Bisphenol A Activated Both Breast Cancer Cells And Vascular Endothelial Cells Through Shared GPER-dependent Pathway In Hypoxia

Bisphenol A(BPA)behaviors as a kind of environmental disrupt chemicals(EDCs)and is able to exert estrogen-like activity.In our daily life,this xenoestrogen has been widely used in food cans or basic appliances.Recently,numerous scientific works reported that BPA played a significant role in tumor development.It is true that malignant cells may recruit various constituents such as vascular endothelial cells(VECs)into solid mass surface to support tumor growth in hypoxia.Alough numerous studies reported that BPA triggered cellular proliferation in normal cells or tumor cells,little referece focused both on cancer cells and cancer-related stromal cells and on its interaction,neither on underlying mechanism.Given that most breast cancer cells and VECs present both estrogen and cytokines-sensitive,we here propose there is close reciprocity between them in tumor microenvironment from para secreting growth factor via shared molecular mechanism affected by estradiol or environmental endocrine disruptors(EDCs),leading to neoplasm progression.In present study,we evaluated the effect of BPA in vitro and in vivo model on activation of breast cancer cells and VECs and explored underlying molecular mechanisms.The entire results provide a novel insight in illustrating the contribution of EDCs with respect to carcinogenesis process.Section 1Objective: With simulating hypoxic model in vitro,we desire to observe the biological actions exerted by BPA in VECs and explore related mechanism.Methods: 1)Cobalt chloride(Co Cl2)was used to build a chemical hypoxic state in vitro.2)Cellular proliferation,migration and angiogenesis were assessed respectively by CCK-8,wound healing,transwell assays and tube formation experiment.3)RT-PCR and Western blotting were used to measure the expression of GPER,HIF-1α and VEGF in transcript or translation level.4)Western blot was used to detect the activation of rapid signaling cascades.5)Immunofluorescence and Immunoprecipitation assays were performed to observe the interaction of GPER,Cav-1 and HSP90.Result: 1)In hypoxia,1μm BPA was able to remarkably enhance the ability of cellular proliferation,migration and angiogenesis for VECs(P<0.05)while antagonist G-15 abolished the positive impact.2)BPA and GPER agonist G-1 induce the expression of GPER,HIF-1α and VEGF in transcription and translation level(P<0.05).3)GPER activated by BPA induced the translation of HIF-1α by orchestrating rapid signaling pathway such as PI3K/AKT/m-TOR and ERK1/2,following by competitively binding to caveolin-1(Cav-1)and facilitating the release of heat shock protein 90(HSP90).However,this prompting impact exerted by BPA was abolished when using ERK inhibitor U0126,PI3 K inhibitor Wortamanin and m-TOR inhibitor Rapamycin or HSP90 antagonist 17-AAG and Cav-1 inhibitor cyclodextrin,as well as knocking down the expression of HSP90 or Cav-1.Conclusions: Our results indicated that GPER acted as a receptor target of BPA,which stimulated the expression of HIF-1α and VEGF via PI3K/AKT/m-TOR and ERK1/2 at short term and GPER/Cav-1/HSP90 reciprocity for long term exposure,leading to the activation of VECs.Section 2Objective: With simulating hypoxic model in vitro,we desire to observe whether BPA exerted biological actions in breast cancer cells through shared molecular mechanism with VECs.Methods: 1)Cellular proliferation and migration were assessed respectively by CCK-8 and wound healing experiment.2)RT-PCR and Western blotting were used to measure the expression of GPER,HIF-1α and VEGF in transcript or translation level.3)Western blot was used to detect the activation of rapid signaling cascades.4)Immunoprecipitation assays were performed to observe the interaction of GPER,Cav-1 and HSP90.Result: 1)In hypoxia,1μm BPA was able to remarkably enhance the ability of cellular proliferation and migration for Sk Br-3 and MDA-MB-231 cell lines while antagonist G-15 abolished the positive impact.2)BPA and GPER agonist G-1 induce the expression of GPER,HIF-1α and VEGF in transcription and translation level(P<0.05).3)GPER activated by BPA induced the translation of HIF-1α by orchestrating rapid signaling pathway such as PI3K/AKT/m-TOR and ERK1/2,following by competitively binding to caveolin-1(Cav-1)and facilitating the release of heat shock protein 90(HSP90)in breast cancer cells.However,this prompting impact exerted by BPA was abolished when using ERK inhibitor U0126,PI3 K inhibitor Wortamanin and m-TOR inhibitor Rapamycin or HSP90 antagonist 17-AAG and Cav-1 inhibitor cyclodextrinConclusions: Our results indicated that GPER acts as a receptor target of BPA,which stimulated the expression of HIF-1α and VEGF via common signaling pathway to VECs.Section 3Objective: Base on the model in vitro and in vivo,we testified that BPA enhanced the reciprocity of breast cancer cells and VECs mediated by GPER,resulting in tumor growth.Methods: 1)Co-culturing with Sk Br-3 and VECs was set to observe the cell-to-cell interaction.And then we collected conditioned medium from VECs to incubate breast cancer cell to demonstrate BPA promoted VEGF expression in VECs to induce tumor cell growth.2)Based on MDA-MB-231 to constitute xenograft tumor model of severe combined immuno-deficiency(SCID),the mice were intramuscularly injected with corn oil(vehicle),BPA(150 mg/kg b.w.),G-15(370 μg /kg b.w.)and BPA in combination with G-15(at the concentrations mentioned above).The volumes and body weights were recorded.3)Immunoblotting and Immunochemistry assays were used to detect the expression of GPER,HIF-1α and VEGF.In order to compare angiogenesis progression,the staining of CD34 was performed in paraffin section.Result: 1)BPA significantly increased cellular ability of breast cancer cells when co-culturing with VECs or VECs medium(P<0.05).2)BPA induced neoplasms hyperplasia of vessel in the region of tumor infiltration,and also stimulated xenograft mass growth(P<0.05).While in combination with G-15 was capable to prevent the enhancing effects of BPA.3)BPA promotes the expression of GPER,HIF-1α and VEGF in solid tumor,in consistence with the observation in vitro.Conclusions: The results in this chapter were corresponding to the observation in vitro.In detail,BPA strengthened the interaction of breast cancer cells and VECs through shared signaling cascade in vivo,in other word,BPA prompted xenograft tumor growth and angiogenesis in SICD mice via GPER/HIF-1α/VEGF transduction pathway.Overall,our data provide a novel view to explore the molecular mechanism and extend cross-talk network signaling pathway of GPER,as well as broaden the horizon in understanding the amplified effects of weak xenoestrogen on tumor progression and offering a new receptor target.