Modulation of Estrogen Receptor Alpha (ERα) and Tumor Suppressor Gene BRCA1 in Breast Cancer Cells by Bazedoxifene Acetate (BZA).

Selective estrogen receptor modulators (SERMs) are steroid analogs with dual functionality, acting as partial estrogen receptor agonists to preserve postmenopausal bone density and as estrogen receptor antagonists in breast tissue. Bazedoxifene acetate (BZA) is an FDA-approved, third-generation SERM used in the treatment of osteoporosis in women. It demonstrates potential as a therapeutic option for breast cancer patients undergoing endocrine therapy. Our study aimed to assess BZA's effects on Estrogen Receptor Alpha (ERα) and tumor suppressor gene BRCA1 in T-47D and MCF-7 breast cancer cells, using Western blots, cellular viability, apoptosis assays, and RT-qPCR. Cells were cultured in 5% charcoal-stripped fetal bovine serum for six days to deplete endogenous steroids. Following a 24 h exposure to 2 µM BZA (optimal concentration determined from 1 nM-2 µM studies), Western blot analyses revealed reduced ERα and BRCA1 protein levels in both cell lines. ERα decreased by 48-63% and BRCA1 by 61-64%, indicating sensitivity to antiestrogens. Cytolocalization of ERα and BRCA1 remained unchanged after BZA and 17-ß-estradiol (E2) treatment. ESR1 mRNA expression correlated with Western blot findings. Image cytometric analysis using the stain, propidium iodide, detected decreased cellular proliferation in T-47D and MCF-7 cells following a 6-day treatment ranging from 1 nM to 2 µM BZA. BZA treatment alone led to a tenfold reduction in cellular proliferation compared to estrogen-treated cells, suggesting antiproliferative effects. Understanding BZA's modulation of BRCA1 and ERα, along with their mechanistic interactions, is vital for comprehending its impact on breast cancer tumor suppressors and hormone receptors.

Hormone-Like Effects of Bisphenol A on p53 and Estrogen Receptor Alpha in Breast Cancer Cells.

Bisphenol A (BPA) is a polymerizing agent commonly found in plastics that has been linked to xenoestrogenic activity. In this study, we analyzed the estrogen-like effects of BPA on the expression of estrogen receptor (ER)α and p53 with hormonal and antihormonal treatments in T-47D and MCF-7 cells. Cells were cultured in medium containing 5% charcoal-stripped fetal bovine serum for 6 days to deplete any endogenous steroids or effectors. The cells were then treated for 24 h with 600 nM BPA, which was determined to be the optimal value by a concentration study of BPA from 1 nM to 2 µM. Extracted cellular proteins were quantified and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blot analysis. The cell proliferation assays were quantified upon exposure to BPA. Laser confocal microscopy was performed to determine the cytolocalization of p53 and ERα upon treatment with BPA. Western blot analysis revealed that BPA caused an increase in the cellular protein p53 in a concentration-dependent manner. While treatment with BPA did not affect the cytolocalization of p53, an increase in cell proliferation was observed. Our studies provide interesting leads to delineate the possible mechanistic relationship among BPA, ER, and tumor suppressor proteins in breast cancer cells.

The antiestrogenic effects of black cohosh on BRCA1 and steroid receptors in breast cancer cells.

BACKGROUND: Black cohosh (BC) is an herbal remedy often used by women to treat symptoms associated with menopause. Research has shown that the molecular activity of BC is associated with estrogen receptor alpha (ER-α) regulation. Progesterone receptor (PR) expression is found to be consistent with ER expression and mutations in the BRCA1 gene, a tumor-suppressor gene, are known to be responsible for about 40%-45% of hereditary breast cancers. PURPOSE: The objective of this study was to determine the effects of BC alone, as well as in combination with hormones and antihormones, on cell viability and expression of ER-α, PR, and BRCA1 in both T-47D and MCF-7 cell lines. METHODS: Cells were cultured in charcoal-stripped serum prior to their treatment and subsequent protein extraction. Western blot analyses were performed following a Bio-Rad Bradford protein assay and SDS-PAGE gel electrophoresis, with ECL luminescence and Image Studio Lite software. Cellular viability assays were performed using propidium iodine (PI) staining, and the distribution of fluorescent structures was evaluated through confocal microscopy. RT-qPCR analysis was performed on extracted cellular RNA. All statistical analyses were performed using SPSS software, and data was subjected to Kruskal-Wallis testing, followed by post-hoc analysis using the Mann-Whitney U-test to determine the statistical significance of all findings. RESULTS: Western blot analysis displayed significant alterations of ER-α, PR, and BRCA1 protein levels after 24-hour treatment with 80-500 µM BC. BC displayed a concentration-dependent decrease on ER-α and BRCA1 expression, with an 87% reduction of ER-α expression and a 43% of BRCA1 expression in T-47D cells compared to control. After six days of treatment with 400 µM BC, a 50% decrease in cell proliferation was observed. Following 24 hours of co-treatment with 400 µM BC and 10 nM E2, ER-α was downregulated by 90% and BRCA1 expression was reduced by 70% compared to control. The expression of PR, following the same treatment, exhibited similar effects. The proliferative effect of E2 was reduced in the presence of BC. CONCLUSION: Black Cohosh demonstrates substantial anti-cancer properties, and this study may significantly aid in the understanding of the molecular effects of BC on ER-α, PR, and BRCA1 in breast cancer cells.

