Cancer Research AACR Conference on Molecular Diagnostics - 2008 Cancer Health Disparities Conference 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, S. O.
Right arrow Articles by Gao, A. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, S. O.
Right arrow Articles by Gao, A. C.
Related Collections
Right arrow Preclinical Intervention
Right arrow Preclinical Intervention: In Vitro: Drugs, Mechanisms
[Cancer Research 65, 3487-3492, April 15, 2005]
© 2005 American Association for Cancer Research

Epidemiology and Prevention

Selenium Disrupts Estrogen Signaling by Altering Estrogen Receptor Expression and Ligand Binding in Human Breast Cancer Cells

Soo Ok Lee1, Nagalakshmi Nadiminty1, Xiu Xian Wu1, Wei Lou1, Yan Dong2, Clement Ip2, Sergio A. Onate3 and Allen C. Gao1

Departments of 1 Medicine, Pharmacology and Therapeutics, 2 Cancer Prevention and Population Sciences, and 3 Urologic Oncology, Roswell Park Cancer Institute, Buffalo, New York

Requests for reprints: Allen C. Gao, Grace Cancer Drug Center, Departments of Medicine, Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-1201; Fax: 716-845-8857; E-mail: allen.gao{at}roswellpark.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cancer prevention studies suggest that selenium is effective in reducing the incidence of cancers including prostate, colon, and lung cancers. Previous reports showed that selenium inhibits premalignant human breast MCF-10AT1 and MCF10AT3B cell growth in vitro and reduces mammary tumor incidence after exposure to carcinogens in tumor models. Because estrogen is critical to the development and differentiation of estrogen target tissues, including the breast, the present study was designed to examine the effect of selenium on estrogen receptor (ER) expression and activation using methylseleninic acid (MSA), an active form of selenium in vitro. Selenium decreased the levels of expression of ER{alpha} mRNA and protein and reduced the binding of labeled estradiol to estrogen receptor in MCF-7 cells. Selenium inhibited the trans-activating activity of estrogen receptor in MCF-7 cells expressing functional estrogen receptor using a luciferase reporter construct linked to estrogen responsive element. Selenium decreased the binding of estrogen receptor to the estrogen responsive element site using an electrophoretic mobility gel shift assay. Selenium suppressed estrogen induction of the endogenous target gene c-myc. In contrast to the effect on ER{alpha} in MCF-7 cells, selenium increased ERß mRNA expression in MDA-MB231 human breast cancer cells. Thus, differential regulation of ER{alpha} and ERß in breast cancer cells may represent a novel mechanism of selenium action and provide a rationale for selenium breast cancer prevention trial.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The growth of breast cancer cells is known to be regulated by estrogen through binding to estrogen receptor (ER), which affects cell growth by inducing cell proliferation (1–3) and preventing apoptotic cell death (4, 5). Estrogen receptor is a hormone-dependent transcription factor that belongs to the steroid/thyroid hormone receptor superfamily. Estrogen receptor is activated by ligand binding, followed by receptor conformational changes and dimerization and binding to estrogen response elements located in the promoter of estrogen regulated genes. The majority of the actions of estrogen are mediated by ER{alpha} and ERß. Since estrogen effects can be modulated by the agonists and antagonists of these receptors, compounds that block estrogen signaling have proven useful in the treatment of estrogen receptor–positive breast cancer patients. Tamoxifen is widely used for the treatment of breast cancer (6).

