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Articles

Indole-3-Carbinol Is a Negative Regulator of Estrogen Receptor- Signaling in Human Tumor Cells¹

Department of Radiation Oncology and ^* Department of Otolaryngology, Long Island Jewish Medical Center, New Hyde Park, NY 11040

²To whom correspondence should be addressed.

	ABSTRACT

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Estrogen, via its binding to the estrogen receptor (ER), plays an important role in breast cancer cell proliferation and tumor development. Indole-3-carbinol (I3C), a compound occurring naturally in cruciferous vegetables, exhibits a potent antitumor activity via its regulation of estrogen activity and metabolism. This study was designed to determine the effect of I3C on the potential to inhibit the ER-. Using a reporter gene driven by the estrogen receptor, I3C (10–125 µmol/L) significantly repressed the 17ß-estradiol (E2)-activated ER- signaling in a dose-dependent manner. I3C and breast cancer susceptibility gene 1 (BRCA1) synergistically inhibited transcriptional activity of ER-. Moreover, I3C down-regulated the expression of the estrogen-responsive genes, pS2 and cathepsin-D, and up-regulated BRCA1. The inhibitory effects of I3C did not contribute to its cytotoxic effects because these activities were observed at less than toxic concentrations. These results further suggest that antitumor activities of I3C are associated not only with its regulation of estrogen activity and metabolism, but also its modulation of ER transcription activity.

KEY WORDS: • indole-3-carbinol • estrogen receptor • human breast cancer • breast cancer susceptibility gene 1 (BRCA1)

	INTRODUCTION

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Indole-3-carbinol (I3C)³ is a naturally occurring phytochemical present in cruciferous vegetables such as cabbage, cauliflower, broccoli and brussel sprouts (Loub et al. 1975 ). A number of in vitro and in vivo studies have found that I3C prevents development of estrogen-enhanced cancers including breast, endometrial and cervical cancers (Bradlow et al. 1991 , Jin et al. 1999 , Kojima et al. 1994 ). I3C exerts profound antitumor activities, including blocking cell cycle progression, triggering apoptosis and reducing tumor invasion and metastasis (Bradlow et al. 1991 and 1995 , Hudson et al. 1998 , Liu et al. 1994 , Meng et al. 2000a and 2000b , Niwa et al. 1994 , Telang et al. 1992 and 1997 , Tiwari et al. 1994 , Wong et al. 1997 ). I3C exerts antiestrogenic activities, which undoubtedly contribute to its prevention of estrogen-enhanced cancers. I3C induces 2-hydroxylation of estradiol, resulting in metabolites that are not estrogenic (Michnovicz and Bradlow 1990 ). I3C and its condensation product indolo (3,–2b) carbazol bind the estrogen receptor, albeit with a low affinity (Liu et al. 1994 , Yuan et al. 1999 ). I3C and tamoxifen, one of the most commonly used drugs in breast cancer therapy to reduce estrogenic activities, synergistically inhibit proliferation of breast cancer cells (Cover et al. 1999 ). Chang et al. (1999) recently reported that 2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major active component of I3C in vivo, inhibited the proliferation of breast cancer cells associated with blocking estrogen receptor function. Therefore, it is believed that antiestrogenic activity may be an important mechanism of I3C in prevention of breast cancer and other hormonal organ cancers (Jin et al. 1999 , Kothari et al. 1995 , Michnovicz and Bradlow et al. 1990 , Yuan et al. 1999 ).

I3C is not considered the active component responsible for the biological effects. A series of oligomeric products that are formed from I3C, such as 2-(indol-3-ylmethyl)-3,3'-diindolylmethane, are the active compounds (Broadbent and Broadbent 1998a and 1998b , Chang et al. 1999 ). Oligomeric products arise rapidly in acidic environments such as in the stomach and form more slowly during neutral conditions.

