Effects Of Ros-autophagy Loop On Imatinib Resistance In Chronic Myeloid Leukemia

Background Chronic myeloid leukemia(CML)is a malignant disease involving multifunctional hematopoietic stem cells.CML may be divided into the chronic,accelerated and blast phases,and the majority of the patients are in the chronic phase at the time of diagnosis.Imatinib mesylate(IM)was the first tyrosine kinase inhibitor(TKI)to be used for the treatment of CML in clinical settings,and has provided a survival benefit by restoring normal hematopoiesis and achieving hematological,cytogenetic and molecular remission.However,despite the satisfactory efficacy of IM and second-and third-generation TKIs,a proportion of patients display varying degrees of resistance.Studies have shown that the high level of ROS in cells,which can cause oxidative stress damage and genomic instability,and may eventually lead to the formation of drug resistance.The role of autophagy in hematological tumors has also been confirmed.However,the role and interrelationship of them in drug resistance of chronic myeloid leukemia need to be further explored.Therefore,it is crucial to further investigate the molecular mechanism underlying the development of drug resistance and identify new targets to overcome this resistance.Objective In this study,K562 and IM-resistant K562/G cell lines were used as the research object.Global proteomic analysis was carried out to study the proteins related to TKI resistance in CML.In addition,we focus on these differential molecules and try to have an overall understanding of the signal transduction pathways and potential upstream mechanisms involved in CML resistance.Further explore was performed to study the effect of ROS-autophagy loop.Method(1)The human CML cell line K562 was used as the object of study.K562/G cells(IM-resistant K562 cells)were established by maintaining cells in increasing concentrations of IM.(2)After K562 and K562 cells were treated with imatinib at different concentrations,the proliferation inhibition rate of the two cell lines was detected by CCK-8 method.The drug resistance ratio was calculated by IC50 value.(3)The cells were collected and the protein was extracted according to the manufacturer's instructions.TMT/i TRAQ Labeling,HPLC Fractionation and LC-MS/MS Analysis were applied to investigate the differences in proteomics.(4)GO enrichment was carried out to unveil the differences in biological process,cellular component,and molecular function,and to explain the biological effects of differential proteins from different angles.(5)We used wolfpsort a subcellular localization predication soft to predict subcellular localization.(6)In order to clarify the interaction between intermolecular metabolic pathways,complexes and biochemical reactions,KEGG enrichment was carried out,and the main pathways involved in drug resistance were analyzed.(7)The proteins related to redox balance and autophagy were enriched and analyzed.(8)The mRNA expression levels of autophagy-associated protein(Atg7,Atg8,Atg12,Beclin1,P62)and oxidation-associated protein(GSTM3,GSTM2,MGST1,ALDH2,IDH2,GSTP1,GSTO1,ALDH1A1)in CML cell line K562 and IM drug-resistant cell line K562/G were detected by real-time fluorescence quantitative PCR(qRT-PCR).(9)The working concentration of imatinib was determined by pre-experiment.Western blotting method was used to detect the protein levels of P62,LC3-?,c-PARP and ?-Actin in CML cell line K562 and K562/G and at different time(0h,4h,8h,16 h,24h)after treated with 5.0?mol /L imatinib.(10)The level of reactive oxygen species(ROS)in K562 and K562/G cells was detected by flow cytometry.(11)The working concentration and action time of ROS inhibitor NAC in K562/ G were determined by pre-experiment.K562/G was treated with NAC and IM.The levels of autophagy-related protein P62,LC3-? was detected by western blotting method.(12)The working concentration and action time of chloroquine(CQ),an autophagy inhibitor,were determined by pre-experiment.K562/G was treated with CQ and IM.The protein level of c-PARP,P62,LC3-? were detected by western blotting method.(13)Flow cytometry was used to detect the level of ROS and apoptosis after treatment with IM and CQ for 24 hours.