A landmark cancer prevention trial showed that selenium supplementation was effective in reducing the incidence of cancers including prostate, lung, and colon cancers (7). This trial did not detect a statistically significant change in breast cancer risk by selenium supplementation (7). A likely explanation is the very small number of breast cancer cases in both the placebo and treatment groups, since women accounted for only about 25% of the cohort. A number of prospective case-control studies have also failed to observe decreases of breast cancer risk in women with higher blood or toenail selenium levels (8, 9). A recent epidemiologic study indicated that lower serum concentrations of selenium in women with breast cancer compared to healthy women seemed to be a consequence, rather than a cause, of cancer (10). It should be noted that these epidemiologic studies were designed to evaluate the impact of selenium within the range of dietary intake, i.e., without supplementation. Although the effect of selenium within the range of dietary intake on breast cancer risk was not observed, pharmacologic concentrations of selenium effect were detected in mammary tumor models (11, 12). Several studies showed the efficacy of selenium in the reduction of mammary tumor incidence after exposure to carcinogens, including 2-acetylaminofluorene, methylnitrosourea, and 7,12-dimethylbenz(a)anthracene (13–16). In addition, selenium inhibited the growth of two premalignant human breast cell lines by blocking cell cycle progression at the G0-G1 phase and inducing apoptotic cell death (17). Methylseleninic acid (CH3SeO2H, abbreviated as MSA) was developed specifically for in vitro studies (18), since cultured cells respond poorly to selenomethionine (a commonly used selenium reagent) due to very low levels of ß-lyase activity, which is required for conversion of selenomethionine to the active methylselenol (19). The effect of physiologic concentrations of MSA on cultured cells has been documented in several publications (18, 20–22). Although selenium is an effective chemopreventive agent in mammary models, the effect of selenium on estrogen receptor signaling is currently unknown. In the present study, the effect of MSA on estrogen receptor signaling in breast cancer cells was examined. MCF-7 cells are hormone-dependent breast cancer cells that express high levels of ER{alpha} but very low levels of ERß. MDA-MB231 human breast cancer cells are ER{alpha}-negative but ERß-positive. MSA inhibited ER{alpha} expression and estrogen receptor–mediated gene activation and reduced the binding of 3H-labeled estradiol to the estrogen receptor in MCF-7 cells. In contrast to ER{alpha} in MCF-7 cells, selenium increased ERß expression in MDA-MB231 human breast cancer cells. Differential regulation of ER{alpha} and ERß expression in breast cancer may represent a novel mechanism by which selenium functions as a chemopreventive agent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Selenium reagent, cell culture, and cell growth analysis. MSA was synthesized as described previously (18). ß-Estradiol was purchased from Sigma (St. Louis, MO). The MCF-7 and MDA-MB231 human breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 100 units/mL of penicillin and 100 µg/mL of streptomycin (17). In some experiments, cells were cultured in an estrogen-defined condition by using charcoal-stripped FBS in the presence of 10 nmol/L ß-estradiol. For cell growth analysis by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (23), cells were seeded in 24-well plates at a density designed to reach 70% to 80% confluency at the time of assay. Cells were treated with 1, 2.5, 5, or 10 µmol/L MSA in triplicate 48 hours after seeding. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done after 24, 48, or 72 hours of treatment. For the quantitative determination of estrogen receptor transcripts and proteins, cells were exposed to MSA for much shorter periods of time, usually 24 hours or less. Total RNA and protein were isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA), and stored at –80°C for subsequent real-time reverse transcription-PCR analysis.

Real-time reverse transcription-PCR. First-strand cDNA was synthesized from total RNA by SuperScript II reverse transcriptase (Invitrogen) following the manufacturer's protocol. Briefly, 400 ng of total RNA was mixed with 150 ng of random hexamers in a final volume of 100 µL containing 1x first-strand buffer [50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2], 10 mmol/L DTT, 500 µmol/L of each deoxynucleotide triphosphate, and 200 units of SuperScript II reverse transcriptase. The samples were incubated at 42°C for 50 minutes and the reverse transcriptase was inactivated by heating at 70°C for 15 minutes.

The PCR primers and TaqMan probes for ß-actin, ER{alpha}, and ERß were Assays-on-Demand products from Applied Biosystems (Applied Biosystems, Foster City, CA). Two microliters of first-strand cDNA was mixed with 25 µL of 2x TaqMan Universal PCR Master Mix (Applied Biosystems) and 2.5 µL of 20x primer/probe mixture in a final volume of 50 µL. Temperature cycling and real-time fluorescence measurement were done using an ABI prism 7300 Sequence Detection System (Applied Biosystems). The PCR conditions were as follows: initial incubation at 50°C for 2 minutes, denaturation at 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute.

The relative quantitation of gene expression was done using the comparative CT ({Delta}{Delta}CT) method (21). Briefly, the threshold cycle number (CT) was obtained as the first cycle at which a statistically significant increase in fluorescence signal was detected. Data was normalized by subtracting the CT value of ß-actin from that of the target gene. There was a match control sample for each treatment sample. Each reaction was done in duplicate and the CT values were averaged. The {Delta}{Delta}CT was calculated as the difference of the normalized CT values ({Delta}CT) of the treatment and control samples: {Delta}{Delta}CT = {Delta}CT treatment – {Delta}CT control. {Delta}{Delta}CT was converted to fold of change by the following formula: fold of change = 2–{Delta}{Delta}CT.

