This Article Abstract Full Text (PDF) All Versions of this Article: 20/12/3070    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager NURSA Molecule Pages Link Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sundar, S. N. Articles by Firestone, G. L. Search for Related Content PubMed PubMed Citation Articles by Sundar, S. N. Articles by Firestone, G. L.

Indole-3-Carbinol Selectively Uncouples Expression and Activity of Estrogen Receptor Subtypes in Human Breast Cancer Cells

Department of Molecular and Cell Biology and the Cancer Research Laboratory (S.N.S., V.K., C.N.E., V.B.D., G.L.F.), and Department of Nutritional Sciences and Toxicology (L.F.B.), University of California at Berkeley, Berkeley, California 94720-3200

Address all correspondence and requests for reprints to: Gary L. Firestone, Department of Molecular and Cell Biology, 591 LSA, University of California at Berkeley, Berkeley, California 94720-3200. E-mail: glfire{at}berkeley.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Estrogen-responsive breast cancer cells, such as MCF7 and T47D cells, express both estrogen receptor (ER)- (ER) and ERß. Indole-3-carbinol (I3C) strongly down-regulated ER protein and transcript levels, without altering the level of ERß protein, in both cell lines. In cells transfected with the ER promoter linked to a luciferase gene reporter, I3C ablated ER promoter activity. Propyl pyrazole triol (PPT) is a highly selective ER agonist, whereas, 17ß-estradiol activates both ER and ERß. I3C treatment inhibited the PPT- and 17ß-estradiol-induced proliferation of breast cancer cells, disrupted the PPT and 17ß-estradiol stimulation of estrogen response element (ERE)-driven reporter plasmid activity as well as of endogenous progesterone receptor transcripts. Using an in vitro ERE binding assay, I3C was shown to inhibit the level of functional ER and stimulated the level of ERE binding ERß even though the protein levels of this receptor remained constant. In ER–/ERß+ MDA-MB-231 breast cancer cells, I3C treatment stimulated a 6-fold increase in binding of ERß to the ERE. I3C also induced ERE- and activator protein 1-driven reporter plasmid activities in the absence of an ER agonist, suggesting that ERß is activated in indole-treated cells. Taken together, our results demonstrate that the expression and function of ER and ERß can be uncoupled by I3C with a key cellular consequence being a significantly higher ERß:ER ratio that is generally highly associated with antiproliferative status of human breast cancer cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
ESTROGENS ARE A class of steroid hormones that play a critical role in the development of the normal breast and in the genesis of breast cancer (1, 2). Estrogens mediate their cellular responses through two distinct intracellular receptors, estrogen receptor (ER)- (ER) and ERß, which are encoded by separate genes (3). In the cell systems tested to date, after activation by ligand binding, each ER regulates the transcription of unique but overlapping sets of target genes (4), although both receptors are capable of activating synthetic reporter plasmids driven by estrogen response elements (EREs) or activator protein 1 (AP-1) sites. The two ERs have been shown to play different roles in the reproductive physiology of mammals (5, 6). For example, a high ER:ERß ratio correlates well with high levels of cellular proliferation, whereas predominance of functional ERß over ER correlates with lower levels of proliferation (7, 8, 9). Ectopic expression of ER in estrogen-sensitive breast cancer cell lines, such as MCF7 and T47D, leads to no major changes in proliferation rate, whereas expression of exogenous ERß in these cells leads to a drastic reduction in cell growth rates in culture as well as in nude mouse xenografts (10, 11). This correlation with the proliferative state also holds in a physiological setting in that during the pre and peripubertal phases of rodents, which are periods of high proliferation, ER expression predominates in the mammary epithelium, whereas, during lactation, in which there is minimal mammary epithelial mitogenesis, the amount of ERß protein is 5 times that of ER in the mammary epithelium (12, 13). Additionally, in the ER null mouse mammary gland, even pharmacological doses of estrogen are ineffective in inducing robust growth of mammary epithelium, indicating that ERß does not mediate proliferative effects of estrogens in the mammary epithelium (2).

Although the role of ERß in the etiology of human breast cancer has not been well characterized, ER expression and function appears to be an important factor for prognosis of human breast cancer. ER-positive breast cancers correlate well with the proliferation of differentiated tumor phenotypes, and the majority of these tumors respond well to antihormonal therapy (14). Tamoxifen, a mixed antiestrogen, is clinically useful in the management of ER-positive metastatic breast cancer and in the prevention of breast cancer, and has improved the survival of women with ER-positive breast cancer (15, 16). However, long-term management of breast cancer with tamoxifen is associated with tamoxifen resistance, increased endometrial cancer risk, and other undesirable side effects (15, 16, 17). Tamoxifen-resistant breast cancers respond well to the pure steroidal antiestrogen ICI 182,780 (Faslodex), which has been shown to down-regulate the levels of ER by causing proteasome-dependent degradation (18, 19, 20, 21). Thus, a key issue in the characterization of promising therapeutic molecules to treat breast cancers is to determine their specific effects on ERand ERß responsiveness.