(14)Western blot method was used to detect the expression of cell cycle-related proteins after IM treatment for the corresponding time.Result Results of proteomic analysis(1)A total of 5732 proteins were identified in our study,of which 4990 proteins comprised quantitative information.Protein level was considered as significant difference when the fold change between two cells was?0.6 or?1.5,and t-test p-value<0.05.In this study,149 proteins were up-regulated and 277 proteins were down-regulated in K562/G,compared to K562.(2)Differentially expressed proteins from proteomics are mainly involved in transmembrane receptor,oxygen transporter and glutathione transferase activity,and play crucial roles in myeloid leukocyte activation,immune response,and regulated exocytosis.Some of these proteins are intrinsic structural components of plasma membrane.Among various subcellular structures,the differential proteins mainly located in mitochondria,endoplasmic reticulum,and peroxisome.(3)These differentially expressed proteins between two cell lines were classified functionally into 7 categories.The KEGG analysis for differentially expressed proteins showed that glutathione(GSH)metabolism and necroptosis were significantly enriched.The above two pathways have the close correlation with redox balance and autophagy signal.After screening and enrichment analysis,18 autophagy-related proteins,such as SQSTM(p62)and MAP1LC3 B,and 28 proteins involved in redox balance and GSH metabolism,including ALDH2,ALDH1A1,GSTP1 and GSTO1 were found in those differentially expressed proteins.Results of basic cell experiment(1)The K562/G cells exhibited significantly higher resistance to IM,with >50?fold increase of the IC50 value,compared with K562 cells.The growth curve of K562 and K562/G were almost similar without treatment with IM.However,the growth of K562 was more obviously inhibited by IM of 5.0?mol/L,compared to K562/G.When cells were subjected to IM,the more significant increase of apoptosis signal was detected in K562,not only in early apoptosis,but also in late apoptosis.(3)QRT-PCR results showed that the mRNA levels of ATG7,Beclin-1,GSTP1,GSTO1,ALDH1A1 and RFK were significantly increased,while the mRNA levels of GSTM3,GSTM2,MGST1,ALDH2,IDH2 and PRDX2 were significantly decreased(P<0.05).(4)The autophagy signals of K562 and K562/G cells were analyzed by western blotting.Compared with K562,the LC3-? of K562/G cells increased and p62 decreased,which indicated that K562/G cells had a higher level of basal autophagy.When K562 cells were treated with 5.0 ?mol /L imatinib,the time-dependent increase of LC3-? signal and the decrease of p62 time-dependent were observed.Subsequently,similar results were found in K562/G cells.The autophagy activation of K562/G cells seems to be stronger and earlier than that of K562 cells.However,c-PARP was only detected in K562/G cells at 24 hours,which was significantly later than that in K562 cells(P<0.05).Flow cytometry analysis showed that the ROS of K562 cells was also higher than that of K562 cells(P<0.05).(5)After treated with NAC,a specific quenching agent of ROS,it was found that p62 increased and LC3-? decreased after pretreatment with 5mmol/L NAC(P<0.05).K562/G cells were treated with autophagy inhibitors CQ and IM.No matter whether IM,CQ was added or not,it could inhibit the autophagy activation of K562/G cells,promote the production of c-PARP.The level of ROS was significantly increased when K562/G cells were pretreated with 10.0 ?mol /L CQ,which had nothing to do with IM.In addition,flow cytometry analysis showed that apoptosis was significantly increased in both early and late stage when K562/G was treated with CQ and IM for 24h(P<0.05).(6)Compared to K562,K562/G exhibited higher expression of several relative markers,such as PHGDH,CDK4(Cyclin-dependent kinase 4),CDK6(Cyclin-dependent kinase 6),cyclin D1,p-Rb,p-p38 and p-ERK1/2.Conclusion The activation of autophagy and the level of ROS in drug-resistant chronic myeloid leukemia cells were higher than those in K562 cells.Autophagy and ROS interact with each other to form a negative feedback loop that affects the drug resistance of cells.In the presence of IM,there were significant differences in cell cycle regulation between the two cell lines.The study provides a new idea for imatinib resistance in chronic myeloid leukemia and provides scientific basis for exploring multi-target therapy.