Study Of ERp57 On Enhancing The Anti-breast Cancer Activity And Attenuating The Cardiomyocyte Toxicity Of Doxorubicin In Vitro

Objective:Doxorubicin(Dox)is a chemotherapeutic agent widely used for the treatment of breast cancer.However,the clinical use of Dox is limited by its unwanted cardiotoxicity.In previous studies,we found that BAA(a Danshensu derivative)had the effect of enhancing Dox on anti-breast cancer as well as attenuating myocardial cytotoxicity.And ERp57 was found to be one of the therapeutic targets of BAA.This study aims to discuss the effects of ERp57 on anti-breast cancer and myocardial cytotoxicity of Dox in vitro.Finally,the mechanism would also be investigated.Methods:1.Effect of ERp57 on enhancing the anti-breast cancer and attenuating the cardiomyocyte toxicity of doxorubicin in vitroFirstly,MDA-MB-231 cells and H9c2 cells overexpressing ERp57 were validated,as well as confirming the overexpression by Western Blot.Then the cells were treated with different concentrations of Dox(1?M,2?M,4?M,8?M)for 24 h,the cytotoxicity and cell viability were detected by lactate dehydrogenase kit and MTT assay.MDA-MB-231 cells and H9c2 cells(EV and ERp57~H group)were treated with 2?M Dox for 24 h,then the cells were fixed,nuclei were stained with DAPI.Wild-type MDA-MB-231 cells and H9c2 cells were transiently transfection of siRNA silenced the ERp57 gene,the cell survival rate was measured by MTT method.2.Mechanism of ERp57 on enhancing the anti-breast cancer and attenuating the cardiomyocyte toxicity of doxorubicin in vitroMDA-MB-231 cells and H9c2 cells(EV and ERp57~H group)were treated with doxorubicin(2?M)for 24 h,and then proteins were extracted and analyzed for proteomics,mainly using data-independent acquisition(DIA).To determine possible signaling pathways,differential proteins were analyzed by enrichment.Western Blot validation was performed based on proteomics results.Results:1.ERp57 has an intervention effect on Dox-induced anti-breast cancer and cardiomyocyte toxicity in vitroMDA-MB-231 cells were treated with different concentrations of Dox for 24 h,with the continuous increase of Dox concentration,the cell survival rate gradually decreased in a concentration-dependent manner.ERp57 overexpression increased the cell survival of Dox-induced breast cancer cells and reduced the release of lactate dehydrogenase.In the DAPI staining experiment,2?M Dox was added to treatment for 24 h,the results showed that the overexpression of ERp57 reduced the apoptosis of Dox in breast cancer MDA-MB-231 cells.In the experiments of transiently transfected,ERp57 gene silencing can further promote the apoptosis of Dox in MDA-MB-231 cells.On the contrary,in H9c2 cells,after the cells were treated in the same way for 24 h,ERp57 gene overexpression further reduced the survival rate of cardiomyocytes and enhanced the toxicity of cardiomyocytes.The experimental results of DAPI staining confirmed that the overexpression of ERp57 could promote the apoptosis of Dox in H9c2 cells.In the ERp57-siRNA interference assay,apoptosis induced by Dox in H9c2 cells was significantly reduced after ERp57 gene silencing.2.The mechanism of ERp57 on enhancing the anti-breast cancer and attenuating the cardiomyocyte toxicity of doxorubicin in vitroAnalysis of proteomics pathway enrichment and differential protein showed,the mechanism of ERp57 on enhancing the anti-breast cancer and attenuating the cardiomyocyte toxicity of Dox in vitro may be related to Unfolded protein response(UPR)in endoplasmic reticulum.After Western Blot verification,the results showed that in breast cancer MDA-MB-231 cells,ERp57 was overexpressed and Dox was added,the expression levels of Sec61?,ERp57,BAP31,GRP78,p-IRE1?,TRAF2,CHOP,p-eIf2?,Cleaved caspase 3 and Bax/Bcl-2 were decreased,while the protein expression of p-PERK,and ATF4 are not significantly changed.The result showed that overexpression of ERp57 can inhibit the expression of UPR-related and apoptotic-related proteins in MDA-MB-231 cells.In rat cardiac H9c2 cells,the same method was used to detect expression changes in UPR and apoptosis related proteins.Western Blot analysis showed that ERp57 overexpressing could further up-regulation the expression of Sec61?,ERp57,BAP31,GRP78,p-IRE1?,TRAF2 and p-PERK,p-eIf2?,ATF4,CHOP as well as apoptosis related proteins Cleaved caspase 3 and Bax/Bcl-2.The result indicated that the expression levels of proteins in the UPR and apoptotic signaling pathways were significantly up-regulated after ERp57overexpression in H9c2.Conclusion:1.ERp57 gene silenced and overexpression have different effects on the regulation of Dox-induced anti-breast cancer and cardiomyocyte toxicity in vitro.ERp57overexpression have Pro-survival effect in MDA-MB-31 cells and have Pro-apoptotic effect in H9c2 cells.The silencing of ERp57 gene have pro-apoptotic effect in MDA-MB-31 and protective effect in H9c2 cells.Therefore,the inhibition of the ERp57 function has the effect of enhancing anti-breast cancer and attenuating the cardiomyocyte toxicity of Dox in vitro.For the treatment of breast cancer with anthracyclines,ERp57 may be a potential drug target on enhancing the anti-breast cancer and attenuating the cardiomyocyte toxicity of Dox in vitro.2.After ERp57 overexpressing and Dox was added,in MDA-MB-231 cells and H9c2cells,the expression of proteins in UPR and apoptotic signaling pathways had different changes.In MDA-MB-231 cells,it may inhibit the UPR to promote cell survival,and in H9c2 cells,it may be possible to further promote apoptosis by enhancing the UPR.Therefore,the mechanism of ERp57 on enhancing the anti-breast cancer and attenuating the cardiomyocyte toxicity of Dox in vitro may be related to the URP and apoptotic signaling pathways.