Western blot analysis. Details of the procedure were described previously (24). Immunoreactive bands were quantitated using volume densitometry and normalized against {alpha}-actin. The following monoclonal antibodies were used (source): anti-{alpha}-actin (Sigma), anti–estrogen receptor (BD Transduction Laboratory, San Jose, CA) and anti-c-myc (Santa Cruz Biotechnology, Santa Cruz, CA).

Transfection and luciferase assay. An aliquot of 3 x 105 cells was placed in a six-well plate and transfected with a total amount of 5 µg of DNA using Superfect (Qiagen, Valencia, CA) according to the manufacturer's instructions. The estrogen response element-luciferase reporter plasmid was used (25). The total amount of plasmid DNA was normalized to 5 µg per well by adding empty plasmid. The DNA/liposome mixture was removed 3 hours later, and cells were treated with 10 nmol/L ß-estradiol in charcoal-stripped FBS condition and different concentrations of MSA. Cell extracts were obtained after 24 hours and luciferase activity was assayed using the Luciferase Assay System (Promega, Madison, WI). Protein concentrations in cell extracts were determined using the Coomassie Plus protein assay kit (Pierce, Rockford, IL). Luciferase activities were normalized using the protein concentration of the sample. All transfection experiments were done in triplicate wells and repeated at least four times. The relative luciferase activity was averaged from at least four independent experiments each with triplicate wells. The results were expressed as the percentage of untreated control.

Nuclear lysate preparation. Nuclear protein extract was prepared as described previously (26). Cells were harvested, washed with PBS twice and resuspended in a hypotonic buffer [10 mmol/L HEPES-KOH (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, and 0.1% NP40] and incubated on ice for 10 minutes. Nuclei were precipitated by 3,000 x g centrifugation at 4°C for 10 minutes. After washing once with the hypotonic buffer, the nuclei were lysed in a lysis buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, and 1% Triton X-100] and incubated on ice for 30 minutes. The nuclear lysate was precleared by 10,000 x g centrifugation at 4°C for 15 minutes. Protein concentration was determined using the Coomassie Plus protein assay kit.

Electrophoretic mobility shift assay. Twenty micrograms of nuclear protein extract were incubated in a 20 µL solution containing 10 mmol/L HEPES (pH 7.9), 80 mmol/L NaCl, 10% glycerol, 1 mmol/L DTT, 1 mmol/L EDTA, 100 µg/mL poly(deoxyinosinic-deoxycytidylic acid), and the radiolabeled double-stranded estrogen receptor consensus binding motif 5'-GGATCTAGGTCACTGTGACCCCGGATC-3' (Santa Cruz Biotechnologies). The protein-DNA complexes were resolved on a 4.5% nondenaturing polyacrylamide gel containing 2.5% glycerol in 0.25x Tris-borate EDTA at room temperature and the gels were autoradiographed. Quantitation of estrogen receptor DNA-binding activity in the "protein-DNA" bandshift was measured using the Molecular Imager FX System (Bio-Rad, Hercules, CA). Two- and 4-fold molar excess of cold oligonucleotide were used for competition analysis.

In vitro estrogen receptor binding activity. MCF-7 cells were plated at 1 x 106 cells per dish in 60 mm dishes in DMEM (phenol red–free) + 10% charcoal-stripped FBS and incubated at 37°C for 2 days. On the third day, dishes were either treated or left untreated with 5 µmol/L MSA in 2 mL of media each and incubated at 37°C for 4 hours. Cells were scraped off dishes and homogenized in TEDG buffer [10 mmol/L Tris, 1.5 mmol/L EDTA, 1 mmol/L DTT, 10% glycerol (pH 7.4)]. Cell suspensions were passed 10 to 15 times through a 26-gauge needle and incubated on ice for 10 minutes. The homogenate was centrifuged at 13,000 x g for 30 minutes at 4°C. The supernatant was collected and used as the cytosol. Total protein was estimated in both the MSA-treated and untreated cell lysates and equal amounts of protein were used in the subsequent assay.

The reaction mixtures contained 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, and 1 nmol/L [3H]estradiol with or without 1 x 10–6 mol/L cold estradiol and equal amounts (100-200 µg) of protein in a total volume of 250 µL. The tubes were incubated overnight at 4°C. Dextran-coated charcoal suspension (500 µL) was added to each tube and incubated for 10 minutes with vigorous shaking at 4°C. The tubes were centrifuged at 3,000 x g at 4°C for 10 minutes and 500 µL of the supernatant was counted in a Beckman LS 9100 liquid scintillation counter with 5 mL of scintillation fluid. The difference between cpm with [3H]estradiol only and cpm with [3H]estradiol + cold estradiol was calculated and taken as the amount of bound [3H]estradiol. The data was analyzed by Scatchard analysis.

