Inhibition of autophagy has been accepted as a promising therapeutic strategy in cancer,but its clinical application is hindered by lack of effective and specific autophagy inhibitors.We previously identified cepharanthine(CEP)as a novel autophagy inhibitor,which inhibited autophagy/mitophagy through blockage of autophagosome-lysosome fusion in human breast cancer cells.In this study we investigated whether and how inhibition of autophagy/mitophagy by cepharanthine affected the efficacy of chemotherapeutic agent epirubicin in triple negative breast cancer(TNBC)cells in vitro and in vivo.In human breast cancer MDA-MB-231 and BT549 cells,application of CEP(2 μM)greatly enhanced cepharanthine-induced inhibition on cell viability and colony formation.CEP interacted with epirubicin synergistically to induce apoptosis in TNBC cells via the mitochondrial pathway.We demonstrated that co-administration of CEP and epirubicin induced mitochondrial fission in MDA-MB-231 cells,and the production of mitochondrial superoxide was correlated with mitochondrial fission and apoptosis induced by the combination.Moreover,we revealed that co-administration of CEP and epirubicin markedly increased the generation of mitochondrial superoxide,resulting in oxidation of the actin-remodeling protein cofilin,which promoted formation of an intramolecular disulfide bridge between Cys39 and Cys80 as well as Ser3 dephosphorylation,leading to mitochondria translocation of cofilin,thus causing mitochondrial fission and apoptosis.Finally,in mice bearing MDA-MB-231 cell xenografts,co-administration of CEP(12 mg/kg,ip,once every other day for 36 days)greatly enhanced the therapeutic efficacy of epirubicin(2 mg/kg)as compared with administration of either drug alone.Taken together,our results implicate that a combination of cepharanthine with chemotherapeutic agents could represent a novel therapeutic strategy for the treatment of breast cancer.