Effect Of Bisphenol A On The GPR30-EGFR-STAT3Pathway In Breast Cancer Cells

[Objective]Bisphenol A(BPA)is one of the environmental estrogens and is widely used in thecarbonate plastics, such as food containers, beverage bottles and dental fillings. BPAgenerously exists in our living environments and can be digested and absorbed byhuman beings. Small amounts of BPA can be transferred from polymerizedpolycarbonates to food and water, especially when it is heated. Followed by theincreasing aggravated environmental pollutions, the human beings are exposed in anenvironment that can be contacted with BPA at any time. At present, the mechanism ofhow BPA induces the onset of estrogen-depended tumors such as breast cancers isunclear. BPA can simulate estrogen and promote cell's proliferation, most current studiesare focused on the estrogen receptors(ER), chemoresistance and the apoptosis proteinsBcl-2and Bax, which are related to the BPA-induced proliferation effect. But itspotential effects on signal pathways in estrogen depended tumors have been seldomreported．Only one report suggested that the activation of ERK1/2or p38in the ovariancancer cells by the EEs is not related with the cell proliferation. In the most pathwayswhich mediated breast cancers, GPR30-EGFR-STAT3is one of the most important one.Our research aims to explore the effect of BPA on the GPR30-EGFR-STAT3signalingpathway and provides the basis for the mechanism of how BPA induced breast cancers.[Methods]The study explores the BPA's proliferation promotion effect of estrogenreceptor-positive breast cancer MCF-7and T47D cells and estrogenreceptor-negative breast cancer MDA-MB-231cell by contrast reaction study. Weapplied3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT)cytotoxicity assay to determine the optimal dose and time point of BPA-promoted proliferation effect on the MCF-7and T47D cells．Gene expressions of GPR30,EGFR and STAT3were assayed on both the transcriptional and translational level byReal-time PCR and Western blot at the optimal dose of BPA in a time dependentmanner. We explored the mechanism of BPA on breast cancer cells proliferationthrough inhibiting the candidate genes of STAT3with RNA interference techniqueand EGFR with its inhibitor AG1478, and further explored the role of EGFR-STAT3signal pathway in the BPA-promoted cell proliferation effect and its mechanism ofcarcinogenesis．[Results]1．We determined the optimal concentration and time point of how BPA used incell proliferation study with MCF-7and T47D cells. We found that the BPA-inducedproliferation in MCF-7and T47D cells were obvious after the treatment with1μMBPA for24h and the effect lasted till48h, the most obvious proliferation effectappeared after BPA treatment for48h(P＜0.05). However, the BPA-inducedproliferation in MDA-MB-231cells was not obvious after the treatment with BPA for24h,48h and72h.2．Combining the results of Real-time PCR and Western blotting, the expressionof STAT3and EGFR were up-regulated remarkably after1μM BPA treatment for48h,and the inducing effects of BPA were statistically significant(P＜0.05).Theexpression of GPR30was downregulated potently after BPA treatment for48h, andthe inhibiting effect of BPA was statistically significant(P＜0.05).3．Our research applied AG1478to block EGFR signaling in breast cancer cells.The stimulation of proliferation in MCF-7and T47D cells by the combinedtreatment with BPA and AG1478for48h resulted in similar effects with thetreatment of BPA alone since the statistical differences were not significant(P＞0.05).4．Our data demonstrated that AG1478mediated EGFR inhibition in MCF-7cellsresults in an obvious downregulation of STAT3gene expression(P＜0.05).5．The STAT3siRNA5mediated the most potent inhibition of STAT3gene expression among the STAT3siRNAs pool(P＜0.05). BPA failed to induceproliferation of MCF-7and T47D cells interfered with STAT3siRNA5since thestatistical differences were not significant between the two groups(P＞0.05).[Conclusions]1．Our study further confirmed BPA's proliferation effect in breast cancer cells.We also found the optimal concentration and time point of BPA for promoting breastcancer cell proliferation are1μM and48h．2．EGFR-STAT3signaling pathway are effectively activated in the BPA-inducedproliferation of MCF-7cells with the evidence of strong induction of targetingprotein by the continuous treatment of BPA. The maximal inducing effect occurredat the48h time point of BPA treatment. However, the expression of GPR30decreased obviously as well with the potential reason that the GPR30protein isreleased after binding with BPA then transmits the signals into the cells.3．In our study,EGFR inhibitor AG1478did not neutralized the stimulating effectof BPA in the cell proliferation of breast cancer cells. So we conclude that EGFR isnot a necessary way in the BPA-induced proliferation effect, BPA can interact withSTAT3directly or through other pathways.4．Our report also shows that EGFR-STAT3pathway is highly correlated andSTAT3can be downregulated obviously after the inhibition of EGFR. However,theinhibition has no effect on the BPA-induced proliferation, BPA can activate theuninhibited gene of STAT3without through the EGFR pathway to promote breastcancer cell's proliferation.5．STAT3is a very vital element and plays an important role in BPA-inducedbreast cancer cell's proliferation．6．GPR30-EGFR-STAT3is an important signal pathway in the development ofbreast cancer since BPA can interact with the three elements among which STAT3isthe most important one. BPA can act directly on STAT3even if the STAT3gene isdownregulated after the inhition of EGFR, and BPA can act on the remain STAT3topromote cell's proliferation.