Both epidemiological and physiological studies suggest that phytochemicals from vegetables and fruits represent a largely untapped source of potential anticancer molecules (22, 23, 24, 25, 26, 27). One such phytochemical is indole-3-carbinol (I3C), an autolysis product of glucosinolates present in Brassica vegetables such as broccoli, cabbage, and cauliflower. Dietary exposure to I3C reduced tumor occurrence and decreased the multiplicity of spontaneous as well as carcinogen-induced mammary tumor formation in rodent model systems (28, 29). I3C also tested positive as a chemopreventative agent in several short-term bioassays relevant to carcinogen-induced DNA damage, tumor initiation and promotion, and oxidative stress (30). We and others have demonstrated that I3C and its natural dimer, 3'-3'-dindolylmethane (DIM) have direct antiproliferative effects on human reproductive cancer cells (31, 32, 33, 34, 35). For example, I3C induces a G1 cell cycle arrest of both estrogen-sensitive MCF7 and estrogen-insensitive MDA-MB-231 breast cancer cells (36) that is accompanied by a transcriptional down-regulation of cyclin-dependent kinase (CDK) 6 (39), and an inhibition of CDK2 activity in which the size distribution of associated cyclin E forms and subcellular localization of the CDK2 protein complex is altered (37). I3C also cooperates with tamoxifen to more effectively induce a G1 cell cycle arrest and ablated phosphorylation of the retinoblastoma protein (Rb) in MCF7 breast cancer cells (32). This indole has also been shown to elicit antiestrogenic effects and decrease the level of phosphorylated ER protein (38), although the underlying mechanism was not examined.

In this study, we report that at concentrations of I3C effective in causing a G1 cell cycle arrest, I3C differentially regulates the expression and function of ER and ERß. This indole strongly down-regulated ER expression due to a loss of promoter activity, which disrupts the regulated expression of exogenous and endogenous genes by 17ß-estradiol (E) and by propyl pyrazole triol (PPT), a highly selective agonist for ER, without altering ERß expression. Furthermore, we report that the loss of ER expression is accompanied by an increase in functional ERß that can bind to an ERE and increase ERß-associated ERE and AP-1-driven transcriptional activity. Thus, our study demonstrates that treatment with I3C results in a significantly higher ERß:ER ratio of functional ER subtypes that is highly associated with antiproliferative status of human breast cancer cells.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
I3C Down-Regulates ER Protein Levels without Altering ERß Protein Levels in MCF7 and T47D Human Breast Cancer Cells
Estrogen-responsive breast cancer cells express two ER subtypes (ER and ERß) that are encoded by distinct genes (3). ER responsiveness is generally associated with proliferative mechanisms, whereas dominance of ERß responsiveness has been linked to antiproliferative processes (2, 3, 11, 12). To examine the relative expression levels of ER and ERß under I3C-mediated growth-arrested conditions (36), estrogen-responsive MCF7 and T47D human breast cancer cells were treated with various doses of I3C for 48 h, and Western blots of total cell extracts were probed for either ER or ERß protein. The levels of ß-actin were used as a gel loading control. As shown in Fig. 1A, I3C strongly down-regulated the level of ER protein in both cell lines in a dose-responsive manner, without causing any effect on the level of ERß protein. Western blots showed that 1 µM E was unable to reverse the I3C-mediated down-regulation of ER protein (data not shown), suggesting that I3C does not act as an ER antagonist in agreement with our previous report demonstrating that I3C does not bind ERs (32). Figure 1B (top panel) shows the quantification by densitometric measurement of ER and ERß protein levels in cells treated at various I3C concentrations in comparison to the G1 cell cycle arrest induced by I3C in MCF7 cells as determined by flow cytometry. The overall dose-dependent profile of down-regulation of ER protein levels strongly correlated with that of the increase in G1 phase arrested cells and down-regulation of S phase cells. The half-maximal indole responses were approximately 100 µM I3C, and the maximal responses without any apoptotic effects were observed at 200 µM I3C. The data shown in Fig. 1B are for MCF-7 cells, although the same effects were observed with T47D human breast cancer cells (data not shown). Taken together, these data show that the maximal indole concentrations, which induce a G1 cell cycle arrest, result in a predominantly ERß-responsive cellular background, and therefore 200 µM I3C was the dose used throughout the remainder of this study.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Dose-Dependent Effects of I3C Production of ER and ERß Protein in MCF7 and T47D Human Breast Cancer Cells A, MCF7 and T47D cells were treated with the indicated concentrations of I3C for 48 h. Isolated protein extracts were fractionated using SDS-PAGE and electrophoretically transferred to nitrocellulose membranes, and immunoblots were probed with antibodies specific for ER and ERß. ß-Actin protein was used as a gel loading control. The band intensities were quantified using densitometry, and the bar graph represents relative values for ER and ERß normalized to actin values. B, MCF7 cells were exposed to DMSO or indicated doses of I3C for 48 h, hypotonically lysed in a solution containing propidium iodide, and subjected to flow cytometric analysis based on DNA content. This analysis is depicted as a bar graph with SE bars.

View larger version (44K): [in this window] [in a new window]   Fig. 2. Time Course of I3C Regulation of ER and ERß Protein Expression in MCF7 and T47D Breast Cancer Cells MCF7 and T47D breast cancer cells were treated with either DMSO or 200 µM I3C for varying durations as indicated. Isolated total cell extracts were electrophoretically fractionated, and immunoblot analysis for ER and ERß was carried out as described in Materials and Methods. CDK2 protein levels have been shown to remain unaltered with I3C treatment and were used as a protein loading control.

I3C Selectively Down-Regulates ER Transcript Levels and Promoter Activity

View larger version (37K): [in this window] [in a new window]   Fig. 3. I3C Down-Regulates ER Transcript Levels in MCF7 Cells MCF7 cells were treated with DMSO, 200 µM I3C, 1 µM tamoxifen (Tam), or 200 µM Tryptophol (Tryp) for 48 h, and 2 µg of isolated total RNA was reverse transcribed using random primers and Mu-MLV. cDNA (400 ng) was in the PCR using specific primers for ER and ERß. The PCR products were electrophoretically fractionated in 1% agarose gels, and GAPDH levels were used as a loading control.