Estrogen is clearly responsible for the regulation of many genes involved in the regulation of proliferation and chemosensitivity in estrogen-sensitive tissues. Estrogen deprivation causes regression of many breast tumors (McKeon 1997 ). 17ß-estradiol (E2) binds to the estrogen receptor (ER) with high affinity. In turn, the ER (ER- and ER-ß) bind estrogen response elements (ERE) located in promoters of estrogen-responsive genes and regulate their transcription (Evans 1988 , Katzenellenbogen 1996 ). The action of the ER is regulated not only by ligand, but also by some coregulatory proteins (Torchia et al. 1998 ). Recently, we found that the breast cancer susceptibility gene 1 (BRCA1) interacts physically with ER- and inhibits the expression of an ER- transcription cofactor, p300 (Fan et al. 1998 and our unpublished data). As a result of these effects, the hormone-activated transcription activity of the ER- is suppressed by BRCA1 (Fan et al. 1999 ). In addition, we found that I3C up-regulates BRCA1 expression in a dose-dependent manner (Meng et al. 2000a ) in breast cancer cells and in cervical cancer cells (unpublished). Therefore, these findings compelled us to question whether I3C directly modulates the transcription activity of ER- and whether I3C gives rise to any effects on BRCA1 functions as an inhibitor of ER- signaling.

	MATERIALS AND METHODS

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Cell culture, vectors and indole-3-carbinol (I3C).

The cell lines used in this study were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained as monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mmol/L glutamine, 0.1 mg/L streptomycin and 1000 U/L penicillin G (BioWhittaker, Walkersville, MD). All of the vectors used in this study were described previously (Fan et al. 1999 ), including the ER expression plasmid containing both the T7 and cytomegalovirus promoters, the ERE-TK-LUC reporter composed of the vitellogenin A2 ERE controlling a minimal thymidine kinase promoter (TK81), and luciferase in plasmid pGL2, the expression of wild-type BRCA1 in pcDNA3 plasmid, pCMV-Sp1 (Sp1) and Sp1-TK-LUC. I3C was purchased from Sigma (St. Louis, MO), dissolved in 95% ethanol and stored at -20°C before use. The MTT assay of I3C toxicity showed that IC3 (>150 µmol/L, 24 h treatment) resulted in a significant toxicity in these cell lines (data not shown). Therefore, 10–125 µmol/L doses of I3C were employed in the present study.

Determination of the 17ß-estradiol–activated ER-–mediated transcriptional activity.

Assay of E2-activated ER-–mediated transcriptional activity was performed as described previously (Fan et al. 1999 ). Subconfluent proliferating cells plated in 24-well culture dishes were cotransfected with the luciferase reporter plasmid containing ERE (ERE- TK-LUC; 0.5 µg), ER expression plasmid (0.5 µg/L) and pCMV-ß-gal (0.5 µg/L). pBluescript DNA (Stratagene, La Jolla, CA) was used to adjust the total DNA concentration to an equal amount per well. The transfection was carried out using the transfection reagent, lipofectin (Gibco-BRL, Gaithersburg, MD) according to the manufacture’s instructions. Cells were cultured for an additional 24 h in DMEM (phenol red free, and charcoal-stripped FCS and 1 µmol/L E2) with different concentration of l3C. Cells were harvested with a luciferase lysis buffer (Promaga, Madison, WI). Lysates were analyzed for luciferase activity using a liquid scintillation counter (Model LS60001C, Beckman, Fullerton, CA) and the data were normalized by protein concentration.

