The Molecular Mechanism Of Combination With Cepharanthine And Doxorubicin Mediating Reversal Of Multidrug Resistance And Induction Of Apoptosis In K562/ADR Cells

BackgroundMultidrug resistance（MDR）is the major obstacle in clinical chemotherapy.Recently,some chemotherapeutic strategies to reverse MDR have been developed.However,the leukemia patients still exhibit poor survival with higher recurrence rate.Thus,defining new treatment for leukemia remains a challenge and better therapeutic tools are urgently required.Autophagy inhibition has been widely accepted as a promising therapeutic strategy in cancer including leukemia,while lack of effective and specific autophagy inhibitors hinders its application.Therefore,development of novel efficient autophagy inhibitors with low toxicity has important clinical significance.Cepharanthine（CEP）,a benzylisoquinoline alkaloid extracted from Stephania cepharantha Hayata,has been used to treat patients with a low level of white blood cells for many years.In this study,we found that CEP potently blocked the degradation of autophagy in K562/ADR cell line.We also found for the first time that co-treatment with CEP markedly decreased the viability and increased apoptosis in K562/ADR cells treated with doxorubicin（DOX）.Our findings suggest that CEP could potentially be further developed as a novel autophagy inhibitor,and a combination of CEP with classical chemotherapeutic drugs like DOX could represent a novel therapeutic strategy for reversing drug resistance in human leukemia.ObjectiveTo elucidate the mechanism of combination with CEP and DOX in reversing multidrug resistance of K562/ADR cells and provide a theoretical basis for reversing CML resistance.MethodsCell viability was assessed by MTT assay.Cell apoptosis and generation of reactive oxygen species（ROS）were determined by flow cytometry.The expression of diverse proteins was examined by using western blot analysisResults1.Identification of drug-resistant CML cell line.The half survival rate(IC₅₀)of K562/ADR cells treated with DOX was significantly greater than that in K562 cells.The expression of Mdr-1 in K562/ADR cells was significantly higher than that in K562 cells.Treatment with DOX at same concentration resuleted in significant increases in apoptosis in K562 cells,but had no effect on apoptosis in K562/ADR cells.2.Combination of CEP and DOX resulted in marked decreases in cell viability and increases in apoptosis in K562/ADR cells in a dose-dependent manner.Western blot analysis showed that combination of CEP and DOX resulted in degradation of PARP-1,cleavage/activation of caspase 3 and cytochrome c release.Mechanistically,combination of CEP and DOX caused dephosphorylation and mitochondrial translocation of Drp1（Ser637）in K562/ADR cells.3.Combination of CEP and DOX resulted in significant increases in levels of the autophagy marker protein LC3 in both whole cell lysates and mitochondria fraction ofK562/ADR cells.Pretreatment of K562/ADR cells with autophagy inhibitor 3-MA or knockdown of ATG7 with shRNA significantly abrogated accumulation of autophagosomes and apoptosis mediated by combination.These results suggest that excessive accumulation of autophagosomes is involved in the combination-mediated apoptosis in K562/ADR cells.4.Combination of CEP and DOX resulted in marked increases in generation of ROS.Inhibition of ROS with TBAP significantly attenuated the combination-mediated dephosphorylation and mitochondrial translocation of Drp1,mitochondrial fission,and apoptosis.These findings suggest that production of ROS(particularly O₂^·-)is primarily responsible for dephosphorylation and mitochondrial translocation of Drp1,mitochondrial fission and apoptosis mediated by combination treatment.ConclusionCombination of CEP and DOX reverses multidrug resistance of K562/ADR through excessive accumulation of autophagosomes,generation of ROS,dephosphorylation and mitochondrial translocation of Drp1,mitochondrial fission and apoptosis.