cancer. 2001;41(1-2):57-63.

 

Quantitative determination of 3,3'-diindolylmethane in urine of individuals

receiving indole-3-carbinol.

 

Sepkovic DW, Bradlow HL, Bell M.

 

Institute for Biomedical Research, Hackensack University Medical Center,

Hackensack, NJ 07601, USA.

 

Indole-3-carbinol (I3C) or, more correctly, its acid condensation products act as

chemoprotective agents via several mechanisms. It induces the expression of

cytochrome P-450 1A1, which shifts the estrogen metabolic pathway in favor of C-2

hydroxylation and away from the formation of 16 alpha-hydroxyestrone, a suspected

endogenous carcinogen. Increased 16 alpha-hydroxylation of estrogen is associated

with greater risk of cancer of the cervix, breast, endometrium, and larynx. The

production of 4-hydroxyestrone is also inhibited by I3C. I3C can induce a G1 cell

cycle arrest in human MCF-7 breast cancer cells. It can suppress aberrant crypt

foci. I3C significantly inhibits the cell adhesion, spreading, and invasion

associated with an upregulation of PTEN (a tumor suppressor gene) and E-cadherin

(a regulator of cell-cell adhesion) expression in T47-D human breast cancer

cells. Thus I3C exhibits anticancer activities by suppressing breast tumor cell

growth and metastatic spread. A gas chromatography-mass spectrometry method for

the quantitation of diindolylmethane, the principal acid condensation product of

I3C, has been developed for use in determining compliance in subjects who have

been treated with I3C. The method utilizes a 1-ml urine sample. We have used this

method to correlate I3C ingestion with regression of cervical intraepithelial

neoplasia in a population of women at risk for cervical cancer. The assay

provides an objective marker of consumption using a noninvasive biological fluid

and illustrates that diindolylmethane may be used as a marker of compliance in

I3C dietary intervention studies.

 

PMID: 12094629 [PubMed - indexed for MEDLINE]

 

Antivir Ther. 2002 Mar;7(1):1-9.

 

Therapy for recurrent respiratory papillomatosis.

 

Auborn KJ.

 

Department of Otolaryngology, Long Island Jewish Medical Center, Albert Einstein

College of Medicine, New Hyde Park, NY, USA.

 

Human papillomaviruses types 6 or 11 are aetiological agents of recurrent

respiratory papillomatosis, a disease characterized by benign exophytic tumours

usually on the vocal cords. Surgery debulks the tumours, but these growths

generally recur at regular intervals. Adjunct medical treatments, aimed at

containing the virus and growth of tumours, include indole-3-carbinol or its

dimer diindolylmethane, interferon, photodynamic therapy and others. Preventive

and therapeutic vaccines hold promise for eliminating the virus.

 

PMID: 12008782 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

   

Carcinogenesis. 2001 Nov;22(11):1809-17.

 

Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells

result from an estrogen receptor-mediated increase in transforming growth

factor-alpha expression.

 

Leong H, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of

California-Berkeley, Berkeley, CA 94720, USA.

 

3,3'-Diindolylmethane (DIM), a major in vivo product of indole-3-carbinol (I3C),

is a promising anticancer agent derived from vegetables of the Brassica genus

including broccoli, Brussels sprouts and cabbage. We report here that DIM has a

potent cytostatic effect in cultured human Ishikawa endometrial cancer cells. A

combination of northern blot and quantitative PCR analyses revealed that DIM

induced the level of TGF-alpha transcripts by approximately 4-fold within 24 h of

indole treatment. DIM also induced a 4-fold increase in the activity of the

estrogen response marker, alkaline phosphatase (AP). Co-treatment of cells with

the estrogen receptor (ER) antagonist ICI, or with the inhibitor of PKA-mediated

activation of the ER, H89, ablated the DIM induction of both TGF-alpha expression

