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

doi:10.1016/S0006-2952(00)00307-5

Biochemical Pharmacology

Volume 60, Issue 2, 15 July 2000, Pages 167-177

https://doi.org/10.1016/S0006-2952(00)00307-5 Get rights and content

Abstract

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 17β-estradiol (E₂). DIM weakly inhibited the E₂-induced proliferation of ER-containing MCF-7 cells and induced proliferation of these cells in the absence of steroid, by approximately 60% of the E₂ 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 E₂- and diethylstilbesterol-responsive reporter construct pATC2. The estrogen antagonist ICI 182780 (7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3,17β-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 E₂, 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.

Section snippets

Materials

DMEM, Opti-MEM, and Lipofectamine were supplied by Gibco/BRL. Phenol red-free DMEM, FBS, calf serum, tamoxifen, and E₂ were supplied by the Sigma Chemical Co. ICI 182780 (7α- 9, 4, 5sulfonyl]nonyl]-estra-1,3,5 [10]-triene-3,17β-diol) was supplied by Tocris. [γ-³²P]ATP and [³H]acetyl-CoA were supplied by New England Nuclear. SKF-82958 [(±)6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepinehydrobromide] was purchased from RBI, and H-89 (N-[2-(p

DIM-induced growth of MCF-7 cells in E₂-depleted medium

The effects of DIM on cell proliferation were examined in estrogen-treated and untreated MCF-7 cells over a 7-day time course. Treatment of cells with DIM in E₂-stripped medium produced a concentration-dependent increase in cell proliferation that at 10 μM reached a maximum of 60% of the growth produced in the absence of DIM in cells grown in complete, E₂-rich medium (Fig. 2). Co-treatment of cells with DIM (10 μM) in the E₂-rich medium, however, weakly inhibited estrogen-stimulated cell

Discussion

Our results show that at concentrations of up to 10 μM, DIM exhibited primarily agonist activity on cell proliferation and transcriptional activation of E₂-responsive endogenous and transfected reporter genes. Although DIM did not interact with the ligand-binding site of the ER, DIM activated the ER to a form that binds in vitro to the consensus ERE. In the absence of E₂, DIM promoted a level of cell proliferation in the E₂-responsive MCF-7 cell line that was approximately 60% of the maximum

Acknowledgements

This work was supported by the Department of Defense, Army Breast Cancer Research Program Grant DAMD17–96-1–6149, and by Grant CA69056 from the National Institutes of Health.

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Citation Excerpt :
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Molecular and cellular pharmacologyLigand-independent activation of estrogen receptor function by 3,3′-diindolylmethane in human breast cancer cells

Abstract

Section snippets

Materials

DIM-induced growth of MCF-7 cells in E2-depleted medium

Discussion

Acknowledgements

Chem Biol Interact

J Biol Chem

J Biol Chem

Biochem Pharmacol

Gene

J Steroid Biochem Mol Biol

Biochem Biophys Res Commun

Biochem Pharmacol

Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles

Cancer Res

Protection by indole-3-carbinol against covalent binding of benzo[a]pyrene metabolites to mouse liver DNA and protein

Food Chem Toxicol

Mechanisms of anti-carcinogenesis by indole-3-carbinolDetailed in vivo DNA binding dose-response studies after dietary administration with aflatoxin B1

Carcinogenesis

Chemoprevention of chemically-induced mammary carcinogenesis by indole-3-carbinol

Anticancer Res

Effects of dietary indole-3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice

Carcinogenesis

Screening of potential chemopreventive agents using biochemical markers of carcinogenesis

Cancer Res

Enhancement of carcinogenesis by the natural anticarcinogen indole-3-carbinol

J Natl Cancer Inst

Multiple dietary factors in the enhancement of dimethylhydrazine carcinogenesisMain effect of indole-3-carbinol

J Natl Cancer Inst

Enhancement by indole-3-carbinol of liver and thyroid gland neoplastic development in a rat medium-term multiorgan carcinogenesis model

Carcinogenesis

Structure-activity relationships of dietary indolesA proposed mechanism of action as modifiers of xenobiotic metabolism

J Toxicol Environ Health

Molecular and cellular pharmacology
Ligand-independent activation of estrogen receptor function by 3,3′-diindolylmethane in human breast cancer cells

DIM-induced growth of MCF-7 cells in E₂-depleted medium