Reversal Of Drug-resistance By Inhibiting Aerobic Glycolysis In Multidrug-resistent Leukemia K562/ADM Cells

Multi-drug resistance（MDR）is one of the major causes of tumor treatment failure.Many factors are thought to contribute to drug-resistance,but the occurrence and mechanisms remain incompletely understood.Extensive recent research has demonstrated a relationship between aerobic glycolysis in tumors and the occurrence of drug-resistance.However,although aerobic glycolysis has been proposed for more than 90 years,its molecular mechanisms and role in tumor drug-resistance are also still unclear.It is therefore necessary to study the molecular mechanisms of MDR from the point of tumor metabolism,and to explore specific ways of overcoming MDR to provide new potential targets for clinical treatment.In this study,we investigated differences in glucose metabolism between the human multi-drug resistant acute myeloid leukemia cell line K562/ADM（adriamycin）and its parental,drug-sensitive K562 cell line.K562/ADM cells demonstrated increased aerobic glycolysis compared with drug-sensitive K562 cells under normoxic conditions.K562/ADM cells consumed more glucose and produced more lactic acid than the parental cells,and also demonstrated dependence on lactic acid.This reprogramming of glucose metabolism in drug-resistant cells might help to fight against the drugs by reducing their absorption through acidification of the cell microenvironment,resisting oxidant injury by decreasing oxygen free radicals,increasing ATP（adenosine triphosphate）-dependent drug efflux,or by other mechanisms.Moreover,experiments to detect the expression and activity of key proteins involved in glucose metabolism demonstrated increased activities of hexokinase（HK）and pyruvate kinase and increased expression of glucose transporter 4（GLUT4）and HK-II associated with aerobic glycolysis in K562/ADM cells.We used 2-deoxy-D-glucose and oxamate as pharmacological tools to inhibit glycolysis and found that glycolytic inhibition improved the therapeutic effect of ADM in both cell lines and re-sensitized ADM-resistant cells,with K562/ADM cells exhibiting a stronger response to chemotherapy than their counterparts.These results suggest that ADM resistance of K562/ADM cells may be associated with enhanced aerobic glycolysis.Gene sequencing technology continues to improve,and analysis of differential gene expression at the RNA level detected 79 genes associated with glucose metabolism that were differentially expressed between K562 and K562/ADM cells.Among these,genes involved in toxicant efflux,channel regulation,and enzyme regulation showed the highest differential enrichment.Given the up-regulation of glycolysis and down-regulation of the tricarboxylic acid cycle in K562/ADM multidrug-resistant cells compared with drug-sensitive K562 cells,metabolic pathway annotation may thus provide more significant evidence for a role of aerobic glycolysis in enhancing MDR.Furthermore,many signaling pathways related to glucose metabolism involved differentially enriched genes,including the PI3K-AKT signaling pathway,which showed the highest enrichment.It is suggested that up-regulation of the PI3K-AKT pathway may mediate MDR in K562/ADM cells by increasing aerobic glycolysis.We therefore analyzed up-and down-stream proteins in the PI3K-AKT pathway by western blotting and revealed that ADM-stimulated upregulation of the AKT-mTOR-c-Myc/p-GSK-3β（Ser9）pathway was an important factor in ADM resistance.Glycolysis inhibitors counteracted ADM-stimulated up-regulation of AKT-mTOR-c-Myc and dramatically down-regulated the AKT-mTOR-HIF-GLUT4/HK-II pathway in K562/ADM cells,thereby improving inhibition by ADM.We therefore suggest that interference with the AKT-mTOR pathway represents a key to overcoming ADM resistance in leukemic K562/ADM cells.In addition,the present view of suppressor genes or oncogenes of glycogen synthase kinase-3β（GSK-3β）to tumor cells is not conform.Our results indicated that GSK-3β may act as a suppressor factor during leukemic cell growth,and that sensitization of ADM-resistant cells by glycolytic inhibition may be related to GSK-3β phosphorylation at Ser9 and enhanced GSK-3β activity.Leukemic K562/ADM cells show cross-resistance to many chemotherapeutic drugs.As₂O₃ can decrease the expression and phosphorylation levels of many molecules in the AKT-mTOR-HIF pathway,and we found that multidrug-resistant K562/ADM cells were more sensitive to As₂O₃ than drug-sensitive K562 cells.Moreover,As₂O₃ can restrain the expression of HK-II,LDH（lactate dehydrogenase）-A,and GLUT4,reduce the activity of LDH-A and strengthen that of LDH-B,suppress glucose consumption,and lead to pyruvic acid accumulation.As₂O₃ thus appears to inhibit the flow of aerobic glycolysis,which may be one reason why drug-resistant cells are more sensitive to As₂O₃ than sensitive cells.Further studies are needed to clarify the role of As₂O₃ in aerobic glycolysis.In conclusion,the present study demonstrated that K562/ADM MDR leukemia cells exhibited increased aerobic glycolytic activity than K562 parental sensitive cells,and the inhibition of glycolysis could reverse the resistance of MDR cells to anticancer agents.Drug resistance via increasing aerobic glycolysis in MDR leukemia cells was related to the AKT-mTOR-c-Myc/HIF/GSK-3β pathway.Down-regulation of AKT-mTOR-HIF-1a by As₂O₃ in multidrug-resistant K562/ADM cells could inhibit aerobic glycolysis and ATP supply,and that this might be responsible for increased sensitivity to As₂O₃ in K562/ADM leukemia MDR cells.We analyzed the molecular mechanisms of drug resistance in K562/ADM cells from the point of cell metabolism in this study.Inhibition of aerobic glycolysis and down-regulation of the signaling pathways involved in aerobic glycolysis represents a potential chemotherapeutic strategy for sensitizing leukemic cells and thereby overcoming MDR.These results provide a theoretical and material basis for the development of therapies aimed at relapsed and refractory leukemia/cancers.