Resveratrol Induces Autophagic Apoptosis Via Lysosomal Cathepsin D Pathway In Multidrug-resistant Leukemia Cells

Background and objective: Autophagy is a programmed cell death that delivers cell contents to lysosome for degradation.The induction or inhibition of autophagy both resulted in the death of cancer cells,and the regulation of specific genes and proteins also play an important role in autophagic regulation.However,a mass studies promt that autophagy is the protective effect for tumor cells towards chemotherapeutic drugs,thereby promoting cell survival.Yet some researchers believe that hyperactive autophagy will seriously disturb the coordination of cell metabolism,and finally causes the cell death.In this study,a kind of polyphenols antitoxin drug-resveratrol was used to observe the autophagic activity and autophagy-related genes expression of drug-resistance cell line-K562/ADM and drug-sensitive cell line-K562 cells.Our study was also aimed to detect the role of resveratrol in the induction of leukemia and the relationship between resveratrol induced apoptosis and autophagic cell death.Thus,a theoretical basis can be provided to improve the sensitivity of drug-resistant leukemia cells.Methods:A well drug-resistant cell line has been established by K562 cells,which was named K562/ADM cell.Cell viability of K562/ADM cells and K562 cells was determined with the MTT assay.The formation of autophagy vacuoles was detected using electron microscopy and monodansylcadaverine(MDC)staining.Western blotting was used to detect the expression of autophagy-related proteins LC3,Beclin-1 and P62.Cell cycle and cell apoptosis was evaluated using flow cytometry.Also used Western blotting and flow cytometry to measure the expression of caspase-3,Bcl-2,Bax and Cyt c,Cath D and P-gp and the change of reactive oxygen species.Results: 1.The results of cell proliferation inhibition test showed that different concentrations of resveratrol could inhibit the proliferation of K562/ADM and K562 cells.The IC50 of K562/ADM cells was 57.71±0.67?mol/L,and K562 cells was 46.67±0.38 ?mol/L,which showed us that the drug-resist leukemia K562/ADM cells has a high resistant to resveratrol.2.Electron microscopy and MDC staining showed that the number of autophagic bubbles and fluorescence intensity was increased significantly after treated by resveratrol,and the K562/ADM cells has more autophagic bubbles than K562 cells.After treated with resverotrol for 24 hour,the expression of autophagy marker protein LC3 and Beclin-1 were up-regulated significantly in K562/ADM cells when compared with the control group,and the expression of P62 protein was obvioustly lower than that of the control group(P <0.05).The expression of LC3 and Beclin-1 in K562 cells was also slightly up-regulated,but lower than that of drug-resistant cell lines;the change of P62 was not obvious.3.Our results showed that resveratrol induced apoptosis in K562/ADM and K562 cells.The total apoptosis rate were significantly increased and the two cell lines underwent cell cycle arrest after treated with resveratrol.At the same time,the detection of apoptosis-related proteins expressions also showed that the drug could promote the activation of caspase-3 and decrease the ratio of Bcl-2/Bax,increase the release of Cyt c and the production of ROS.These results were consistent with the western-blotting results.It is interesting to note that the expression of Cathepsin D in drug-resistant cells K562/ADM was significantly up-regulated,and the expression level was significantly higher than that of the control group after treated with drugs for 72 hours.The released Cath D may trigger caspase-dependent cell death through the Bcl-2 family of proteins.Resveratrol did not reduce the expression of P-glycoprotein in drug-resistant cells.4.The inhibitory on autophagy by chloroquine(CQ)can decrease the sensitivity of K562/ADM cells to resveratrol.After pretreated with chloroquine,the apoptosis rate of K562/ADM cells was lower than resteratrol treated alone.The results of western-blotting showed that the expression of autophagy marker protein LC3 and Beclin-1 significantly lower than that of resveratrol induced alone,and the activation of caspase-3 decreased,the ratio of Bcl-2/Bax protein expression increased.Conclusion: 1.Resveratrol can induce the change of autophagic activity in K562/ADM cells and K562 cells,and the autophagic activity in the drug-resistant K562/ADM cells is significantly higher than that in K562 cells.2.Resveratrol induce apoptosis by enhancing the autophagic activity in the drug-resistant cells.The cytotoxic effect of resverotrol leads to an increase in lysosomal membrane permeability,then the release of CathD into cytoplasm increased,the Bax-mediated mitochondrial membrane permeability and the release of cytochrome C increased,the Caspase cascade reaction is activated and eventually leads to cell apoptosis.Resveratrol also can induce autophagy and apoptosis in drug-sensitive cells,but the activation of autophagy is only a concomitant phenomenon of apoptosis.3.The level of intracellular ROS in the drug-resistant cell lines is significantly lower than that of sensitive cell lines,and the ROS level induced by resveratrol is also lower than that of K562 cells.The high basal autophagic activity and low ROS level in K562/ADM cells may be one of the mechanisms of drug resistance,promt that to improve the level of ROS in drug-resistant cells may be a new strategy to overcome the resistance of tumor cells.4.P-gp maybe not participate in the regulation of autophagic activity induced by resveratrol to mediate cell apoptosis.