View larger version (19K): [in this window] [in a new window]   Fig. 4. I3C Decreases ER Promoter Activity A, MCF7 cells were transfected with luciferase reporter plasmids constructs containing either the –3.2 kb upstream region of the ER gene promoter (–3200-luc) or the –700 bp upstream region of the ER gene promoter (–700-luc). The two ER transcription start sites are denoted as B and A; the B site is 1994 bp upstream of the A site. Cells were treated 24 h post transfection with DMSO or 200 µM I3C for 24 h, and the relative luciferase activity was evaluated in lysed cells using the Promega luciferase assay kit. Two controls (data not shown) employed were the empty control pGL2 (RLU = 0.1–0.5) and constitutive CMV promoter (RLU in millions). Bar graphs indicate relative luciferase activity normalized to the protein input with SE. The level of observed promoter activity in the absence of I3C was approximately 1020 RLU per µg protein for the –3200-Luc reporter plasmid and approximately 800 RLU per µg protein for the –700-Luc reporter plasmid. B, MCF7 cells were transfected with plasmids containing ER cDNA for ectopic overexpression. Control and ER overexpressing MCF7 cells were treated with DMSO or 200 µM I3C for 48 h. Total RNA isolated from these cells was subjected to reverse transcription followed by PCR with primers specific for ER or GAPDH. PCR products were electrophoresed and visualized using a UV transilluminator.

I3C Disrupts ER-Responsive ERE Activity and Estrogen-Induced Expression of Endogenous Progesterone Receptor

View larger version (10K): [in this window] [in a new window]   Fig. 5. I3C Inhibition of ERE Activity Induced by E or the ER-Specific Agonist PPT MCF7 cells transfected with pgl2 reporter plasmid containing the vitellogenin-3xERE or with an empty pGL2 vector, were grown in steroid-deficient media supplemented with 10% dextran charcoal-stripped fetal bovine serum. Cells were then pretreated with either DMSO or 200 µM I3C for 24 h and then incubated for 24 h with the indicated combinations of 200 µM I3C (I), 10 nM E, and/or 100 nM PPT. Cell lysates were evaluated for relative luciferase activity using the Promega luciferase assay system. Bar graphs indicate relative luciferase activity (RLU) normalized to protein input with SE.

View larger version (30K): [in this window] [in a new window]   Fig. 6. I3C Disrupts E and PPT Stimulation of Progesterone Receptor (PR) Expression MCF7 cells were grown in steroid-deficient media supplemented with 10% dextran charcoal-stripped fetal bovine serum. Cells were pretreated with DMSO or 200 µM I3C for 24 h. Cells were then treated with DMSO and 200 µM I3C in the presence and absence of 10 nM E or 100 nM PPT for 24 h. Cells were harvested in trizol, and 2 µg of total RNA was subjected to reverse transcription using random primers and Mu-MLV. This cDNA (400 ng) was subjected to PCR with primers specific for progesterone receptor. GAPDH transcript levels served as loading control.

Differential Effects of I3C on the Binding of ER and ERß to the ERE

View larger version (72K): [in this window] [in a new window]   Fig. 7. Effects of I3C on the Levels of ERE Binding Forms of ERß and ER MCF7 ER-positive breast cancer cells and MDA-MB-231 ER-negative breast cancer cells were treated with DMSO or 200 µM I3C, and 2 mg of protein lysates were subjected to affinity column chromatography using biotinylated ERE conjugated to streptavidin agarose beads. After elution with high-salt solution, eluates or total cell lysates were subjected to immunoblot analysis for ER and ERß. A (Left panel), ERE binding assay using MCF7 cell lysates and final eluates were immunoblotted for ER and ERß. A (Right panel), ERE binding assay using MDA-MB-231 cell lysates and final eluates were immunoblotted for ER and ERß. B (Left panel), MCF7 cells total cell lysates immunoblotted for ER, ERß, and ß-actin (loading control). B (Right panel), MCF7 and MDA-MB-231 total cell lysates immunoblotted for ER, ERß, and ß-actin (loading control).

I3C Treatment Increases ERß-Mediated ERE and AP-1 Activity in MDA-MB-231 Breast Cancer Cells
To assess I3C regulation of ERß activity in cells expressing ERß in the absence of ER, MDA-MB-231 cells were transfected with luciferase reporter plasmids containing highly estrogen-responsive DNA elements, Vit-3xERE or AP-1. Cells were then cultured in media supplemented with steroid-deficient fetal bovine serum in the presence of the indicated combinations of 10 nM E and 200 µM I3C for 24 h, and relative luciferase-specific activity was measured as described in Materials and Methods. A parallel experiment was performed with 1 µM tamoxifen instead of E. As shown in Fig. 8A, I3C treatment caused a reproducible activation of the ERE-containing reporter plasmid activity (DMSO vs. I3C) in the absence or presence of 17ß-estradiol. As expected, E had no effect on ERE activity in this ER-deficient cell line. As shown in Fig. 8B, I3C treatment also increased AP-1-driven luciferase activity independent of E treatment. As shown in Fig. 8C, the robust I3C-mediated increase in AP-1 activity was partially reversed by tamoxifen, in accordance with previous reports that suggest that in breast cancer cells, tamoxifen can act as a pure ERß antagonist (47). These results, together with the increased binding of ERß to the ERE, suggest that I3C enhances ERß responsiveness even in the absence of an ER agonist.

