Hydroxychloroquine Reverses Drug Resistance Of Leukemic K562/ADM Cells By Inhibition Of Autophagy

Background Autophagy is an essential metabolic pathway mediated by lysosomal degradation, and it is involved in scavenging and therefore recycling senescent or damaged organelles and biological macromolecules in eukaryotic cells. In tumor initiation and progression, however, autophagy has shown multifaceted roles. Increasing evidences corroborate a role of autophagy as a suppressor to prevent the tumor initiation. Conversely, autophagy also maintains metabolic pathways in cancer cells and promotes survival in response to diverse anticancer therapies. Therefore,the autophagy is able to promote the resistance in tumor cells in order to survive under the circumstances of drug- and radiation-induced cytotoxicities. The investigation of the relationship between autophagy and drug resistance of cancer cells can be conducted by interfering with drug resistance of tumors through modulating the autophagic activity.Objective Our study attempts to comparatively investigate the differences of basic and induced autophagic activity between drug-sensitive and multidrug-resistant leukemia cells and explore whether drug-resistance of leukemic cells can be overcome by autophagy inhibitor hydroxychloroquine(HCQ)-mediated repression of autophagic activity, and discusses the correlation of autophagy and leukemic drug resistance.Methods The leukemia drug-sensitive K562 cells and drug-resistant K562/ADM cells were used as cell model, the basic and starvation- or Adriamycin(ADM)-induced autophagy of the cells and the sensitivity of hydroxychloroquine-pretreated leukemia cells to ADM were examined. Moreover, the morphology of cellular autophagy was observed by using transmission electron microscope and fluorescent staining of MDC, the morphology of cellular apoptosis was observed by using AO/EB co-staining, the cell proliferation was detected by MTT assay. The beclin1、bax、bcl-2 and mdr1 m RNAs were measured by real time RT-PCR, flow cytometry was employed to detect the expression levels of apoptosis-related proteins Bax, Bcl-2, P-glycoprotein(P-gp) and Caspase-3 activity, and the expressions of LC3I/II, Beclin1, Bax, Bcl-2, Caspase-3, P-gp and P62 proteins were measured by western blot.Results K562/ADM presented strong resistance to ADM and the IC50 values for 12h、24h and 48 h were 20.6、44.7 and 62.9 fold higher than that of K562 cells, respectively. The data detected by flow cytometry and western blot showed that drug resistant protein P-gp was extremely lowly expressed in K562 cells, whereas P-gp was highly expressed in nearly 100% of K562/ADM cells. The level of basic autophagy in K562/ADM cells was higher than that in K562 cells, which could be characterized by more cytosolic contents-packaged autophagic vacuoles in K562/ADM cell when compared to that in K562 cells. The observation of MDC staining showed that the fluorescent intensity of autophagosome in K562/ADM cells was stronger than that in K562 cells. The expression of Beclin1 and the ratio of LC3-II to LC3-I were distinctly higher in K562/ADM cells, however, the P62 protein was relatively lower in K562/ADM cells. When K562 and K562/ADM cells were cultured in serum- and amino acid-free EBSS balance buffer for 0, 1, 2, 4 and 6 hours or induced with different concentration of ADM for 12 or 24 hours, numerously typical autophagic vacuoles appeared with different degree in cells, and both the expression of Beclin1 and the ratio of LC3-II to LC3-I reached the peak value in 1hour of starvation and then gradually decreased while the degradation of P62 reached the peak value post 2~4 hours of starvation. ADM was capable of inducing the autophagic activity of K562 and K562/ADM cells, however, the autophagic alteration in K562 cells appeared earlier and ADM-induced autophagic activity could be restrained by autophagy inhibitor hydroxychloroquine. In order to demonstrate the correlation between autophagic activity and the resistance of cancer cells to chemotherapy drugs and the possibility that leukemic drug resistance could be overcome by using autophagy inhibitors, we treated K562 and K562/ADM cells with different concentration of hydroxycholoquine to inhibit the cellular autophagy and detected the sensitivity of the cells to ADM. The results showed that the sensitivity of co-treated K562 cells to ADM was enhanced and the IC50 values of 12、24 and 48 hours were 0.98、0.84 and 0.42 folds of that in cells treated with ADM alone, respectively. Yet, the sensitivity of K562/ADM cells to ADM was extremely strengthened and the IC50 values were 0.8、0.46 and 0.21 folds of that in ADM-treated cells, respectively. Meanwhile, more typical morphological changes of apoptosis appeared, the ratio of Bax/Bcl-2 and the activity of Caspase-3 were markedly increased in K562/ADM cells. Interestingly, the expression of mdr1 m RNA and P-gp in drug-resistant K562/ADM cells was markedly enhanced as increased autophagic activity induced by nutriment deprivation, and HCQ significantly reduced the increased expression of P-gp via inhibition of autophagic activity.Conclusion Multi-drug resistant K562/ADM cells own higher basic autophagy activity than the parental drug-sensitive K562 cells, and the elevated autophagic activity can be induced by starvation and cytotoxicitic drugs. Autophagy inhibitor hydroxychloroquine significantly promotes the sensitivity of K562/ADM to ADM via restraining autophagy and facilitating apoptosis. Furthermore, the promotion or inhibition of autophagic activity in K562/ADM cells upregulates or downregulates the expression of P-gp, respectively, which indicates that P-gp potentially sustainsdrug resistance through participating in the regulation of autophagic activity in drug resistant cells.