and AP activity. Furthermore, DIM increased the maximum stimulatory effect of

estrogen on TGF-alpha expression. Co-treatment with the protein synthesis

inhibitor, cycloheximide, abolished the inductive effects of DIM, indicating

differences in the mechanistic requirements of DIM and estrogen. DIM treatment

also stimulated levels of secreted TGF-alpha protein by >10-fold. The ectopic

addition of TGF-alpha inhibited the growth of Ishikawa cells, whereas incubation

with a TGF-alpha antibody partially reversed the growth inhibitory effects of

DIM. Taken together, these results extend our previous findings of the ligand

independent estrogen receptor agonist activity of DIM, and uncover an essential

role for the stimulation in TGF-alpha expression and the TGF-alpha activated

signal transduction pathway in the potent cytostatic effects of DIM in

endometrial cancer cells.

 

PMID: 11698343 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

Cancer Lett. 2000 Apr 14;151(2):169-79.

 

Inhibition of carcinogen-induced rat mammary tumor growth and other

estrogen-dependent responses by symmetrical dihalo-substituted analogs of

diindolylmethane.

 

McDougal A, Sethi Gupta M, Ramamoorthy K, Sun G, Safe SH.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University,

College Station, TX 77843-4466, USA.

 

90%) by the haloDIMs at concentrations of 5 or 10 microM, and only 4,

4'-dichloroDIM alone increased cell proliferation. With the exception of

5,5'-difluoroDIM, the remaining compounds also inhibited E2-induced growth of

MCF-7 human breast cancer cells. DihaloDIMs (100 mg/kg/dayx3) were not estrogenic

in the immature female B6C3F1 mouse uterus; however, in animals co-treated with

E2 (0.02 microg/mouse), 5,5'-dichloro- and 6,6'-dichloroDIM inhibited uterine

progesterone receptor (PR) binding and uterine peroxidase activity, whereas

5,5'-dichloro- and 5,5'-dichloro-2,2'-dimethylDIM inhibited only the latter

response. The antitumorigenic activities of the dihaloDIMs were determined by

their inhibition of carcinogen-induced mammary tumor growth in female

Sprague-Dawley rats. 4,4'-Dichloro-, 5,5'-dibromo- and 6,6'-dichloroDIM,

significantly inhibited mammary tumor growth at doses of 1 mg/kg every second

day, and no significant changes in organ weights or liver and kidney

histopathology were observed. These three compounds were more active than DIM in

the same in vivo assay.

 

PMID: 10738111 [PubMed - indexed for MEDLINE]

   

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

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 [PubMed - indexed for MEDLINE]

 

Biochem Pharmacol. 1999 Sep 1;58(5):825-34.

 

Cytostatic and antiestrogenic effects of

2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major in vivo product of dietary

indole-3-carbinol.

 

Chang YC, Riby J, Chang GH, Peng BC, Firestone G, Bjeldanes LF.

 

Division of Nutritional Sciences and Toxicology, University of California,

Berkeley 94720, USA.

 

Under acidic conditions, indole-3-carbinol (13C) is converted to a series of

oligomeric products thought to be responsible for the biological effects of

dietary 13C. Chromatographic separation of the crude acid mixture of 13C, guided

by cell proliferation assay in human MCF-7 cells, resulted in the isolation of

2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr-1) as a major antiproliferative

component. LTr-1 inhibited the growth of both estrogen-dependent (MCF-7) and

-independent (MDA-MB-231) breast cancer cells by approximately 60% at a

non-lethal concentration of 25 microM. LTr-1 had no apparent effect on the

proliferation of MCF-7 cells in the absence of estrogen. LTr-1 was a weak ligand

for the estrogen receptor (ER) (IC50 70 microM) and efficiently inhibited the

estradiol (E2)-induced binding of the ER to its cognate DNA responsive element.

The antagonist effects of LTr-1 also were exhibited in assays of endogenous pS2

gene expression and in cells transiently transfected with an estrogen-responsive

reporter construct (pERE-vit-CAT). LTr-1 activated both binding of the aryl

hydrocarbon (Ah) receptor to its cognate DNA responsive element and expression of

the Ah receptor-responsive gene CYP1A1. LTr-1 was a competitive inhibitor of

CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity. In summary, these

results demonstrated that LTr-1, a major in vivo product of I3C, could inhibit

the proliferation of both estrogen-dependent and -independent breast tumor cells

and that LTr-1 is an antagonist of estrogen receptor function and a weak agonist

of Ah receptor function.