View larger version (12K): [in this window] [in a new window]   Fig. 8. I3C Increases ERE and AP-1-Driven Promoter Activity in ER-Negative MDA-MB-231 Human Breast Cancer Cells MDA-MB-231 breast cancer cells transfected with a pgl2 reporter plasmid containing either the vitellogenin-3-ERE (panel A) or a consensus AP-1 site (panel B), were grown in steroid-deficient media supplemented with 10% dextran charcoal-stripped fetal bovine serum. Cells were pretreated with DMSO (D) or 200 µM I3C (I) for 24 h, and then treated with DMSO (D) or 200 µM I3C (I) in the presence and absence of 10 nM E. C, MDA-MB-231 cells transfected with AP-1-luciferase reporter construct was treated as described in panels A and B but with 1 µM tamoxifen (T) instead of E. The relative luciferase activity was examined using the Promega luciferase assay system. Bar graphs indicate the relative ERE-luciferase activity normalized to protein input with SE.

View larger version (25K): [in this window] [in a new window]   Fig. 9. I3C Suppresses E and ER-Specific Agonist PPT-Induced Proliferation of MCF7 Breast Cancer Cells A, MCF7 cells were grown in steroid-deficient media supplemented with 10% dextran charcoal-stripped fetal bovine serum. Cells were pretreated with DMSO or 100 µM I3C for 24 h, and then treated with DMSO or 100 µM I3C (I) in the presence or absence of 10 nM E or 100 nM PPT for 24 h. Cells were harvested in PBS and stained with a hypotonic solution containing propidium iodide. Stained nuclei were subjected to flow cytometry analysis as described in Materials and Methods. B, The flow cytometry results from three independent experiments were quantified. Bar graph indicates percent cells in G1, S, and G2 phases with SE bars.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

Strong correlation has been found between cellular proliferation in normal mammary glands and mammary cancers to the relative levels of ER and ERß. ERß expression levels were higher in normal human mammary epithelial cells in vivo, and in nonproliferative benign breast disease compared with its levels in more malignant preinvasive lesions, such as ductal carcinoma in situ, which usually overexpress ER (7, 8, 14). The roles of ER and ERß in mammary epithelial cell proliferation have been evaluated in knock out mouse models (2, 3), as well as in human breast cancer cell lines in culture (10, 11). The proliferative effects of estrogens are thought to be mediated primarily by ER, because ablation of ER in mouse mammary glands leads to a severely underdeveloped mammary epithelium. The ER null epithelium still expresses ERß, and the mammary epithelial growth impairment cannot be rescued by administration of pharmacological levels of estrogens (2). This genotype is also associated with a major decrease in the development of estrogen-unresponsive mammary cancers indicating that estrogen signaling through ER plays a major role in proliferation of normal mammary epithelial cells in vivo as well as in hormone-responsive and hormone-unresponsive mammary carcinogenesis (1, 2). Current breast cancer therapeutic drugs that inactivate ER, such as tamoxifen, have been shown to prevent development of carcinogen-induced rat mammary tumors (16) and prevent breast cancer in humans (42), which further establishes a positive correlation between ER and cellular proliferation as well as mammary carcinogenesis. In contrast to the information currently available on ER, the precise significance of ERß in human breast cancers remains unclear, although ERß is thought to play a role in inhibiting mammary epithelial cell proliferation during lactation and involution in the normal mammary gland. ERß null mice show normal development of the mammary gland until lactation. Lactating wild-type mice show a marked cessation of mammary epithelial cell proliferation, whereas ERß null mice show significant levels of mammary epithelial cell proliferation during lactation (43).

Our observation that I3C down-regulates ER gene expression by an inhibition of promoter activity is consistent with the results of our previous studies demonstrating that I3C strongly down-regulates CDK6 promoter activity in human breast cancer cells that undergo a G1 cell cycle arrest (36, 39). I3C disrupts the function of the Sp1 and ETS transcription factors at a composite element in the CDK6 promoter (31, 39). Interestingly, the kinetics of ER down-regulation by I3C is strikingly similar to that of CDK6 (39), suggesting that an analogous regulatory mechanism may be operating on ER. Consistent with this concept, I3C inhibited a luciferase reporter plasmid containing a 3200-bp ER promoter fragment, but had no effect when the reporter plasmid was linked to a 700-bp 5'-deleted promoter fragment. The larger ER promoter fragment contained both the transcription start sites (B at –1994 bp and A), suggesting the existence of an I3C-regulated transcriptional element in the ER promoter between –3200 and –700. This region contains binding sites for Sp1, the binding of which is disrupted by I3C leading to transcriptional down-regulation of CDK6 (39). We are currently in the process of determining the precise location of the I3C-regulated element and of identifying which transcription factor(s) binding is/are targeted by I3C.

Several lines of evidence show that a key consequence of the I3C down-regulation of ER transcription is the loss of ER responsiveness. I3C ablates the stimulation of growth and activation of an ERE reporter plasmid by PPT, a highly selective ER agonist that possesses a 400-fold higher affinity for ER than ERß (4). Furthermore, I3C treatment blocks the E and PPT-stimulated expression of progesterone receptor, a known ER-responsive gene. Interestingly, PPT stimulates growth of MCF7 and T47D cells to the same extent as 17ß-estradiol. I3C also appears to activate ERß by inducing a significant increase in the DNA binding form of ERß, and by stimulating vitellogenin ERE reporter plasmids in MDA-MB-231 breast cancer cells that express ERß, but not ER. It is interesting to note that I3C is effective in causing a cell cycle arrest in MDA-MB-231 breast cancer cells (36), and this correlates well with activation of ERß. These breast cancer cells are unlikely to express any unusual ER isoforms because this gene is heavily silenced by methylation (40). We are currently attempting to elucidate the mechanism by which I3C activates ERß function in human breast cancer cells.

