3, 3’-diindolylmethane

Clinical Studies
References


Diindolylmethane may be used for preventing breast and uterine cancer. It may also be used to prevent benign prostatic hypertrophy (BPH) and for premenstrual syndrome (PMS).

Diindolylmethane is a major active metabolite of indole-3-carbinol, which is a constituent of cruciferous vegetables such as cabbage, brussels sprouts, cauliflower and broccoli. Indole-3-carbinol is thought to be one of the agents responsible for the cancer-protective properties of these vegetables. In stomach acid indole-3-carbinol is unstable and readily converted to a variety of compounds including diindolylmethane.

A typical diet provides 20-120mg of indole-3-carbinol per day. Approximately 10-20% is converted to diindolylmethane, providing 2-24mg daily. Researchers are interested in diindolylmethane due to potential activity against breast cancer cells. Diindolylmethane has partial estrogen receptor agonist and antagonistic activity. There is preliminary evidence that diindolylmethane can inhibit the growth of mammary tumors in animal models. Diindolylmethane induces apoptosis (programmed cell death) in human breast cancer cells in vitro.

Diindolylmethane may be beneficial for estrogen balance, cancer prevention and anti-aging.

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Published Clinical Studies
3, 3’-diindolylmethane


Ligand-independent activation of estrogen receptor function by 3, 3'-diindolylmethane in human breast cancer cells.

Riby JE, Chang GH, Firestone GL, Bjeldanes LF.

Biochem Pharmacol. 2000 Jul 15; 60(2):167-77.

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]

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3, 3’-Diindolylmethane induces apoptosis in human cancer cells.

Ge X, Yannai S, Rennert G, Gruener N, Fares FA.

Biochem Biophys Res Commun. 1996 Nov 1; 228(1):153-8.

Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa, Israel.

3, 3'-Diindolylmethane is a dimer of indole-3-carbinol formed both in vivo and in vitro. In this study, human cancer cells MCF-7 (with wild-type p53), T47-D (mutant p53), and Saos-2 (deficient in p53 gene), were used to examine the anticancer activities of 3, 3'-diindolylmethane. The dose-dependent growth inhibitory effect was found in all these cell lines. Exposure of the cells to 50 microM solution of 3,3'-diindolylmethane for 48 h, apoptosis (programmed cell death) was evidenced by the characteristic morphology of cell nuclei under fluorescence microscope and the DNA "ladder" in agarose gel electrophoresis. The percentage of apoptotic cells in each cell line was found to be 12% for MCF-7, 14% for T47D and 13% for Saos2 cells. Exposure of MCF-7 cells to 100 microM 3, 3'-diindolylmethane for 24 h, 19% of apoptotic cells were detected by flow cytometry analysis. The lowest dose required for induction of apoptosis in MCF-7 cells was found to be 10 microM after 72 h incubation. Western blot showed that wild-type p53 protein was unchanged after MCF-7 cells had been exposed to 50 microM 3, 3'-diindolylmethane for 8 h. This study provides evidences that 3, 3'-diindolylmethane induces apoptosis in human cancer cells and that the induction of apoptosis is independent of p53 pathway.

PMID: 8912651 [PubMed - indexed for MEDLINE]

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The anticarcinogen 3, 3’-diindolylmethane is an inhibitor of cytochrome P-450.

Stresser DM, Bjeldanes LF, Bailey GS, Williams DE.

J Biochem Toxicol. 1995 Aug; 10(4):191-201.

Department of Food Science and Technology, Oregon State University, Corvallis 97331-6602, USA.

Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several mechanisms of inhibition may exist. We reported previously that 3, 3'-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent ethoxyresorufin O-deethylase with Ki values in the low micromolar range. We now report a similar potent inhibition by 3, 3'-diindolylmethane of rat and human CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential activity assays. 3, 3'-Diindolylmethane also inhibited in vitro CYP-mediated metabolism of the ubiquitous food contaminant and potent hepatocarcinogen, aflatoxin B1. There was no inhibition of cytochrome c reductase. In addition, we found 3, 3'-diindolylmethane to be a substrate for rat hepatic microsomal monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These observations indicate that 3, 3'-diindolylmethane can inhibit the catalytic activities of a range of CYP isoforms from lower and higher vertebrates in vitro. This broadly based inhibition of CYP-mediated activation of procarcinogens may be an indole-3-carbinol anticarcinogenic mechanism applicable to all species, including humans.

PMID: 8568833 [PubMed - indexed for MEDLINE]

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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.

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

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]

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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.

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

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]

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Bcl-2 family-mediated apoptotic effects of 3, 3’-diindolylmethane (DIM) in human breast cancer cells.

Hong C, Firestone GL, Bjeldanes LF.

Biochem Pharmacol. 2002 Mar 15; 63(6):1085-97.

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]

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Indole-3-carbinol and 3, 3’-diindolylmethane induce apoptosis in human prostate cancer cells.

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

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

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 haracterize 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]

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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.

Carcinogenesis. 2002 Aug; 23(8):1297-305.

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 transcriptions 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]

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References

  1. Natl Inst Health, Natl Inst Environmental Health Sci. Indole-3-carbinol. URL: http://ntp-server.niehs.nih.gov/htdocs/Chem_Background/ExecSumm/IndoleCarbinol.html#CONTENTS (Accessed 5 March 2001).

  2. Ge X, Yanni S, Rennert G, et al. 3'3-diindolylmethane induces apoptosis in human cancer cells. Biochem Biophys Res Com 1996;228:153-8.

  3. Lake BG, Tredger JM, Renwick AB, et al. 3'3-diindolylmethane induces CYP1A2 in cultured precision-cut human liver slices. Xenobiotica 1998;28:803-11.

  4. Chen I, McDougal A, Wang F, Safe S. Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis 1998 Sep;19(9):1631-9.

  5. McDougal A, Gupta MS, Ramamoorthy K, et al. Inhibition of carcinogen-induced rat mammary tumor growth and other estrogen-dependent responses by symmetrical dihalo-substituted analogs of diindolylmethane. Cancer Lett 2000;151:169-79.

  6. Riby JE, Chang GHF, Firestone GL, Bjeldanes LF. Ligand-independent activation of estrogen receptor function by 3,3'-diindolylmethane in human breast cancer cells. Biochem Pharmacol 2000;60:167-77.

  7. Balk JL. Indole-3-carbinol for cancer prevention. Altern Med Alert 2000; 3:105-7.

  8. Chen I, Safe S, Bjeldanes L. Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah) receptor agonists and antagonists in T47D human breast cancer cells. Biochem Pharmacol 1996;51:1069-76.