The Aerobic Glycolysis-mediated Drug Resistance And Dihydroartemisinin-induced Ferroptosis In K562/ADM Cells

The relationship of aerobic glycolysis and multidrug resistance in K562/ADM cellsBackground and objective One of the major causes of cancer chemotherapy failure is the multi-drug resistance(MDR).Enhanced aerobic glycolysis has been identified as an important biological hallmark of tumor cells.Recent studies show that enhanced aerobic glycolysis is closely linked with tumor malignancy and resistance to chemoradiotherapy,but its mechanism and regulation are not clear.In our study the leukemia sensitive K562 cells and MDR cells K562/ADM derived from K562 cells are employed as a cell model to compare the differences of glucose metabolism of drug-sensitive and-resistant cells,demonstrate the characteristics of glucose metabolism of resistant cells,probe the relationship between the multi-drug resistance and the aerobic glycolysis in K562/ADM cells,and ulteriorly investigatethe the feasibility and molecular mechanism of reversing multi-drug resistance by inhibiting aerobic glycolysis in K562/ADM cells.The new strategies for overcoming multi-drug resistance in clinical cancer treatment will be provided by bringing these researches to success.Methods MTT colorimetry was used to measure the proliferative activity of cells,Enzymologic methods to employed to assess consumption of Glucose,lactic acid export,activities of glucose metabolism-related enzymes,and content of glutathione(GSH).Adenosine triphosphate(ATP)was detected by bioluminescence method,and mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)were tested by flow cytometry.The expression of glycolysis-associated proteins was monitored by Western-blotting assay.Results1.In normoxia environment,the glucose consumption and lactic acid production of K562/ADM cells were significantly higher than the parental K562 cells(P<0.05),but there was no significant difference in ATP content.Compared with K562 cells,the enzyme activities ofhexokinase(HK),lactate dehydrogenase(LDH)and pyruvate kinase(PK)significantly were increased(P<0.05-0.001)and phosphofructokinase(PFK)was decreased(P<0.01).Western-blotting assay showed that the expression of glucose transporter 4(GLUT4)and LDH-A in K562/ADM cells were higher than that in K562 cells(P <0.01-0.001).K562/ADM cells' MMP and GSH level were decreaed and increased,respectively,compared with K562 cells and(P<0.01-0.001),but there was no significant difference in ROS content between K562 and K562/ADM cells.2.After administration with glycolysis inhibitor 2-deoxy-D-glucose(2-DG),the sensitivity of K562/AMD cells and K562 cells to Adriamycin(ADM)was improved,and the inhibitory rate increased by 15% and 30% in K562 cells and K562/ADM cells,respectively.2-DG was able to reduce the ATP content of cells,when 2-DG concentration reaches 4m M,the ATP content of K562/ADM cells dropped more than doubled that of K562 cells.ADM inhibited glucose consumption,lactic acid output and the activity of HK,and combined 2-DG and ADM treatment,the inhibition of glucose consumption and lactic acid production in K562 cells and K562/ADM cells increased by 30%,10% and 40%,30% respectively.Inhibition of glucose metabolism the HK activity in K562/ADM cells decreased by 10 units,and hardly changed in K562 cells.In addition,2-DG stimulated ROS increase and GSH decrease in K562/ADM cells.3.Compared with K562 cells,the T-m TOR-c Myc and Mek-MAPK signaling pathways were high-activated in K562/ADM cells.2-DG significantly lowered AKT-m TOR-c Myc and Mek-MAPK pathway in K562/ADM cells,and 2-DG combined ADM treatment,the expression of AKT was significantly decreased in K562/ADM cells but no changed in K562 cells.The m TOR expression and phosphorylation were decreased by more than 70% in K562/ ADM cells,while less than 30% in K562 cells.K562/ADM cells' c-Myc expression decreased 14%,but K562 cells did not change.The expression and phosphorylation of Mek and MAPK in K562/ADM cells decreased by more than 40%,and almost unaffected in K562 cells.That 2-DG inhibited the glucose metabolism-related AKT-m TOR-c Myc pathway in K562/ADM cells gave rise to the expression of downstream glucose metabolic proteins including GLUT4,LDH-A and GSK-3?was suppressed significantly(P <0.05-0.01).Conclusions1.Multi-drug resistant K562/ADM cells have significantly stronger ability of aerobic glycolysis than K562 cells,and the glycometabolic reprogramming with enhanced activities of HK,PK and LDH,increased expression of