The Role And Mechanism Of Exosomes In Imatinib Resistance Of Chronic Myelogenous Leukemia

?Background?Chronic myelogenous leukemia?CML?is a malignant clonal proliferative disease of hematopoietic stem cells.Formation of Philadelphia?Ph?chromosome,t?9;22?,and BCR-ABL fusion gene is the most likely cause of this disease.The fusion protein expressed by BCR-ABL gene has continuously activated tyrosine kinase activity,and plays a role in promoting proliferation and inhibiting apoptosis.Tyrosine kinase inhibitor?TKI?,such as Imatinib?IM?,can bind the ATP-binding domain of fusion protein,block Tyrosine kinase activity,prevent its downstream signal transmission,and down-regulate its function,which has a significant therapeutic effect on CML patients.However,IM cannot cure CML completely,and disease progression caused by drug resistance is the most important factor in the death of CML patients.Mechanisms for drug resistance include:1.CML cells are not sensitive to IM;2.BCR-ABL fusion gene mutation in CML cells leads to conformational changes of fusion protein,resulting in steric hindrance and decreased affinity with IM.3.BCR-ABL gene amplification increased the expression of BCR-ABL protein,so that the original drug dose is relatively insufficient.4.The expression of drug-resistant related proteins such as ABCG2 is increased and intracellular drugs are excreted.Various mechanisms of drug resistance suggest that insufficient intracellular drug concentration is the key to drug resistance and a risk factor for drug resistance.Therefore,it is of great significance to explore how to improve the intracellular IM drug concentration to improve the prognosis of patients with CML drug resistance.Exosomes are 30-150nm diameter vesicles that are formed from multiple vesicle bodies?MVB?by endocytosis and then fuse with the cell membrane.They can carry lots kinds of intracellular substances out of the cell and participate in many important biological functions such as communication between distant cells and transport of substances.Tumor cells secrete more exosomes than normal tissue cells,which play an important role in a variety of tumor activities such as tumorigenesis,drug resistance and metastasis.However,whether exosome secretion is involved in directly reducing CML intracellular IM concentration remains to be studied.Abnormal cholesterol metabolism has an important effect on the development of drug resistance in various tumors.The increased cholesterol in tumor cells provides a material basis for its rapid proliferation and also creates favorable conditions for exosome secretion.When the cholesterol content is increased,the endosome tended to form MVB and release exosomes.On the contrary,endosomes tend to fuse with lysosomes to degrade and exosome secretion decreases.The effects of cholesterol metabolism on exosomes and its role in CML cell resistance are worthy of further study.?Objective?Through the intervention of cholesterol metabolism and exosome secretion in K562G and K562 cells,the influences of cholesterol level and exosome secretion on cell drug resistance were discussed respectively.The correlation between cholesterol level and exosome secretion was also discussed,so as to clarify the cholesterol-exosome-drug resistance pathway.?Methods?1.Imatinib resistant cell line K562G was cultured from K562 cells;2.qRT-PCR was used to detect the expressions of proteins related to the cholesterol metabolism pathways,and to evaluate the differences in cholesterol metabolism between the two cells;The cells were treated with Atorvastatin,GW3965,methyl-?-cyclodextrin and water-soluble cholesterol.The cholesterol was detected by cholesterol detection kit or by stained with filipin III to observe the effect of cholesterol intervention.3.Exosomes were extracted by density gradient centrifugation,and the amount of MVB in cells was observed by electron microscopy.The difference in the amount of exosomes secreted by drug-resistant cells and sensitive cells was observed.GW4869 and Rab27a gene interference was used to inhibit exosome secretion,and the effect of inhibiting exosome secretion on cell IM resistance was observed.4.The intracellular and extracellular IM contents were determined by LC-MS?liquid chromatography tandem mass spectrometry?,and the cell resistance was evaluated by IC₅₀determined by CCK-8 assay and flow cytometry.5.RNA-seq found differential expressed miRNAs in K562 and K562G cells.Cells were transfected with candidate miRNA mimics to observe their effects on ABCA1expression and exosome secretion.?Results?1.The apoptosis rate of K562G cells was significantly decreased when the two cells were treated with IM at the same concentration.The content of IM in K562G cells was lower than that in K562 cells as determined by LC-MS.Further studies showed that both K562 and K562G cells could secrete exosomes containing IM.Compared with K562 cells,the number of exosomes secreted by IM-resistant K562G cells was significantly increased,but there was no statistical difference in IM content per unit of exosome.It indicated that K562G secreted more IM through exosome pathway.Inhibition of K562G exosome secretion by GW4869 or by interfering Rab27a gene significantly improved its sensitivity to IM.2.The expression of cholesterol synthesis related enzymes in K562G cells was lower than that in K562 cells,while the cholesterol absorption,excretion and esterification related protein expression were enhanced.However,the cholesterol level in K562G cells was lower than that in K562 cells.Increasing exogenous cholesterol significantly increased the K562cells resistance to IM,while lowering cholesterol levels by Atorvastatin,GW3965 or methyl-?-cyclodextrin could not enhance the IM sensitivity of K562.3.Compared with K562,K562G cells exhibited increased exosome secretion,significantly increased ABCA1 expression,and decreased miR-106-5p expression which regulates ABCA1 expression.However,increasing intracellular miR-106-5p inhibited the expression of target gene ABCA1 and upregulated exosome secretion.?Conclusions?1.Exosome secretion is involved in the development of drug resistance,and inhibition of exosome secretion can partially improve the sensitivity of K562G cells to IM.2.Compared with K562 cells,K562G cells had no significant difference in cholesterol synthesis related enzyme metabolism,while other cholesterol metabolism pathways were significantly enhanced,but the cholesterol content of K562G cells was lower than that of K562 cells,suggesting that the cholesterol balance point of K562G cells had been changed.Atorvastatin,GW3965 and methyl-?-cyclodextrin could not significantly improve the sensitivity of resistant cells to IM by lowering cholesterol,suggesting that?1?lowering cholesterol has little effect on cells,or?2?cholesterol compensation ability is so strong that single drug cannot effectively reduce cholesterol level.3.Increasing the level of miR-106-5p in the cells inhibited the expression of ABCA1and promoted the secretion of exosomes,suggesting that reducing the expression of ABCA1will block the excretion of cholesterol,and compensatory increase the secretion of K562and K562G exosomes.It was also suggested that ABCA1 did not assist exosomes secretion,but was also a pathway for cell cholesterol excretion.The results also explain the phenomenon that simple intervention of cells'cholesterol metabolism has little effect on IM sensitivity.Therefore,the role of abnormal cholesterol metabolism in IM resistance and its complex relationship with exosome excretion and the specific mechanism still need to be further studied.