Protein analysis.

pS2, cathepsin-D and BRCA1 proteins ware assayed using a Western blot assay as described previously (Fan et al. 1998 ). Conditioned media from the cultures of I3C-untreated and -treated cells were collected and concentrated using a Centriprep-3 device (Amicon, Beverly, MA). Conditioned medium (50 µL) was electrophoresed on a 15% SDS-polyacrylamide gel and transferred to membranes via electroblotting. In addition, cells were harvested by lysing PBS containing 1% Nonidet P-40 and protease inhibitors, 10 ng/L leupeptin, 10 ng/L aprotinin, 2 mmol/L 4-(2- aminoethyl)-benzene-sulfonyl fluoride, 10 mmol/L sodium fluoride, 1 mmol/l sodium -orthovanadate and 5 mmol/L sodium pyrophosphate. Equal aliquots of total protein (50 µg per lane) were electrophoresed on a 6% SDS-polyacrylamide gel and transferred to membranes by electroblotting. The membranes were incubated with primary antibodies, pS2 (V3030, monoclonal, Biomedia, CA), cathepsin-D (06–467, polyclonal, Upstate Biotechnology, Lake Placid, NY) and BRCA1 (C-20, polyclonal; Santa Cruz, Hercules, CA), and then incubated with secondary antibodies after being washed extensively. Antibody reaction was revealed using an enhanced chemiluminescence detection system (Amersham Life Science, Arlington Heights, IL) as instructed by the manufacturer. Equal protein loading and transfer were confirmed by immunoblotting for -actin protein using a goat polyclonal -actin antibody (I-19; Santa Cruz). A colored marker (Bio-Rad Laboratories, Hercules, CA) was used as a molecular size standard.

Statistical analysis.

The statistical analyses were carried out by the Statistica (Stat-Soft, Tulsa, OK) software system. The differences in luciferase activity were assessed by Student’s t test. P < 0.05 was considered to be significant and 0.05 P < 0.1 was considered to be marginally significant.

	RESULTS

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The hormone-activated transcriptional activity of the ER- is repressed by I3C.

First, we examined the effect of I3C on estrogen-activated transcriptional activity of the ER- in MCF-7, an ER-positive human breast cancer cell line. The cells were cotransfected overnight with an ERE-containing luciferase reporter (ERE-TK-LUC) plasmid and an ER expression plasmid. The ER- expression plasmid controlled by the CMV promoter was provided to ensure a high level expression of ER- under all assay conditions. As shown in Figure 1A , I3C inhibited the E2-stimulated reporter activity. There was a dose-dependent decrease up to an 50% reduction compared with the positive control level (without I3C treatment). Similar results were also observed in the human breast cancer cell lines T47D and MDA-MB-231 (Fig. 1B ) and three human cervical cancer cell lines, CaSki, SiHa and C33-A (Fig. 1C ). We also tested the capability of I3C to regulate the activity of a reporter gene driven by the Sp1 transcription factor (Fig. 1D ). I3C had neither an inhibitory nor an enhancing effect on the ability of Sp1 to regulate this reporter gene.

View larger version (47K): [in this window] [in a new window]   Figure 1. Indole-3-carbinol (I3C) selectively represses 17ß-estradiol (E2)-activated estrogen receptor (ER)-a transcriptional activity in human cancer cells. Subconfluent proliferating human breast cancer MCF-7, T-47D and MDA-MB-231 (A and B) and cervical cancer CaSki, Siha and C33-A (C) cells were cotransfected overnight with indicated vectors and then postincubated for a further 24 h in serum- and phenol-free Dulbecco’s modified Eagle’s medium containing E2 (1 µmol/L) and I3C (10–125 µmol/L). (D) Subconfluent proliferating MCF-7 and MDA-MB-231 cells were cotransfected with pCMV-Spl (Spl, 0.5 µg) and SP1-thymidine kinase (TK)-luciferase (LUC) (0.5 µg) and incubated for 24 h in the presence or absence of I3C (50 or 100 µmol/L). The cells were harvested, lysed and analyzed for the luciferase activity. Values of luciferase are the means ± SEM of four determinations. *P < 0.05; **0.05 P < 0.1 vs. control without I3C.