The effects of black cohosh on the regulation of estrogen receptor (ERα) and progesterone receptor (PR) in breast cancer cells.

The North American plant Cimicifuga racemosa, also known as black cohosh (BC), is a herb that recently has gained attention for its hormonal effects. As the usage of hormone replacement therapy is declining due to its adverse effects in women with cancer, many are turning to herbal remedies like BC to treat menopausal symptoms. It is crucial to determine whether the effects of BC involve estrogen receptor-alpha (ERα). Previous studies from our laboratory have shown ERα to be a possible molecular target for BC. In this study, we examined the effects of BC (8% triterpene glycosides) alone and in combination with hormones and antihormones on the cellular viability, expression of ERα and progesterone receptor (PR)-A/B, and cytolocalization of ERα in ER (+) and PR-A/B (+) T-47D breast cancer cells. Cells were cultured and proteins were extracted and quantified. Western blot analysis revealed alterations in the expression of ERα and PR after treatment with BC (5-100 µM). BC induced a concentration-dependent decrease in ERα and PR protein levels when compared to the control. Image cytometric analysis with propidium iodide staining was used to enumerate changes in T-47D cell number and viability. A decrease in T-47D cell viability was observed upon treatment with 5-100 µM BC. The ideal concentration of BC (100 µM) was used in combination with hormones and antihormones in an effort to further understand the possible similarities between this compound and other known effectors of ERα and PR. After a 24-hour concomitant treatment with and/or in combination of BC, estradiol, ICI 182, 780, and Tamoxifen, downregulation of ERα and PR protein levels was observed. Delineating the role of BC in the regulation of ERα, PR, as well as its mechanisms of action, may be important in understanding the influence of BC on hormone receptors in breast cancer.

Effects of Atrial Natriuretic Peptide on p53 and Estrogen Receptor in Breast Cancer Cells.

The atrial natriuretic peptide (ANP) hormone is secreted by cardiac atrial myocytes and acts to regulate blood pressure homeostasis in humans. Previous research indicates ANP treatment significantly decreases the proliferation of human prostate cancer cells, pancreatic adenocarcinoma, and breast cancer cells. Minimal studies have been conducted with regard to ANP regulating tumor suppressor genes and steroid hormone receptors in breast cancer cells. Our study analyzed the effects of ANP in combination with 17ß-estradiol (E2) and antiestrogen treatments on p53 and ERα levels in T-47D breast cancer cells. Preliminary studies through Western blot analysis showed that ANP treatment decreases p53 and ERα expression levels in a concentration-dependent (10-100 nM) manner. Treatment with ANP alone, at a 100 nM concentration, causes a decrease of p53 and ERα expression compared with Cs (control stripped), but with E2 and antiestrogen combinations, expression of both protein levels decreased compared with treatments without ANP. Combined treatment with E2, an estrogen antagonist, and ANP decreased cellular proliferation compared with treatments without ANP, except in the case of raloxifene (RAL). Our studies indicate that ANP has potential as a therapeutic breast cancer treatment and should inspire further studies on the molecular mechanism of ANP in T-47D breast cancer cells.

The regulation of steroid receptors by epigallocatechin-3-gallate in breast cancer cells.

It has been reported that phytoestrogen epigallocatechin gallate (EGCG) suppresses cancer cell proliferation and may have antitumor properties. In this study, we analyzed the effects of EGCG on estrogen receptor α (ERα) and progesterone receptor in hormone-dependent T-47D breast cancer cells. Western blot analysis revealed EGCG induced a concentration-dependent decrease in ERα protein levels, with a 56% reduction occurring with 60 µM EGCG when compared to controls. Downregulation of ERα protein levels was observed after 24-hour co-treatment of T-47D cells with 60 µM EGCG and 10 nM 17ß-estradiol (E2). The proliferative effect of E2 on cell viability was reversed when treated in combination with EGCG. In contrast, the combination of EGCG with the pure ER antagonist, ICI 182, 780, showed no further reduction in cell number as only 5% of the cells were viable after 6 days of treatment. These studies may provide further understanding of the interactions among flavonoids and steroid receptors in breast cancer cells.