Statistical analysis. Student's t test (two-tailed) was used to determine the significance between treatments and untreated controls, and P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Methylseleninic acid inhibits MCF-7 cell growth. Table 1 shows the results of the effect of MSA treatment on cell growth. The data were expressed as percentages of the untreated control. A concentration of 1 µmol/L MSA did not alter cell growth, even after 3 days of treatment. MSA did not affect cell growth at concentrations between 1 and 5 µmol/L at the 24-hour time point. MCF-7 cell growth at the 24-hour time point was inhibited 24% by 10 µmol/L MSA. Increasing the concentration of MSA to 2.5 µmol/L until the 48-hour time point, but cell growth was inhibited by about 51% at the 72-hour time point. MCF-7 cell growth was inhibited by 5 µmol/L MSA by 32% and 64% at the 48 and 72-hour time points, respectively. Increasing MSA concentration to 10 µmol/L inhibited the growth of MCF-7 cells further by 46% and 68% at the 48- and 72-hour time points, respectively.


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of MSA on the accumulation of MCF-7 cells

 
Methylseleninic acid suppresses ER{alpha} mRNA and protein expression in MCF-7 cells. We next examined the effect of MSA on the expression of estrogen receptor. MCF-7 cells express endogenous ER{alpha}. The expression of ER{alpha} mRNA in response to MSA was measured using real-time reverse transcription-PCR. Figure 1A shows the time course of MCF-7 cells in response to 10 µmol/L MSA. The levels of ER{alpha} mRNA decreased significantly after 2 hours of treatment with 10 µmol/L MSA and continued to decreased to 30% after 16 hours of treatment. The dose response to MSA was done at the 6-hour time point (Fig. 1B). Increasing the concentration of MSA to 5 µmol/L or above decreased ER{alpha} transcripts to 20% or less of controls, although 2.5 µmol/L MSA slightly increased the level of ER{alpha} mRNA. We next examined ER{alpha} protein expression in response to increasing doses of MSA treatment. MSA produced a graded suppression of the estrogen receptor protein in a dose-dependent manner (Fig. 1C). The changes in protein levels were consistent with changes in mRNA levels in MCF-7 cells in response to MSA treatment.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. Effect of MSA on ER{alpha} expression. MCF-7 cells were cultured in phenol red–free DMEM medium in 10% charcoal-stripped FBS in the presence of 10 nmol/L ß-estradiol. The cells were treated either with increasing doses of MSA or variable time points. Total RNAs were extracted for quantitative reverse transcription-PCR analysis and whole cell protein extracted for protein expression assay. A and B, change in ER{alpha} mRNA, as determined by quantitative reverse transcription-PCR, as a function of time of treatment with 10 µmol/L MSA (A), or as a function of MSA concentration at 6-hour treatment (B); C, effect of increasing concentrations of MSA treatment at the 6-hour time point on the levels of ER{alpha} protein expression. The percentage of control is represented as the mean ± SE of four independent experiments; *, significantly different from the control (P < 0.05), which is set as 100%. Bars, SE. Actin as a loading control.

 
Methylseleninic acid increases ERß mRNA expression in MDA-MB231 cells. MCF-7 cells are hormone-dependent breast cancer cells that express high levels of ER{alpha} but very low levels of ERß. To examine the effect of selenium on the expression of endogenous ERß, ER{alpha}-negative but ERß-positive MDA-MB231 human breast cancer cells were tested by quantitative reverse transcription-PCR. Treatment with MSA increased ERß mRNA expression in a dose- and time-dependent manner in MDA-MB231 cells (Fig. 2). Within the first hour, there was a 50% increase in ERß mRNA expression in response to 10 µmol/L MSA (Fig. 2A). The levels of ERß mRNA continued to increase to about 250% after 16 hours of treatment with MSA. Treatment with MSA in a concentration of 2.5 µmol/L or above increased ERß mRNA expression by 2.5-fold compared with the untreated control at the 6-hour time point (Fig. 2B).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 2. Effect of MSA on ERß expression. MDA-MB231 cells were cultured in DMEM medium with 10% FBS. The cells were treated either with increasing doses of MSA or in variable time points. Total RNAs were extracted for quantitative reverse transcription-PCR analysis. A, time course of treatment with 10 µmol/L MSA as determined by quantitative reverse transcription-PCR; B, as a function of increasing MSA concentrations at 6-hour treatment by quantitative reverse transcription-PCR. The percentage of control is represented as the mean ± SE of four independent experiments.