View larger version (23K): [in this window] [in a new window]   Figure 2. Breast cancer susceptibility gene 1 (BRCA1) enhances indole-3-carbinol (I3C) ability in suppression of 17ß-estradiol (E2)-activated estrogen receptor (ER)-a transcriptional activity. (A) Subconfluent proliferating MCF-7 and T47-D cells were cotransfected with an equal amount (0.5 µg) of estrogen response element, thymidine kinase and luciferase in plasmid pGL2 (ERE-TK-LUC), ER and BRCA1 or pcDNA3 plasmid overnight, and then incubated for a further 24 h in serum- and phenol-free Dulbecco’s modified Eagle’s medium (D-MEM) containing E2 (1 µmol/L). (B) Subconfluent proliferating MCF-7 and T47-D cells were cotransfected with ERE-TK-LUC (0.5 µg), ER (0.5 µg) and BRCA1 (0.05 µg) or pcDNA3 (0.5 µg) plasmid overnight. The cells were then incubated for a further 24 h in serum- and phenol-free D-MEM containing E2 (1 µmol/L) and 100 µmol/L I3C. The cells were harvested, lysed and analyzed for the luciferase activity. Values are means ± SEM of four determinations. *P < 0.05; **0.05 P < 0.1 vs. control without I3C.

Finally, we determined that I3C could affect the amount of BRCA1 and pS2 and cathepsin-D. Both the conditioned medium and extracts from the cells of exponentially growing MCF-7 cells cultured in serum-free medium containing E2 (1 µmol/L) and/or I3C (100 µmol/L) were used for Western analysis. As shown in Figure 3A and B, E2 significantly increased the expression of pS2 and cathepsin-D proteins 3.5–4 and 5.5–6.0 fold in 10% FSC medium and serum-free medium, respectively. However, I3C significantly suppressed this estrogen-enhanced protein expression. In contrast to pS2 and cathepsin-D, I3C significantly increased BRCA1 protein expression in the presence and the absence of E2. 17ß-estradiol slightly decreased BRCA1 protein. Similar results were also obtained in T47D cells (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 3. Indole-3-carbinol (I3C) increases breast cancer susceptibility gene 1 (BRCA1) protein and decreases pS2 and cathepsin-D proteins in estrogen-responsive MCF- 7 cells. (A) Subconfluent proliferating MCF- 7 cells were treated with I3C (100 µmol/L) for 24 h in serum- and phenol-free Dulbecco’s modified Eagle’s medium (D-MEM) with or without 17ß-estradiol (E2; 1 µmol/L). E2 (1 µmol/L, 24 h) was also added to the normal culture [D-MEM + 10% fetal calf serum (FCS)] of MCF-7 cells as a control. Medium was concentrated and Western blotted (50 µL/lane) for the pS2 protein expression. The cells were harvested for the analysis of cathepsin-D and BRCA1 proteins (50 µg protein/lane). (B) Protein bands were quantitated by densitometry and expressed relative to the untreated control (D-MEM + 10% FCS).

	DISCUSSION

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This direct link between ER signaling modulation and I3C has important implications in the understanding of antitumor activity of I3C because many tumors are enhanced by estrogen. It is known that estrogen, via its specific binding to ER, stimulates the expression of many genes containing ERE in their regulatory regions. Many such genes are involved in promotion or enhancement of cell proliferation, DNA and protein synthesis and metastatic capability of breast cancer [for reviews, see Dickson and Lippman (1987) and Osborne (1998) ]. Our work and other previous studies showed that I3C exhibited the chemoprevention properties of breast cancer through its antitumor cell proliferation, antitumor cell migration and invasion activities (Bradlow et al. 1991 and 1995 , Chen et al. 1999 , Cover et al. 1999 , Ge et al. 1999 , Hudson et al. 1998 , Liu et al. 1994 , Meng et al. 2000a and 2000b, Niwa et al. 1994 , Telang et al. 1992 and 1997 , Tiwari et al. 1994 , Wong et al. 1997 ). Thus, the negative regulatory role of I3C in the transcriptional activity of the ER- identified in this study provides a plausible mechanism for some of the antiproliferation and metastasis potential of I3C in breast cancer. This notion is supported by other in vitro and in vivo observations, suggesting an inverse correlation between the concentration of I3C and ER expression in cancer cells (Liu et al. 1994 , Yuan et al. 1999 ). In fact, it has been found that 2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major active component of I3C in vivo, inhibits the proliferation of breast cancer cells associated with blocking ER function (Chang et al. 1999 ).