The effects of turmeric (curcumin) on tumor suppressor protein (p53) and estrogen receptor (ERα) in breast cancer cells.

Curcumin (CUR) is a compound that has antibacterial, antiviral, anti-inflammatory, and anticancer properties. In this study, we have analyzed the effects of CUR on the expression of ERα and p53 in the presence of hormones and anti-hormones in breast cancer cells. Cells were cultured in a medium containing charcoal-stripped fetal bovine serum to deplete any endogenous steroids and treated with CUR at varying concentrations or in combination with hormones and anti-hormones. Protein analysis revealed a relative decrease in the levels of p53 and ERα upon treatment with 5-60 µM CUR. In cell proliferation studies, CUR alone caused a 10-fold decrease compared with the treatment with estrogen, which suggests its antiproliferative effects. Delineating the role of CUR in the regulation of p53, ERα, and their mechanisms of action may be important in understanding the influence of CUR on tumor suppressors and hormone receptors in breast cancer.

Isolation and comparative analysis of potential stem/progenitor cells from different regions of human umbilical cord.

Human umbilical cord (hUC) blood and tissue are non-invasive sources of potential stem/progenitor cells with similar cell surface properties as bone marrow stromal cells (BMSCs). While they are limited in cord blood, they may be more abundant in hUC. However, the hUC is an anatomically complex organ and the potential of cells in various sites of the hUC has not been fully explored. We dissected the hUC into its discrete sites and isolated hUC cells from the cord placenta junction (CPJ), cord tissue (CT), and Wharton's jelly (WJ). Isolated cells displayed fibroblastoid morphology, and expressed CD29, CD44, CD73, CD90, and CD105, and showed evidence of differentiation into multiple lineages in vitro. They also expressed low levels of pluripotency genes, OCT4, NANOG, SOX2 and KLF4. Passaging markedly affected cell proliferation with concomitant decreases in the expression of pluripotency and other markers, and an increase in chondrogenic markers. Microarray analysis further revealed the differences in the gene expression of CPJ-, CT- and WJ-hUC cells. Five coding and five lncRNA genes were differentially expressed in low vs. high passage hUC cells. Only MAEL was expressed at high levels in both low and high passage CPJ-hUC cells. They displayed a greater proliferation limit and a higher degree of multi-lineage differentiation in vitro and warrant further investigation to determine their full differentiation capacity, and therapeutic and regenerative medicine potential.

The regulation of tumor suppressor protein, p53, and estrogen receptor (ERα) by resveratrol in breast cancer cells.

Resveratrol (RES) is a natural antioxidant found abundantly in grapes, peanuts, and berries, and is known to possess anti-tumorigenic properties. However, there is a noticeable lack of studies on the mechanistic effects of Resveratrol on tumor suppressors. Previous studies from our laboratory have shown the tumor suppressor protein p53 and estrogen receptor-alpha (ERα) to be possible molecular targets for RES. In this study, the anti-estrogenic effects of RES were analyzed on the expression of ERα and p53. The breast cancer cells grown in stripped serum were treated with 60 µM RES, as the optimum concentration based on data obtained from a concentration study using 1-100 µM RES. Our studies indicate that RES caused a decrease in the levels of protein expression of p53 and ERα as compared to the control. Increasing concentrations of RES caused a four-fold decrease in cell number in comparison to estradiol. RES, in conjunction with ICI 182,780 (ICI), caused a down-regulation of both p53 and ERα as compared to the control. These observed effects on cell proliferation and regulation of both p53 and ERα by RES may lead to further understanding of the relationship between tumor suppressor proteins and steroid receptors in breast cancer cells.

Anti-hormones: mechanism and use in treatment of breast cancer.

Breast cancer is the second-leading cause of cancer-related death in women. Recently, new drugs are being developed based on the molecular mechanisms of receptors, tumor suppressor genes, monoclonal antibodies, tumor markers and antihormone therapy. Anti-hormone therapy is used in the treatment of hormone-dependent breast tumors. Among the anti-hormone therapies, a substantial amount of research has been focused on the development of the ideal selective estrogen receptor modulator to treat metastatic breast tumors and to prevent breast cancer in high risk women.

Effects of estrogen metabolite 2-methoxyestradiol on tumor suppressor protein p53 and proliferation of breast cancer cells.