 
Methylseleninic acid inhibits estrogen receptor trans-activating activity. In an attempt to determine the ability of MSA to affect estrogen receptor trans-activating activity, we tested the effect of MSA on MCF-7 cells transiently transfected with an estrogen response element-luciferase reporter plasmid (25). MSA inhibited the luciferase reporter in a dose-dependent manner (Fig. 3). The estrogen response element promoter activity decreased by 18%, 50%, 64%, or 88% in the presence of 2.5, 5, 10, or 20 µmol/L MSA, respectively.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 3. Effect of MSA on estrogen response element promoter activity. The cells were cultured in phenol red–free media with charcoal-stripped FBS containing 10 nmol/L of ß-estradiol. The results are expressed as percentages of untreated control. *, significantly different from the control (P < 0.05); bars, SE.

 
Methylseleninic acid decreases binding of estrogen receptor to estrogen response element. Electrophoretic mobility shift assay was done using radiolabeled oligonucleotides of estrogen response element with nuclear extracts from MCF-7 cells treated with 10 µmol/L MSA for 30 minutes to determine whether MSA reduces DNA binding activity of estrogen receptor protein to estrogen response element. Estrogen receptor-estrogen response element complex formation decreased with MSA treatment compared with the untreated control (Fig. 4A). Cold competitor oligonucleotides blocked the complex formation (Fig. 4B), indicating that estrogen receptor protein specifically binds the estrogen response element region.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 4. Effect of MSA on estrogen receptor binding to estrogen response element. A, electrophoretic mobility shift assay results of estrogen receptor binding to estrogen response element as a function of different concentrations of MSA at 3-hour treatment; B, competition analysis was done with 2-fold (2x), 4-fold (4x) excess of unlabeled competitor probe or no competitor (0) in the reaction mixture containing the labeled probe.

 
Methylseleninic acid suppresses c-myc protein expression in MCF-7 cells. The expression of c-myc is regulated by estrogen receptor signaling. To examine whether MSA affects the expression of c-myc protein, Western blot analyses were done. Cells were treated with increasing concentrations of MSA (0, 2.5, 5, 10, and 20 µmol/L) for 6 hours, and nuclear proteins were prepared and analyzed. The c-myc protein expression in response to ß-estradiol was blocked by MSA treatment Fig. 5A and B. The level of c-myc protein expression was barely detected after treatment with 20 µmol/L MSA.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 5. Effect of MSA on c-myc protein expression. A, Western blot analysis of the levels of c-myc protein expression in increasing concentrations of MSA at 6 hours of treatment. Twenty micrograms of nuclear proteins were loaded and equal protein loading was confirmed by Ponceau stain of the membrane after blotting; B, c-myc expression normalized quantitative changes compared with the ß-estradiol control value of 100%.

 
Methylseleninic acid reduces the binding of labeled estradiol to the estrogen receptor in MCF-7 cells. MCF-7 cells were subjected to Scatchard analysis in the absence and presence of 5 µmol/L MSA to examine whether MSA affects estradiol binding to the estrogen receptor (Fig. 6). MSA treatment decreased Bmax 44% from 59.1 ± 4.2 to 35.4 ± 3.9 fmol (n = 3; P < 0.05). Treatment with 5 µmol/L MSA altered the kinetics of estradiol binding to estrogen receptor in MCF-7 cells. Kd decreased 50% in the presence of MSA (0.22 ± 0.04 nmol/L) compared with untreated controls (0.41 ± 0.08 nmol/L).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 6. Scatchard analysis of specific estradiol binding to estrogen receptor in MCF-7 cells in the absence and presence of MSA (5 µmol/L). The kinetic constants for the data presented are Kd values for untreated control (0.41 ± 0.08 nmol/L) and with MSA 0.22 ± 0.04 nmol/L, whereas Bmax values for untreated control was 59.1 ± 4.2 fmol and with MSA 35.4 ± 3.9 fmol. Values are mean ± SE for triplicate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed that selenium disrupted estrogen receptor signaling in breast cancer cells in vitro. The anticancer activity of pharmacologic doses of selenium has been shown in mammary tumor models and cell lines (12, 17). Although epidemiologic studies have yet to show the chemopreventive activity of selenium in breast cancer, accumulating epidemiologic and molecular evidence suggest that selenium protects against the development of a variety of cancers including prostate, colon, esophagus, lung, and gastric cardia (7–9, 27). The apparent disconnect between results from studies in cell lines and the epidemiologic studies in breast cancer may be due to the different doses of selenium used in cell lines (pharmacologic doses) and in the epidemiologic studies (nutritional range). To put things in perspective, we need to take our in vitro data and design the appropriate in vivo experiment in order to determine the dose sensitivity of estrogen signaling modulation by selenium treatment.