GLUT4 and LDH-A,and weakened mitochondrial function exists in K562/ADM cells.2.The drug resistance of K562/ADM cells is tightly tied to the enhanced aerobic glycolysis.The efficient inhibition of aerobic glycolysis is capable of reversing the resistance of K562/ADM cells to Adriamycin.3.In addition to directly competitively inhibiting HK activity to reduce glycolysis and ATP supply,the resistance-reversing roles of 2-deoxy-glucose are also linked to the depressed activities of AKT-m TOR-c-Myc and Mek-MAPK pathways,down-regulated expression of metabolic effectors GLUT4,LDH-A and GSK-3? and furhter reduced energy supply in drug-resistant K562/ADM cells.Dihydroartemisinin-induced Ferroptosis in drug-resistant leukemia K562/ADM cellsBackground and objective Ferroptosis is a recently discovered iron-dependent non–apoptotic type of cell death,which possesses the morphological,biochemical and genetic characteristics differing from apoptosis,autophagy and necrosis.Recent studies show that Ferroptosis is closely related to autophagy,mitochondrial dysfunction,oxidative stress and energy metabolism.Dihydroartemisinin(DHA)is one of artemisinin derivatives and its anti-tumor activity has become a hot-spot issue in recent years.The anti-tumor mechanisms of DHA have been clarified including promoting apoptosis,arresting cell-cycle,inhibiting angiogenesis and inducing iron-dependent death,etc.The iron-dependent celullar cytotoxicity of DHA are associated with Ferroptosis.In this study,we take multidrug-resistant leukemia K562/ADM cells as the target to explore whether DHA can induce drug-resistant cells to Ferroptosis.Methods MTT colorimetric method was used to detect cell proliferation-inhibition caused by DHA,The cell morphological changes were investigated by transmission electron microscopy(TEM)and Wright-Gimsa staining method.The contents of intracellular glutathione were measured by biochemical-enzymic method.Intracellular ROS levels was detected with fluorescence probe by flow cytometry.The expression of Ferroptosis associated proteins glutathione S-transferase(GST)and transferrin receptor(Tf R)were detected by Western-blotting assay.Results1.Different concentrations of dihydroartemisinin had toxic effects on both K562 sensitive and K562/ADM drug-resistant cells,and at 12 h,24h and 48 h,the IC50 values were 50.22±2.48,31.89±1.66,7.43±0.09 ?mol/L for K562 cells,and 71.18±3.19,40.54±1.75,20.01±1.37 ?mol/L for K562/ADM cells,respectively.These showed that the sensitivity of K562/ADM cells to DHA was lower than that of K562 cells.2.After DHA treatment,Wright-Giemsa staining analysis showed that both K562 and K562/ADM cells appeared typical changes of Ferroptosis such as cell volume shrinkage,condensed protoplasm,and foaming cellular membrane with normal nuclear chromatin;and the shrinked mitochondria,condensed or densed mitochondrial membrane,and fused,decreased or disappeared mitochondrial cristae were also observed under TEM.Western-blotting assay showed that DHA reduced the expression of GST and Tf R.These data suggested that DHA inducedFerroptosis in K562 and K562/ADM cells.3.Ferroptosis inducer ferric ammonium citrate(FAC)could enhance the DHA-induced cytotoxicity and ferroptosis,and K562/ADM cells displayed more distinct ferroptotic features such as mitochondrial shrinkage,increased mitochondrial membrane densities and reduced mitochondrial crista,decreased GSH content and increased ROS accumulation.The DHAinitiated cell proliferation-inhibition and ferroptosis could be partially reversed by specific ferroptosis-inhibitor Ferrostatin-1,and the decreased GSH and ROS accumulation in DHA-treated cells re-increased and subsided,respectively.These demonstrated that DHA-mediated cellular proliferation inhibition of drug-resistant leukemic cells was precipitated by inducing ferroptosis.4.The expression of GST and Tf R was down-regulated significantly in DHA-treated K562/ADM and K562 cells,and the DHA-caused down-regulation of GST and Tf R expression was reinforced by FAC and reversed by Ferrostatin-1,respectively.Conclusions1.DHA exhibits anti-leukemia activity in K562 and K562/ADM cells,and the sensitivity of K562/ADM cells to DHA was lower than that of K562 cells.2.Ferroptosis occurs in DHA-induced K562/ADM and K562 cells,which can be strengthened by ferroptosis-stimulator or weakened by ferroptosis-inhibitor.3.Ferroptosis is highlighted as a new form of DHA-induced cell-death in K562/ADM cells and potential therapeutic approaches for drug-resistant leukemia.