 

PMID: 10449193 [PubMed - indexed for MEDLINE]

 

Carcinogenesis. 1998 Sep;19(9):1631-9.

 

Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of

diindolylmethane.

 

Chen I, McDougal A, Wang F, Safe S.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University,

College Station 77843-4466, USA.

 

Phytochemicals such as indole-3-carbinol (I3C) and sulforaphane are components of

cruciferous vegetables which exhibit antitumorigenic activity associated with

altered carcinogen metabolism and detoxification. Diindolylmethane (DIM) is a

major acid-catalyzed metabolite of I3C formed in the gut that binds to the aryl

hydrocarbon receptor (AhR) and treatment of MCF-7 human breast cancer cells with

10-50 microM DIM resulted in rapid formation of the nuclear AhR complex and

induction of CYP1A1 gene expression was observed at concentrations >50 microM.

Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD), a high affinity AhR ligand, inhibits 17beta-estradiol (E2)-induced

responses in MCF-7 cells and growth of E2-dependent 7,12-dimethylbenzanthracene

(DMBA)-induced mammary tumors in female Sprague-Dawley rats. Results of this

study show that like TCDD, DIM inhibits E2-induced proliferation of MCF-7 cells,

reporter gene activity in cells transiently transfected with an E2-responsive

plasmid (containing a frog vitellogenin A2 gene promoter insert) and

down-regulates the nuclear estrogen receptor. Moreover, DIM (5 mg/kg every other

day) also inhibits DMBA-induced mammary tumor growth in Sprague-Dawley rats and

this was not accompanied by induction of hepatic CYP1A1-dependent activity. Thus,

DIM represents a new class of relatively non-toxic AhR-based antiestrogens that

inhibit E2-dependent tumor growth in rodents and current studies are focused on

development of analogs for clinical treatment of breast cancer.

 

PMID: 9771935 [PubMed - indexed for MEDLINE]

 

Xenobiotica. 1998 Aug;28(8):803-11.

 

3,3'-Diindolylmethane induces CYP1A2 in cultured precision-cut human liver

slices.

 

Lake BG, Tredger JM, Renwick AB, Barton PT, Price RJ.

 

BIBRA International, Carshalton, UK.

 

1. The effect of 3,3'-diindolylmethane (DIM), an indole derivative derived from

cruciferous vegetables, on cytochrome P450 (CYP) isoforms in the CYP1A and CYP3A

subfamilies has been studied in 72-h cultured human liver slices. 2. In cultured

human liver slices 50 microM DIM induced 7-ethoxyresorufin O-deethylase and to a

lesser extent 7-methoxyresorufin O-demethylase activities. 3. Western

immunoblotting of liver slice microsomes was performed with antibodies to rat

CYP1A2 and human CYP3A4. Compared with control liver slice microsomes (dimethyl

sulphoxide-only treated), DIM induced levels of CYP1A2 but had little effect on

levels of CYP3A4. The treatment of human liver slices with 2 microg/ml of the

polycholorinated biphenyl mixture Aroclor 1254 also resulted in an induction of

levels of CYP1A2, but had no effect on CYP3A4. 4. These results demonstrate that

DIM induces CYP1A isoforms in cultured human liver slices. Some variability in

the magnitude of induction of enzyme activities by DIM was observed in four human

liver samples examined. For 7-ethoxyresorufin O-deethylase, the magnitude of

induction by 50 microM DIM ranged from 2.3- to 19.3-fold. 5. These results

demonstrate that cultured human liver slices can be used to evaluate the effect

of chemicals derived from cruciferous and other vegetables on CYP isoforms.

 

PMID: 9741959 [PubMed - indexed for MEDLINE]