Estrogen signaling has been shown to be essential for the proliferation of normal mammary epithelial cells and is a key risk factor in the development of estrogen-responsive as well as estrogen-unresponsive breast cancer (1, 3, 14, 17, 42). It has been established that benign lesions showing elevated levels of ER pose a higher risk for development of malignant breast cancer linking ER to estrogen-mediated increase in tumorigenicity in rodents and humans (44, 45). Conceivably, a potential prophylactic use of I3C is to down-regulate ER in high-risk but benign ER overexpressing premalignant cell populations, thus effectively dampening the promotion of premalignant lesions to malignancy by estrogens. Furthermore, I3C has been shown to cooperate with the antiestrogen tamoxifen in that a combination of tamoxifen and I3C caused a more potent growth arrest than either compound alone (32). These findings suggest that this indole could be used as a potential adjuvant therapeutic for estrogen-responsive breast cancer. Taken together, our results implicate I3C as a new class of therapeutic molecules that mediate their anticancer effects in estrogen-responsive cell types through the selective control of ER gene expression.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Materials
DMEM, Iscove’s modified Dulbecco’s medium, fetal bovine serum, calcium- and magnesium-free PBS, L-glutamine, and trypsin-versene mixtures were purchased from BioWhittaker, Inc. (Walkersville, MD). Insulin (bovine), and 17ß-estradiol, dimethyl sulfoxide, and tamoxifen were purchased from Sigma Chemical Co. (St. Louis, MO). PPT was obtained from Tocris (Ellisville, MO). I3C, DIM, and Tryptophol were purchased from Aldrich (Milwaukee, WI). The sources of other reagents are either listed below or were of the highest purity available.

Cell Culture
MCF7 and T47D human breast adenocarcinoma cells were obtained from the American Type Culture Collection (Manassas, VA). MCF7 cells were grown in DMEM supplemented with 10% fetal bovine serum (BioWhittaker), 10 µg/ml insulin, 50 U/ml penicillin, 50 U/ml streptomycin, and 2 mM L-glutamine. T47D cells were grown in Iscove’s modified Dulbecco’s medium containing 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 U/ml streptomycin. The cells were grown to subconfluency in a humidified air chamber at 37 C containing 5% CO₂. Stock solutions of I3C, DIM, Tryptophol (99.9% HPLC grade), 17ß-estradiol, PPT, and tamoxifen were dissolved in DMSO to yield appropriate concentrations. DMSO was used as vehicle control for all experiments.

ER Gene Promoter Transfections and Luciferase Activity Assays
Plasmids containing 5'-deletions of ER gene promoter were a kind gift from Dr. Lisa McPherson, Stanford University. MCF7 cells, grown to confluency in six-well plates, were transfected with 1 µg of plasmid constructs containing either –3.2 kb upstream region of the ER gene with both promoter sites B and A or a 700-bp upstream region containing only start site A. Transfection was performed in serum-supplemented media using Fugene 6 (Roche, Indianapolis, IN) as per manufacturer’s instructions. Cells were treated 24 h post transfection with DMSO or 200 µM I3C for 48 h. Cells were lysed, and relative luciferase activity was evaluated using Promega luciferase assay kit (Promega Corp., Madison, WI) and a luminometer. Relative luciferase activities were normalized to the protein input with SE. Reproducibility of these results was verified with three independent experiments performed with triplicate samples for each treatment.

ERE-Luciferase/AP-1-Luciferase Transfections and Luciferase Assays
Plasmid (1 µg) containing the consensus vitellogenin vit-3xERE in pgl2 vector or the consensus AP-1 sites was transfected according to manufacturer’s instructions using Fugene 6 (Roche). The media were replaced, 24 h later, with media containing 10% dextran charcoal-stripped fetal bovine serum, and then after an additional 24 h, the cells were treated with DMSO or 200 µM I3C for 48 h. The cells were then treated with DMSO, 10 nM 17ß-estradiol, or 100 nM PPT for 24 h, lysed, and subjected to luciferase activity assays using the luciferase assay kit (Promega). The amount of protein was determined using the Lowry method, and the RLUs were normalized to protein input. These transfection assays were carried out three independent times employing triplicate samples for each treatment group.

Western Blotting for ER and ERß Protein
After the indicated treatments, MCF7 and T47D cells were harvested in the media, pelleted by centrifugation at 8000 rpm for 5 min, resuspended in 1 ml PBS, and pelleted again by centrifugation. These cells were then resuspended in radio immunoprecipitation buffer (150 mM NaCl, 0.5% deoxycholate, 0.1% Nonidet P-40, 0.1% SDS, and 50 mM Tris) containing protease inhibitors (50 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 µg/ml NaF, and 10 µg/ml ß-glycerophosphate). These extracts were then quantified using the Lowry Method (Bio-Rad Laboratories, Hercules, CA). Normalized amounts of protein were mixed with sample buffer [25% glycerol, 0.075% SDS, 1.25 ml ß-mercaptoethanol, 10% bromophenol blue, 3.13% 0.5 M SDS, and 0.4% SDS (pH 6.8)], and fractionated on 8% polyacrylamide/0.1% SDS resolving gels using electrophoresis. Prestained protein ladder (Amersham Life Sciences, Arlington Heights, IL) was used as reference for size. Proteins were then transferred electrically to nitrocellulose membranes for 1 h at 4 C. Equal loading was confirmed with Ponceau S incubation of membranes. The blots were then blocked for 1 h in 5% nonfat dry milk dissolved in Western wash buffer [10 mM Tris-HCl (pH 8), 150 mM NaCl, and 0.05% Tween 20] at room temperature. The blots were the rinsed in Western wash buffer and incubated overnight at 4 C in antibodies diluted in Western wash buffer. Rabbit anti-ER and -ERß antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz CA) were diluted 1:1000 in wash buffer. CDK2 immunoblotting was performed as described previously (34). Immunoreactive proteins were detected after incubation with horseradish peroxidase-conjugated secondary antibodies diluted to 3 x 10^–4 in 1% non-fat dry milk in Western wash buffer (goat antirabbit IgG; Bio-Rad Laboratories). Blots were then treated with enhanced chemiluminescence reagents (NEN Life Sciences, Boston, MA), and all proteins were detected by exposure to Hyperfilm (Eastman Kodak, Rochester, NY). Immunoblotting for ER and ERß was repeated using total protein extracts from two independent experiments.

