Ligand-Independent Activation of Estrogen Receptor Function by 3,3'-Diindolylmethane in Human Breast Cancer Cells Jacques E. Riby,* Grace H. F. Chang,* Gary L. Firestone† and Leonard F. Bjeldanes*‡ *DIVISION OF NUTRITIONAL SCIENCES AND TOXICOLOGY, AND †DEPARTMENT OF MOLECULAR AND CELL BIOLOGY, UNIVERSITY OF CALIFORNIA,BERKELEY, CA 94720, U.S.A. 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 17-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 (7-[9-[(4,4,5,5,5-pentafluoropentyl)sulfonyl- ]nonyl]-estra-1,3,5(10)-triene-3,17-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 pro- moter-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. BIOCHEM PHARMACOL 60;2: 167–177, 2000. © 2000 Elsevier Science Inc. KEY WORDS. estrogen receptor; agonist; breast cancer; diindolylmethane; ligand independent I3C§ (Fig. 1), a hydrolysis product of glucobrassicin found in Brassica plants, including turnips, kale, broccoli, Brussels sprouts, and cauliflower, is a potential cancer protective agent. Oral administration of I3C reduced BP-induced neoplasia of the forestomach and total covalent binding of BP and N-nitrosodimethylamine to hepatic DNA in mice [1–3]. In trout, oral I3C reduced aflatoxin B1-induced hepatocarcinogenesis when administered prior to and dur- ing carcinogen treatment [4]. Similarly, in rodents, oral I3C reduced DMBA-induced mammary tumor incidence by 70 – 80% [1, 5], and reduced by 50% the incidence and multiplicity of spontaneous mammary tumors [6]. In a screen of 90 potential chemopreventive agents in a series of six short-term bioassays relevant to initiation and post- initiation phases of carcinogenesis, I3C was found to be one of only eight compounds that tested positive in all assays [7]. Some evidence suggests, however, that whereas I3C mitigates mammary carcinogenesis, it may also enhance tumorigenesis in other organs under certain conditions. Thus, oral administration of high doses of I3C to rodents or trout following exposure to certain organ-selective carcin- ogens resulted in increased tumor incidence of the liver [8], ‡ Corresponding author: Dr. Leonard F. Bjeldanes, Division of Nutritional Sciences and Toxicology, 119 Morgan Hall, University of California, Berkeley, CA 94720. Tel. (510) 642-1601; FAX (510) 642--0535; E-mail: lfb@nature.berkeley.edu § Abbreviations: AhR, aryl hydrocarbon receptor; BP, benzo[a]pyrene; CAT, chloramphenicol acetyltransferase; CDK, cyclin-dependent kinase; CYP, cytochrome P450; DES, diethylstilbesterol; DIM, 3,3'-diindolyl- methane; DMBA, dimethylbenzanthracene; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; E 2 , 17-estradiol; ER, estrogen receptor; ERE, estrogen response element; EROD, ethoxyresorufin O-de- ethylase; FBS, fetal bovine serum; GAPDH, glyceraldehyde phosphate dehydrogenase; I3C, indole-3-carbinol; ICZ, indolo[3,2-b]carbazole; and PKA, protein kinase A. Received 8 September 1999; accepted 29 November 1999. Biochemical Pharmacology, Vol. 60, pp. 167–177, 2000. ISSN 0006-2952/00/$–see front matter © 2000 Elsevier Science Inc. All rights reserved. PII S0006-2952(00)00307-5