Increased expression of the tumor suppressor gene BRCA1 may also play an important role for I3C in the suppression of ER- transcriptional signaling. Mutations in BRCA1 increase the risk of breast and ovarian cancer (Ford et al. 1994 , Miki et al. 1994 ) and prostate cancer in men (Langston et al. 1996 ). BRCA1 exhibits multiple biological functions as a tumor suppressor (Fan et al. 1998 ). Loss of heterozygosity has been found frequently in a region of chromosome 17p at which BRCA1 locates in cervical carcinoma cases (Krul et al. 1999 ). Studies in our laboratory are determining whether papillomavirus oncoproteins (cofactors for cervical cancer) bind and target BRCA1 for degradation. Recent studies from our laboratory showed that BRCA1 functions as a new inhibitor in the modulation of ER- transcriptional signaling through its physical binding to the ER- and its regulation of an ER- cofactor p300/CBP (Fan et al. 1999 and unpublished data). This and previous studies (Meng et al. 2000a ) show that exposure to I3C results in a significant increase in the expression of the BRCA1 protein. Because this up-regulation is independent of estrogen, other mechanisms must account for this. I3C increases expression of some Phase I and Phase II genes via it binding to the Ah receptor [reviewed by Chen et al. (1998) ]. The ability of I3C to increase BRCA1 together with our observations that I3C and BRCA1 synergistically down-regulate ER-dependent gene expression increases the effectiveness of I3C in this activity. Other investigators have determined that I3C promotes the 2-hydroxylation of E2 and decreases the use of the 16-hydroxlation pathway, metabolism that would decrease estrogenic activity [reviewed by Bradlow et al. (1995) ]. Hence, I3C appears to reduce estrogen activity by multiple mechanisms.

Our present findings highlight the potential of this phytochemical for both the prevention and treatment of estrogen-enhanced cancers. In fact, I3C was effective in preventing estrogen-dependent cervical dysplasia in normal mice and estrogen-dependent cervical cancer in mice with papillomavirus transgenes (Jin et al. 1999 ). More recently, I3C was shown to be effective in the treatment of cervical dysplasia in women (Bell et al. 2000 ). Concentrations of I3C in these and other in vivo studies indicate that I3C is effective at concentrations achievable by eating cruciferous vegetables. The higher concentration of I3C used in the cervical dysplasia study was 400 mg/d, a concentration usually obtainable by daily consumption of one third of a head of cabbage. Because the conversion of I3C to the active condensation products is not as efficient in vitro as in the acid environment of the stomach, higher concentrations of this compound are usually required to evaluate its in vivo activities. In addition to the powerful effect of antiestrogenic activities, IC3 has other antitumor activities including induction of enzymes that would detoxify carcinogens [reviewed by Chen et al. (1998) ], induction of apoptosis (Ge et al. 1999 and our unpublished observations in cervical cells), down-regulation of CDK6 (Cover et al. 1998 ) and as an antioxidant (Shertzer et al. 1988 ). Together, these beneficial effects indicate that this phytochemical has tremendous potential in the treatment and prevention of cancer, particularly estrogen-enhanced cancer.

	FOOTNOTES

 
¹ Supported in part by grants from the U.S. Public Health Service (R01-ES09169 and RO1-CA80000) (E.M.R. and S.F.), the USAMRMC BCRP (S.F.) and the CA73385 (K.A.).

³ Abbreviations used: BRCA1, breast cancer susceptibility gene 1; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; ERE-TK-LUC, an ERE, thymidine kinase promoter and luciferase in plasmid pGL2; FCS, fetal calf serum; I3C, indole-3-carbinol.

Manuscript received April 25, 2000. Initial review completed June 27, 2000. Revision accepted August 24, 2000.

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