RNA Extraction and RT-PCRs
MCF7 cells were grown in appropriate media and, after the indicated treatment durations, were harvested in 1 ml of Trizol reagent (Invitrogen, San Diego, CA), and the recommended protocol was followed to extract total RNA. RNA was quantified using spectroscopy, and the quality of RNA was confirmed using A260/A280, and by electrophoresis on 1% agarose gels. Total RNA (2 µg) was subjected to reverse transcription using Mu-MLV Reverse Transcriptase (Invitrogen) with random hexamers, deoxynucleotide triphosphates, and RNAse Inhibitor (Invitrogen). cDNA (4 µl) was then subjected to PCR using primers specific to ER, ERß, PR, and GAPDH as described elsewhere (46) The PCR products were electrophoretically fractionated in 1% Agarose gels along with a 1-kb plus DNA ladder and visualized on a UV transilluminator. RT-PCR was thus performed on total RNA from two independent experiments to confirm the observed expression patterns for ER and ERß.

Affinity Chromatography for ER-ERE Binding
The pBluescript plasmid containing the consensus vitellogenin ERE was subjected to restriction enzyme digest using BglII and HindIII. The products were electrophoresed on a 1% agarose gel containing ethidium bromide. The 550-bp band corresponding to the ERE was excised and extracted using the Qiaex II kit (QIAGEN, Chatsworth, CA). Approximately 16 µg of ERE fragment was biotinylated using Klenow enzyme as per manufacturer’s protocol and was extracted by ethanol/salt extraction. Overall, 7 µg of DNA was then used for the chromatographic assay. MCF7 and MDA-MB-231 cells treated with I3C or DMSO were lysed using a buffer containing 10 mM HEPES, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, and 2 mM EDTA, and a cocktail of protease inhibitors without dithiothreitol. Protein content of cell lysates was then normalized using the Lowry method (Bio-Rad Laboratories). Streptavidin-conjugated agarose beads (Sigma) were resuspended in 400 µl of lysis buffer, and the column was divided into two Eppendorf tubes with 200 µl each. Approximately 2 mg of total protein from DMSO- or I3C-treated cells was added to each column and incubated at 4 C for 1 h with constant rocking using a nutator (Clay Adams, Sparks, MD). The column was then centrifuged at 4 C for 5 min at 5000 rpm. The unbound lysate was stored, and the columns were washed three times using lysis buffer. The elution buffer was prepared by adding 400 mM NaCl to the lysis buffer and added to the beads. After incubation at room temperature for 5 min, the beads were centrifuged, and the supernatant was loaded and subjected to SDS-PAGE. The amount of bound ERs was assayed by immunoblotting as described above. Using different batches of cells and drugs, this procedure was repeated five independent times.

Flow Cytometric Analyses of DNA Content
MCF7 cells were plated onto six-well tissue culture dishes (Nunc Fischer, Hampton, NH). The cells were grown in media containing dextran charcoal-stripped 10% fetal bovine serum. Cells were treated with 10 nM E or 100 nM PPT in the presence and absence of 100 µM I3C. Cells were incubated for 48 h and hypotonically lysed in 1 ml of DNA staining solution (0.5 mg/ml propidium iodide, 0.1% sodium citrate, and 0.05% Triton X-100). Nuclear emitted fluorescence with wavelength of more than 585 nm was measured with a Coulter Elite instrument with laser output adjusted to deliver 15 mW at 488 nm. Nuclei (10,000) were analyzed from each sample at a rate of 300–500 nuclei. The percentages of cells within the G1, S, and G2-M phases of the cell cycle were determined by analysis with the Multicycle computer program provided by Phoenix Flow Systems in the Cancer Research Laboratory Microchemical Facility of the University of California, Berkeley. This experiment was repeated twice to confirm observed results.

	ACKNOWLEDGMENTS

 
We thank Jamin A. Willoughby Sr. and Andrea Steely for their immense help with the manuscript preparation. We also acknowledge the excellent technical assistance of Ms. Payal Patel.

	FOOTNOTES

 
This work was supported by National Institutes of Health (NIH) Public Health Service Grant CA102360 from the National Cancer Institute. S.N.S. is a recipient of a predoctoral fellowship supported by NIH National Research Service Grant CA09041.

First Published Online August 10, 2006

¹ S.N.S. and V.K. contributed equally to this work.

Abbreviations: AP-1, Activator protein 1; CDK, cyclin-dependent kinase; CMV, cytomegalovirus; DIM, 3'-3'-dindolylmethane; DMSO, dimethylsulfoxide; E, 17ß-Estradiol; ER, estrogen receptor; ERE, estrogen response element; GAPDH, Glyceraldehyde phosphate dehydrogenase; I3C, Indole-3-carbinol; PPT, Propyl pyrazole triol; RLU, relative light unit; SDS, sodium dodecyl sulfate.