We have previously shown that pharmacologic doses of selenium causes G1 cell cycle arrest, induction of apoptosis, and modulation of cell signaling molecules such as cyclins A and D1, p16 and p27, which resulted in reduction in the size of intraductal papillary lesions in a rat mammary cancer model (12, 17). Besides induction of apoptosis and inhibition of cell proliferation, other potential mechanisms of the anticancer activity of selenium include suppression of angiogenic activity and modulation of oxidative stress (17, 28, 29). Estrogens exert their proliferative effect on hormone-dependent breast cancer cells by stimulating cell cycle progression and protecting cells from apoptotic death. It is possible that selenium-mediated growth arrest and apoptosis is mediated in part through disruption of the estrogen signaling in estrogen receptor–positive breast cancer cells. This report shows a novel mechanism through modulation of estrogen receptor expression whereby selenium may serve as a chemopreventive agent for breast cancer. Using both MCF-7 (ER{alpha}-positive) and MDA-MB231 (ER{alpha}-negative but ERß-positive) cells, we show that MSA disrupts estrogen receptor signaling by decreasing the levels of ER{alpha} and increasing the levels of ERß expression, inhibiting estrogen receptor trans-activating activity and estrogen receptor–mediated gene expression, and reducing estrogen receptor-ligand binding. It would be interesting to further validate these findings in mammary cell lines that produce approximately equal amounts of ER{alpha} and ERß proteins.

c-Myc is a well-characterized ß-estradiol target gene, which plays a critical role in the ability of ß-estradiol to enhance the proliferation of MCF-7 cells. The effects of selenium on the expression of c-myc were used as a model to assess its activity against endogenous estrogen targets. Treatment with MSA inhibited ß-estradiol-induced expression of c-myc in a dose-dependent manner as early as 6 hours. Since MSA had no effect on MCF-7 cell number at the 6-hour treatment at a concentration of 5 µmol/L, the reduction of c-myc expression was not due to cytotoxicity.

Estrogens signal through two distinct receptor pathways, ER{alpha} and ERß, which show differential tissue distribution (30), affinity for coactivators (31), and responses to hormones (32). ER{alpha} and ERß even shows opposing activation and repression activities (33). It is generally recognized that estrogen transcriptional activities implicated in breast cancer cell proliferation are mediated through ER{alpha} (34, 35). In contrast, ERß inhibits the transcriptional activity of ER{alpha} and negatively affects cell proliferation in breast and uterus (34–36). In this study using cell lines, selenium inhibited expression of ER{alpha} and increased expression of ERß. If selenium modulates ER{alpha} and ERß expression in clinical specimens similar to breast cancer cell lines, these actions on estrogen receptor would characterize an ideal agent for evaluation in breast cancer chemoprevention.


   Acknowledgments
 
Grant support: NIH CA90271 and U.S. Army Medical Research Materiel Command, Prostate Cancer Research Program DAMD17-01-1-0089.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. James Mohler for critical review of this manuscript and Yun Li for excellent technical assistance.

