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.

No abstract

TCEP is an organophosphorus flame-retardant (OPFR) that is used widely in polyurethane foams, furniture, and household products. From these products, TCEP has significant emission into its surrounding environment. Inhalation and dermal absorption are the most common routes of human exposure to OPFRs, which have been measured in human hair, breast milk, and urine. Previous studies of OPFRs have shown their endocrine disrupting actions that have influence on diseases such as cancer. Breast cancer is the second most common cancer among women in the United States, and because of this prevalence, it is imperative to investigate possible causes and treatments for this disease. Due to the endocrine disrupting nature of OPFRs, we are investigating the effects of TCEP on hormone dependent breast cancer cell lines MCF-7 and T-47D. Our study examines the effects of TCEP, alone and in combination with hormones and anti-hormones, on ERα and BRCA1 expression in MCF-7 and T-47D breast cancer cells by utilizing western blot analyses, cellular viability assays, confocal microscopy, apoptosis assays and RT-qPCR analyses. In order to deplete any endogenous steroids or effectors, breast cancer cells were cultured in a medium containing 5% charcoal-stripped fetal bovine serum for six days. Western blot analysis revealed alterations in the expression of ER-alpha after 24 hours of treatment with varying concentrations of TCEP (1µM-2mM). A concentration-dependent decrease of ERα protein levels was noted in the T-47D cell line when compared to the control. BRCA1 protein levels also displayed an altered expression compared to the control through the various concentrations of TCEP. Through our concentration studies, optimum concentrations of TCEP were found to be 100 µM for T-47D and 2mM for MCF-7. For our hormone studies, cell lines were treated with their respective optimum concentration of TCEP as well with combinations of hormones and anti-hormones. After 24-hour treatment of E 2, TCEP, and a combination of E2 with TCEP, a decrease in ERα expression was observed when compared to the control in both MCF-7 and T-47D cell lines. A combination of TCEP with ICI treatment revealed a significant down regulation of ERα expression level compared to the control. The same treatment conditions exhibited an increase with BRCA1 expression compared to the control and these effects were sensitive to the presence of antiestrogens in both cell lines. For cell viability studies, cells were treated for 6 days with TCEP concentrations ranging from 10nm-2µM which displayed an increase (40-50%) in cell proliferation compared to the control in both cell lines. Cytolocalization of ERα remained unaltered with the above treatment conditions. Our studies provide interesting findings about the molecular mechanisms of TECP as a potential endocrine disrupting compound on the steroid receptors and tumor suppressor genes in breast cancer cells. Presentation: No date and time listed

Perfluoroctanesulfonic Acid (PFOS) is a perfluorinated chemical (PFC) and endocrine-disrupting compound (EDC) found in fire-extinguishing foams, food packing, and commonly used household products such as nonstick cookware and cleaning products. PFOS is also an environmental toxicant that has polluted air, soil, and water in past years. Few studies have suggested PFOS may cause endocrine-disrupting effects, therefore it is critical to explore the effects of PFOS on breast cancer cells. This study examined the estrogenic-like effects of PFOS, alone and in combination with hormones and antihormones, on the expression of ERα and BRCA1 in T-47D and MCF-7 breast cancer cell lines by utilizing western blot analyses, cellular viability assays, confocal microscopy, apoptosis assay and RT-qPCR analyses. The cells were cultured with 5% charcoal-stripped fetal bovine serum (FBS) for six days to deplete endogenous steroids or growth factors. In the concentration-dependency study, cells were treated with various concentrations of PFOS (300µM-800µM) for 24 hours. Western blot analysis revealed alterations in the expression of ERα and BRCA1 related with these varying concentrations of PFOS (300 µM-800µM). In comparison to the control, a concentration-dependent decrease of ERα expression levels were seen in both cell lines. A concentration-dependent increase of BRCA1 was seen in both cells. Western blot analysis revealed the optimal concentration of PFOS to be 700 µM in cells, which exhibited the maximal estrogenic-like effect. To further examine the effects of PFOS on breast cancer cells, the optimal concentration (700 µM PFOS) was then used alone and in combination with hormones and anti-hormones in T-47D cells. Treatment with PFOS, E2and the combination of PFOS and E2 for 24 hours showed a significant down-regulation of ERα in T-47D cells when compared to the control. Treatment of PFOS with antiestrogen ICI revealed a significant down-regulation of ERα (approximately 50%) in T-47D cells. Image cytometric analysis with propidium iodide staining was utilized to examine the effects of PFOS on cellular viability in T-47D cells. PFOS alone and in combination with E2 revealed an increase in cellular proliferation compared to the control in T-47D cells and these effects were sensitive to the presence of antiestrogens. RT-qPCR studies revealed a transcriptional expression of ESR1 and BRCA-1 mRNA levels that correlate with the translational data obtained via western blot analyses. Cytolocalization experiments and effects of PFOS with hormones and antihormones on MCF-7 cells studies are in progress. Our studies provide exciting leads to clearly delineate the molecular mechanisms of PFOS as an EDC on the steroid receptors and tumor suppressor gene in breast cancer cells. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Bisphenol A (BPA) is considered to be an endocrine disrupting chemical (EDC), which mimics endogenous hormones and is linked to various cancers. Bisphenol S (BPS) is a BPA analogue, often used in plastics. BPS can leach into food and drink products, exposing humans to these chemicals. Evidence suggests BPS is also an EDC with similar endocrine disrupting effects. Despite hopes for a safer alternative, research has shown BPS possesses estrogenic activity due to its structural similarities with its analogue BPA. Previously we have shown the effects of BPS on estrogen receptor-alpha (ERα) and BRCA-1 in both MCF-7 and T-47D breast cancer cells. The wild-type p53 and BRCA-2 work to prevent cancer by monitoring and repairing DNA damage; however, in breast cancer patients these genes are often mutated. Mutated p53 will induce the cell cycle protein p21 to act as an oncogenic transcription factor. In the present study, we have examined the effects of BPS, alone and in combination with hormones and anti-hormones, on p53, BRCA-2, and p21 in both MCF-7 and T-47D cell lines by utilizing western blot analyses, cellular viability assays, confocal microscopy, apoptosis assay, and RT-qPCR analyses. Western blot studies revealed alterations in the expression of p53, BRCA-2, and p21 related with varying concentrations of BPS (4-20 µM). In comparison to the control, p53 expression increased (65-95%) in the presence of BPS in both MCF-7 and T-47D cells. In addition, BRCA-2 expression revealed a similar increase in both cell lines when treated with BPS. However, p21 expression decreased (approximately 50%) with increasing concentrations in both cell lines. For further evaluation, an optimal concentration of 8 µM BPS was then used in combination with various hormones and anti-hormones. Compared to the control, BPS and E2 were up regulated in a similar fashion to p53. A similar trend in the effects on BRCA-2 expression was depicted in T-47D and MCF-7 cells. However, in p21, BPS and E2 were down regulated in both MCF-7 and T-47D breast cancer cells. In order to determine the influence of BPS on the growth of breast cancer cells, image cytometric analysis with propidium iodide staining was utilized to quantify alterations in T-47D and MCF-7 cell numbers and viability. Upon treatment of BPS concentrations (4-20 µM), an increase in cellular proliferation (12-60% increase) occurred in both cell lines. These cellular proliferative effects of BPS and E2 were sensitive to combination treatments with anti-estrogens. Confocal microscopy was utilized to examine the cytolocalization of p53 upon exposure to BPS alone and in combination with hormones and anti-hormones. The results from this study will yield a greater understanding of the molecular regulation of BPS action via the p53, BRCA-2, and p21 signaling pathways linked with breast cancer.