Received for publication July 1, 2005. Accepted for publication July 21, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Bocchinfuso WP, Korach KS 1997 Mammary gland development and tumorigenesis in estrogen receptor knockout mice. J Mammary Gland Biol Neoplasia 2:323–334[CrossRef][Medline]
2. Bocchinfuso WP, Lindzey JK, Hewitt SC, Clark JA, Myers PH, Cooper R, Korach KS 2000 Induction of mammary gland development in estrogen receptor- knockout mice. Endocrinology 141:2982–2994[Abstract/Free Full Text]
3. Enmark E, Gustafsson JA 1999 Oestrogen receptors—an overview. J Intern Med 246:133–138[CrossRef][Medline]
4. Harris HA, Katzenellenbogen JA, Katzenellenbogen BS 2002 Characterization of the biological roles of the estrogen receptors, ER and ERß, in estrogen target tissues in vivo through the use of an ER-selective ligand. Endocrinology 143:4172–4177[Abstract/Free Full Text]
5. Couse JF, Korach KS 2001 Contrasting phenotypes in reproductive tissues of female estrogen receptor null mice. Ann NY Acad Sci 948:1–8[Abstract/Free Full Text]
6. Couse JF, Korach KS 1999 Estrogen receptor null mice: what have we learned and where will they lead us? Endocr Rev 20:358–417[Abstract/Free Full Text]
7. Roger P, Sahla ME, Makela S, Gustafsson JA, Baldet P, Rochefort H 2001 Decreased expression of estrogen receptor ß protein in proliferative preinvasive mammary tumors. Cancer Res 61:2537–2541[Abstract/Free Full Text]
8. Shaaban AM, O’Neill PA, Davies MP, Sibson R, West CR, Smith PH, Foster CS 2003 Declining estrogen receptor-ß expression defines malignant progression of human breast neoplasia. Am J Surg Pathol 27:1502–1512[Medline]
9. Campbell-Thompson M, Lynch IJ, Bhardwaj B 2001 Expression of estrogen receptor (ER) subtypes and ERß isoforms in colon cancer. Cancer Res 61:632–640[Abstract/Free Full Text]
10. Paruthiyil S, Parmar H, Kerekatte V, Cunha GR, Firestone GL, Leitman DC 2004 Estrogen receptor ß inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest. Cancer Res 64:423–428[Abstract/Free Full Text]
11. Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson JA 2004 Estrogen receptor ß inhibits 17ß-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad Sci USA 101:1566–1571[Abstract/Free Full Text]
12. Saji S, Sakaguchi H, Andersson S, Warner M, Gustafsson J 2001 Quantitative analysis of estrogen receptor proteins in rat mammary gland. Endocrinology 142:3177–3186[Abstract/Free Full Text]
13. Shyamala G, Chou YC, Louie SG, Guzman RC, Smith GH, Nandi S 2002 Cellular expression of estrogen and progesterone receptors in mammary glands: regulation by hormones, development and aging. J Steroid Biochem Mol Biol 80:137–148[CrossRef][Medline]
14. Ali S, Coombes RC 2000 Estrogen receptor in human breast cancer: occurrence and significance. J Mammary Gland Biol Neoplasia 5:271–281[CrossRef][Medline]
15. Burke C 2005 Endometrial cancer and tamoxifen. Clin J Oncol Nurs 9:247–249[CrossRef][Medline]
16. Jang TJ, Park JH, Cho MY, Kim JR 2001 Chemically induced rat mammary tumor treated with tamoxifen showed decreased expression of cyclin D1, cyclin E, and p21(Cip1). Cancer Lett 170:109–116[CrossRef][Medline]
17. Seeger H, Diesing D, Guckel B, Wallwiener D, Mueck AO, Huober J 2003 Effect of tamoxifen and 2-methoxyestradiol alone and in combination on human breast cancer cell proliferation. J Steroid Biochem Mol Biol 84:255–257[CrossRef][Medline]
18. Vergote I, Robertson JF 2004 Fulvestrant is an effective and well-tolerated endocrine therapy for postmenopausal women with advanced breast cancer: results from clinical trials. Br J Cancer 90(Suppl 1):S11–S14
19. Lynn J 2004 Fulvestrant (‘Faslodex’)–a new hormonal treatment for advanced breast cancer. Eur J Oncol Nurs 8(Suppl 2):S83–S88
20. Gradishar WJ 2005 Clinical value of fulvestrant in breast cancer. Clin Breast Cancer 6(Suppl 1):S4
21. Peekhaus NT, Chang T, Hayes EC, Wilkinson HA, Mitra SW, Schaeffer JM, Rohrer SP 2004 Distinct effects of the antiestrogen Faslodex on the stability of estrogen receptors- and -ß in the breast cancer cell line MCF-7. J Mol Endocrinol 32:987–995[Abstract]
22. Teel RW, Huynh H 1998 Modulation by phytochemicals of cytochrome P450-linked enzyme activity. Cancer Lett 133:135–141[CrossRef][Medline]
23. Dreosti IE 1998 Nutrition, cancer, and aging. Ann NY Acad Sci 854:371–377[Abstract/Free Full Text]
24. Singh RP, Tyagi AK, Dhanalakshmi S, Agarwal R, Agarwal C 2004 Grape seed extract inhibits advanced human prostate tumor growth and angiogenesis and upregulates insulin-like growth factor binding protein-3. Int J Cancer 108:733–740[CrossRef][Medline]
25. Silalahi J 2002 Anticancer and health protective properties of citrus fruit components. Asia Pac J Clin Nutr 11:79–84[CrossRef][Medline]
26. Yao LH, Jiang YM, Shi J, Tomas-Barberan FA, Datta N, Singanusong R, Chen SS 2004 Flavonoids in food and their health benefits. Plant Foods Hum Nutr 59:113–122[CrossRef][Medline]