Received 9/10/04. Revised 1/17/05. Accepted 2/ 2/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Prall OW, Sarcevic B, Musgrove EA, Watts CK, Sutherland RL. Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2. J Biol Chem 1997;272:10882–94.[Abstract/Free Full Text]
  2. Prall OW, Rogan EM, Musgrove EA, Watts CK, Sutherland RL. c-Myc or cyclin D1 mimics estrogen effects on cyclin E-Cdk2 activation and cell cycle reentry. Mol Cell Biol 1998;18:4499–508.[Abstract/Free Full Text]
  3. Sutherland RL, Prall OW, Watts CK, Musgrove EA. Estrogen and progestin regulation of cell cycle progression. J Mammary Gland Biol Neoplasia 1998;3:63–72.[CrossRef][Medline]
  4. Kyprianou N, English HF, Davidson NE, Isaacs JT. Programmed cell death during regression of the MCF-7 human breast cancer following estrogen ablation. Cancer Res 1991;51:162–6.[Abstract/Free Full Text]
  5. Wang TT, Phang JM. Effects of estrogen on apoptotic pathways in human breast cancer cell line MCF-7. Cancer Res 1995;55:2487–9.[Abstract/Free Full Text]
  6. Killackey MA, Hakes TB, Pierce VK. Endometrial adenocarcinoma in breast cancer patients receiving antiestrogens. Cancer Treat Rep 1985;69:237–8.[Medline]
  7. Clark LC, Combs GF Jr, Turnbull BW, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA 1996;276:1957–63.[Abstract]
  8. Mannisto S, Alfthan G, Virtanen M, et al. Toenail selenium and breast cancer—a case-control study in Finland. Eur J Clin Nutr 2000;54:98–103.[CrossRef][Medline]
  9. Garland M, Morris JS, Stampfer MJ, et al. Prospective study of toenail selenium levels and cancer among women. J Natl Cancer Inst 1995;87:497–505.[Abstract/Free Full Text]
  10. Lopez-Saez JB, Senra-Varela A, Pousa-Estevez L. Selenium in breast cancer. Oncology 2003;64:227–31.[CrossRef][Medline]
  11. Medina D, Thompson H, Ganther H, Ip C. Se-methylselenocysteine: a new compound for chemoprevention of breast cancer. Nutr Cancer 2001;40:12–7.[CrossRef][Medline]
  12. Ip C, Birringer M, Block E, et al. Chemical speciation influences comparative activity of selenium-enriched garlic and yeast in mammary cancer prevention. J Agric Food Chem 2000;48:2062–70.[CrossRef][Medline]
  13. Mukherjee B, Sarkar A, Chatterjee M. Biochemical basis of selenomethionine-mediated inhibition during 2-acetylaminofluorene-induced hepatocarcinogenesis in the rat. Eur J Cancer Prev 1996;5:455–63.[Medline]
  14. Banner WP, Tan QH, Zedeck MS. Selenium and the acute effects of the carcinogens, 2-acetylaminofluorene and methylazoxymethanol acetate. Cancer Res 1982;42:2985–9.[Abstract/Free Full Text]
  15. Thompson HJ, Becci PJ. Selenium inhibition of N-methyl-N-nitrosourea-induced mammary carcinogenesis in the rat. J Natl Cancer Inst 1980;65:1299–301.
  16. Medina D, Lane HW, Shepherd F. Effect of dietary selenium levels on 7,12-dimethylbenzanthracene-induced mouse mammary tumorigenesis. Carcinogenesis 1983;4:1159–63.[Abstract/Free Full Text]
  17. Dong Y, Ganther HE, Stewart C, Ip C. Identification of molecular targets associated with selenium-induced growth inhibition in human breast cells using cDNA microarrays. Cancer Res 2002;62:708–14.[Abstract/Free Full Text]
  18. Ip C, Thompson HJ, Zhu Z, Ganther HE. In vitro and in vivo studies of methylseleninic acid: evidence that a monomethylated selenium metabolite is critical for cancer chemoprevention. Cancer Res 2000;60:2882–6.[Abstract/Free Full Text]
  19. Ip C. Lessons from basic research in selenium and cancer prevention. J Nutr 1998;128:1845–54.[Abstract/Free Full Text]
  20. Cho SD, Jiang C, Malewicz B, et al. Methyl selenium metabolites decrease prostate-specific antigen expression by inducing protein degradation and suppressing androgen-stimulated transcription. Mol Cancer Ther 2004;3:605–11.[Abstract/Free Full Text]
  21. Dong Y, Lee SO, Zhang H, et al. Prostate specific antigen expression is down-regulated by selenium through disruption of androgen receptor signaling. Cancer Res 2004;64:19–22.[Abstract/Free Full Text]
  22. Zhao H, Whitfield ML, Xu T, Botstein D, Brooks JD. Diverse effects of methylseleninic acid on the transcriptional program of human prostate cancer cells. Mol Biol Cell 2004;15:506–19.[Abstract/Free Full Text]