An endogenous 17ß-estradiol (E(2)) metabolite, 2-methoxyestradiol (2-ME(2)), has been reported to exhibit estrogen receptor (ER)-independent anti-angiogenic and anti-tumor effects. Several mechanisms have been proposed for 2-ME(2) actions, but there is a lack of evidence for a common pathway for all of the cell-types sensitive to this metabolite. We have examined potential alterations in p53 in response to 2-ME(2), E(2) and the microtubule disruptor taxol in T47D breast cancer cells. Cells were cultured for six days in medium depleted of endogenous steroids or effectors. Semi-confluent cells were treated with 2-ME(2) (1 nM - 10 µM), 10 nM E(2) and/or 1 µM taxol and subjected to SDS-PAGE and Western blot analysis, quantitative analysis, or laser-scanning confocal microscopy. Western blot analysis revealed a concentration-dependent biphasic trend in p53 levels. Addition of 10 nM - 1 µM 2-ME(2) induced significant up-regulation in p53, and this response gradually diminished to levels comparable to the control upon treatment with higher concentrations (2.5 - 10 µM). The observed upregulation of p53 induced by 2-ME(2) is inhibited by concurrent treatment with 1 µM taxol. Cell quantitation revealed a significant decrease (50 - 90%) in cell number upon treatment with 1 - 10 µM 2-ME(2) with minimal effect at lower concentrations. No additional effect on cell proliferation was observed when taxol was combined with 10 nM or 1 µM 2-ME(2). In a concentration dependent manner, treatment with 2-ME(2) for 24 h differentially influenced cellular localization of p53. These results may aid in further understanding the relationship between steroid receptors, tumor suppressor proteins, and effects of hormone metabolites on breast cancer cells.

Effects of LY117018 (a SERM analog of raloxifene) on tumor suppressor proteins and proliferation of breast cancer cells.

We have previously shown that presence of estradiol (E2) in the growth medium causes (i) proliferation of T47D breast cancer cells, (ii) elevation of p53 levels, and (iii) hyperphos-phorylation of retinoblastoma protein (pRb). In the present study, we examined the expression of p53, phosphorylation state of pRb and proliferation of T47D cells in the presence of LY117018 (Courtesy of Lilly Research Laboratories), an analog of raloxifene, which is a known selective estrogen receptor modulator (SERM). The cells grown in charcoal-treated serum were treated with 1 nM E2 or different concentrations of LY117018 for 24 h. E2 or LY117018 treatments caused a 2- to 3-fold increase in the level of p53 and hyperphosphorylation of pRb. E2 treatment increased cell proliferation, whereas LY117018 treatment had no such effect but inhibited the E2-dependent cell proliferation. E2 and LY117018 treatments of T47D cells also caused differential effects on intracellular structures. Thus, LY117018 treatment induces changes in the level/activity of p53 and pRb and ultrastructure of T47D cells. Importantly, LY11708 inhibits estrogen-induced cell proliferation while mimicking E2 actions on p53 induction and pRb phosphorylation. The SERM also induced structural alterations in the T47D cells.

Estrogen-like effects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells.

T47D cells represent an estrogen-responsive human ductal carcinoma cell line which expresses detectable levels of estrogen receptor (ER). We have previously shown that estradiol (E(2)) treatment of T47D cells causes an increase in the level of p53 and a concomitant phosphorylation of retinoblastoma protein (pRb). In the present study, we have analysed the expression of p53 and phosphorylation state of pRb and compared the effects of E(2) and triiodothyronine (T(3)) on these phenomena. Cells were grown in a medium containing charcoal-treated serum to deplete the levels of endogenous steroids. Upon confluency, the cells were treated with T(3) (10(-12) to 10(-7) M) for 24 h and the presence of p53 and pRb was detected by Western analysis. E(2) treatment of cells caused a 2-3-fold increase in the level of p53. Presence of T(3) in the medium caused a gradual increase in the level of p53 in a concentration-dependent manner. Under the above conditions, pRb was phosphorylated (detected as an upshift during SDS-PAGE) in the presence of E(2) and T(3). Supplementation of growth medium with T(3) (1 microM) caused an increase in the rate of proliferation of T47D cells and induced hyperphosphorylation of pRb within 4 h; this effect was maintained for up to 12 h. When ICI 164 384 (ICI) (1 microM), an ER antagonist, was combined with E(2) (1 nM) or T(3) (1 microM), effects of hormones on cell proliferation and hyperphosphorylation of pRb were blocked. Western analysis of p53 was supplemented with its cytolocalization by immuno-labeling using laser scanning confocal fluorescence microscopy, which revealed an ICI-sensitive increase in the abundance of p53 in hormone-treated cells. Steroid binding analysis revealed lack of competition by T(3) for the [(3)H]E(2) binding. These results indicate that T(3) regulates T47D cell cycle progression and proliferation raising the p53 level and causing hyperphosphorylation of pRb by a common mechanism involving ER and T(3) receptor (T(3)R)-mediated pathways.