27. Chen MS, Chen D, Dou QP 2004 Inhibition of proteasome activity by various fruits and vegetables is associated with cancer cell death. In Vivo 18:73–80[Medline]
28. Grubbs CJ, Steele VE, Casebolt T, Juliana MM, Eto I, Whitaker LM, Dragnev KH, Kelloff GJ, Lubet RL 1995 Chemoprevention of chemically-induced mammary carcinogenesis by indole-3-carbinol. Anticancer Res 15:709–716[Medline]
29. Bradlow HL, Michnovicz J, Telang NT, Osborne MP 1991 Effects of dietary indole-3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice. Carcinogenesis 12:1571–1574[Abstract/Free Full Text]
30. Zhu CY, Loft S 2003 Effect of chemopreventive compounds from Brassica vegetables on NAD(P)H:quinone reductase and induction of DNA strand breaks in murine hepa1c1c7 cells. Food Chem Toxicol 41:455–462[CrossRef][Medline]
31. Firestone GL, Bjeldanes LF 2003 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. J Nutr 133:2448S–2455S
32. Cover CM, Hsieh SJ, Cram EJ, Hong C, Riby JE, Bjeldanes LF, Firestone GL 1999 Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells. Cancer Res 59:1244–1251[Abstract/Free Full Text]
33. Leong H, Firestone GL, Bjeldanes LF 2001 Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells result from an estrogen receptor-mediated increase in transforming growth factor- expression. Carcinogenesis 22:1809–1817[Abstract/Free Full Text]
34. Zhang J, Hsu BAJ, Kinseth BAM, Bjeldanes LF, Firestone GL 2003 Indole-3-carbinol induces a G1 cell cycle arrest and inhibits prostate-specific antigen production in human LNCaP prostate carcinoma cells. Cancer 98:2511–2520[CrossRef][Medline]
35. Le HT, Schaldach CM, Firestone GL, Bjeldanes LF 2003 Plant-derived 3,3'-diindolylmethane is a strong androgen antagonist in human prostate cancer cells. J Biol Chem 278:21136–21145[Abstract/Free Full Text]
36. Cover CM, Hsieh SJ, Tran SH, Hallden G, Kim GS, Bjeldanes LF, Firestone GL 1998 Indole-3-carbinol inhibits the expression of cyclin-dependent kinase-6 and induces a G1 cell cycle arrest of human breast cancer cells independent of estrogen receptor signaling. J Biol Chem 273:3838–3847[Abstract/Free Full Text]
37. Garcia HH, Brar GA, Nguyen DH, Bjeldanes LF, Firestone GL 2005 Indole-3-carbinol (I3C) inhibits cyclin-dependent kinase-2 function in human breast cancer cells by regulating the size distribution, associated cyclin E forms, and subcellular localization of the CDK2 protein complex. J Biol Chem 280:8756–8764[Abstract/Free Full Text]
38. Ashok BT, Chen YG, Liu X, Garikapaty VP, Seplowitz R, Tschorn J, Roy K, Mittelman A, Tiwari RK 2002 Multiple molecular targets of indole-3-carbinol, a chemopreventive anti-estrogen in breast cancer. Eur J Cancer Prev 11(Suppl 2):S86–S93
39. Cram EJ, Liu BD, Bjeldanes LF, Firestone GL 2001 Indole-3-carbinol inhibits CDK6 expression in human MCF-7 breast cancer cells by disrupting Sp1 transcription factor interactions with a composite element in the CDK6 gene promoter. J Biol Chem 276:22332–22340[Abstract/Free Full Text]
40. Yang X, Phillips DL, Ferguson AT, Nelson WG, Herman JG, Davidson NE 2001 Synergistic activation of functional estrogen receptor (ER)- by DNA methyltransferase and histone deacetylase inhibition in human ER--negative breast cancer cells. Cancer Res 61:7025–7029[Abstract/Free Full Text]
41. Hu K, Zhong G, He F 2005 Expression of estrogen receptors ER and ERß in endometrial hyperplasia and adenocarcinoma. Int J Gynecol Cancer 15:537–541[CrossRef][Medline]
42. Brown K 2002 Breast cancer chemoprevention: risk-benefit effects of the antioestrogen tamoxifen. Expert Opin Drug Saf 1:253–267[CrossRef][Medline]
43. Forster C, Makela S, Warri A, Kietz S, Becker D, Hultenby K, Warner M, Gustafsson JA 2002 Involvement of estrogen receptor ß in terminal differentiation of mammary gland epithelium. Proc Natl Acad Sci USA 99:15578–15583[Abstract/Free Full Text]
44. Medina D, Kittrell FS, Hill J, Shepard A, Thordarson G, Brown P 2005 Tamoxifen inhibition of estrogen receptor--negative mouse mammary tumorigenesis. Cancer Res 65:3493–3496[Abstract/Free Full Text]
45. Shaaban AM, Sloane JP, West CR, Foster CS 2002 Breast cancer risk in usual ductal hyperplasia is defined by estrogen receptor- and Ki-67 expression. Am J Pathol 160:597–604[Abstract/Free Full Text]
46. de Cremoux P, Tran-Perennou C, Elie C, Boudou E, Barbaroux C, Poupon MF, De Rycke Y, Asselain B, Magdelenat H 2002 Quantitation of estradiol receptors and ß and progesterone receptors in human breast tumors by real-time reverse transcription-polymerase chain reaction. Correlation with protein assays. Biochem Pharmacol 64:507–515[CrossRef][Medline]
47. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson J, Nilsson S 1998 Differential response of estrogen receptor and estrogen receptor ß to partial estrogen agonists/antagonists. Mol Pharmacol 54:105–112[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  ERβ

Ligands:   17β-Estradiol

This Article Abstract Full Text (PDF) All Versions of this Article: 20/12/3070    most recent Author Manuscript (PDF)