  23. Lee SO, Lou W, Hou M, et al. Interleukin-6 promotes androgen-independent growth in LNCaP human prostate cancer cells. Clin Cancer Res 2003;9:370–6.[Abstract/Free Full Text]
  24. Lee SO, Lou W, Johnson CS, Trump DL, Gao AC. Interleukin-6 protects LNCaP cells from apoptosis induced by androgen deprivation through the Stat3 pathway. Prostate 2004;60:178–86.[CrossRef][Medline]
  25. De Miguel F, Lee SO, Onate SA, Gao AC. Stat3 enhances transactivation of steroid hormone receptors. Nucl Recept 2003;1:3.[CrossRef][Medline]
  26. Ni Z, Lou W, Lee SO, et al. Selective activation of members of the signal transducers and activators of transcription family in prostate carcinoma. J Urol 2002;167:1859–62.[CrossRef][Medline]
  27. Wei WQ, Abnet CC, Qiao YL, et al. Prospective study of serum selenium concentrations and esophageal and gastric cardia cancer, heart disease, stroke, and total death. Am J Clin Nutr 2004;79:80–5.[Abstract/Free Full Text]
  28. Gallegos A, Berggren M, Gasdaska JR, Powis G. Mechanisms of the regulation of thioredoxin reductase activity in cancer cells by the chemopreventive agent selenium. Cancer Res 1997;57:4965–70.[Abstract/Free Full Text]
  29. Jiang C, Ganther H, Lu J. Monomethyl selenium-specific inhibition of MMP-2 and VEGF expression: implications for angiogenic switch regulation. Mol Carcinog 2000;29:236–50.[CrossRef][Medline]
  30. Palmieri C, Cheng GJ, Saji S, et al. Estrogen receptor ß in breast cancer. Endocr Relat Cancer 2002;9:1–13.[Abstract]
  31. Acevedo ML, Kraus WL. Mediator and p300/CBP-steroid receptor coactivator complexes have distinct roles, but function synergistically, during estrogen receptor {alpha}-dependent transcription with chromatin templates. Mol Cell Biol 2003;23:335–48.[Abstract/Free Full Text]
  32. Paech K, Webb P, Kuiper GG, et al. Differential ligand activation of estrogen receptors ER{alpha} and ERß at AP1 sites. Science 1997;277:1508–10.[Abstract/Free Full Text]
  33. Liu MM, Albanese C, Anderson CM, et al. Opposing action of estrogen receptors {alpha} and ß on cyclin D1 gene expression. J Biol Chem 2002;277:24353–60.[Abstract/Free Full Text]
  34. Sun JM, Chen HY, Davie JR. Effect of estradiol on histone acetylation dynamics in human breast cancer cells. J Biol Chem 2001;276:49435–42.[Abstract/Free Full Text]
  35. Weihua Z, Lathe R, Warner M, Gustafsson JA. An endocrine pathway in the prostate, ERß AR, 5{alpha}-androstane-3ß,17ß-diol, and CYP7B1, regulates prostate growth. Proc Natl Acad Sci U S A 2002;99:13589–94.[Abstract/Free Full Text]
  36. Jensen EV, Cheng G, Palmieri C, et al. Estrogen receptors and proliferation markers in primary and recurrent breast cancer. Proc Natl Acad Sci U S A 2001;98:15197–202.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. L. Kipp, S. M. Kilen, T. K. Woodruff, and K. E. Mayo
Activin Regulates Estrogen Receptor Gene Expression in the Mouse Ovary
J. Biol. Chem., December 14, 2007; 282(50): 36755 - 36765.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
J. Y. Chun, Y. Hu, E. Pinder, J. Wu, F. Li, and A. C. Gao
Selenium inhibition of survivin expression by preventing Sp1 binding to its promoter
Mol. Cancer Ther., September 1, 2007; 6(9): 2572 - 2580.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
I. Chung, M. K. Wong, G. Flynn, W.-d. Yu, C. S. Johnson, and D. L. Trump
Differential Antiproliferative Effects of Calcitriol on Tumor-Derived and Matrigel-Derived Endothelial Cells.
Cancer Res., September 1, 2006; 66(17): 8565 - 8573.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
J. Y. Chun, N. Nadiminty, S. O. Lee, S. A. Onate, W. Lou, and A. C. Gao
Mechanisms of selenium down-regulation of androgen receptor signaling in prostate cancer.
Mol. Cancer Ther., April 1, 2006; 5(4): 913 - 918.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, S. O.
Right arrow Articles by Gao, A. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, S. O.
Right arrow Articles by Gao, A. C.
Related Collections
Right arrow Preclinical Intervention
Right arrow Preclinical Intervention: In Vitro: Drugs, Mechanisms

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online