Study Of Autophagy Inhibitor Bafilomycin A1 Synergizing With Epirubicin To Increase Drug Sensitivity Of Breast Cancer Cells

The clinical therapeutics for breast cancer is mainly based on surgery and chemotherapy,supplemented with radiotherapy,endocrine therapy,targeted therapy and others.Chemotherapy plays an important role in breast cancer treatment,it can improve the survival ratio and prognosis.However,a large number of breast cancer patients,especially for triple-negative breast cancer patients,tend to be resistant to chemotherapeutics during long-term chemotherapy,which lead to unsatisfactory prognosis.In recent years,it has been discovered that autophagy is involved in drug resistance of tumor cells.Dysregulation of autophagy can alter drug sensitivity of certain tumor cells,thereby affect the efficacy of chemotherapy.This study investigated the effect of epirubicin killing tumor cells when breast cancer cells were co-treated with epirubicin and autophagy inhibitors,and further analyzed the molecular mechanism of how autophagy inhibitor Bafilomycin A1（BafA1）modulates the drug sensitivity of breast cancer cells.In this study,the working concentration of three autophagy inhibitors,chloroquine（CQ）,3-methyladenine（3-MA）and BafA1,were determined to ensure the autophagy inhibition on breast cancer cells and keep cell viabilities around 80%.Then each inhibitor was applied in epirubicin-treated breast cancer cell lines MDA-MB-231 and MCF-7respectively.Results showed that the IC₅₀ of epirubicin was markedly decreased when each of two cell lines was co-treated with BafA1 respectively.However,the IC₅₀of epirubicin was markedly decreased when CQ or 3-MA was applied for cotreatment inMDA-MB-231cells,not inMCF-7 cells.These results indicated that BafA1 treatment increased the sensitivity of breast cancer cells against epirubicin.Equivalent graphical analysis showed that BafA1 was able to synergize with epirubicin to kill MDA-MB-231 cells,as well as MCF-7 and BT 549 cells.Mechanism study found that compared to breast cancer cells treated only with epirubicin,mRNA levels of the genes encoding drug-resistant proteins ABCB1 and ABCC1decreased in breast cancer cells co-treated with BafA1 and epirubicin,and there was no significant change in the expression of DNA repair genes XPA and ECRR1.The significant decrease of MDR1 protein expression was observed by western blotting（WB）in breast cancer cells co-treated with BafA1 and epirubicin.WB and puncta fluorescence imaging results showed that epirubicin alone did not affect autophagy level in breast cancer cells,while co-treatment with BafA1 blocked autophagy in epirubicin-treated breast cancer cells significantly.These results suggested that BafA1 may reduce MDR1 expression by blocking the autophagy pathway,thereby increasing the drug sensitivity to epirubicin in breast cancer cells co-treated with BafA1 and epirubicin.In summary,this study illustrated that addition of BafA1 on epirubicin-treated breast cancer cells could increase the drug sensitivity of breast cancer cells to epirubicin through reducing the expression of MDR1 by inhibiting cell autophagy,and thereby BafA1 could synergize with epirubicin to kill breast cancer cells.This study provides a certain reference for combination strategy of epirubicin and improvement of the chemotherapy on breast cancer.