The Mechanism Of STAT5-ROS Pathway Involved In Resistance To Imatinib In Chronic Myeloid Leukemia

Background :Chronic myeloid leukemia(CML)is a malignant hematopoietic stem cell clonal disease that is characterized by myeloid cell proliferation in the peripheral blood and bone marrow,the Ph chromosome by chromosome translocation formation,the positive of BCR-ABL fusion genes.Produced by Ph chromosome gene encoding the BCR-ABL fusion protein is a major cause of lead to CML,has high tyrosine kinase activity.Therefore,Tyrosine kinase inhibitors(TKIs)become the key of CML treatment drugs.Imatinib mesylate(Imatinib,IM)belong to the generation of tyrosine kinase inhibitor,is considered the most effective drug CML treatment at home and abroad,however,we found that 20% to 25% of patients with CML become resistant to imatinib during taking medicine in recent years in turn seriously affect the effects of treatment.The current study of imatinib resistance mechanism has progressed,but is still not fully elucidated.Hence,this study focuses on the STAT5-ROS pathway constitutively activation causes imatinib resistance of chronic myelogenous leukemia.To use the imatinib model and combine model of in-vivo illustrate the role of STAT5-ROS pathways in the mechanism of imatinib resistance for the chronic myelogenous leukemia patients.Objective: the purpose of the study is to research the express of the related markers with the STAT5-ROS pathway,the level of oxidative damage and cells apoptosis and the correlation between treatment response and difference index,then we in-depth understand the mechanism of imatinib resistance of CML.To revel the effect of STAT5-ROS pathways and look for the multiple targets for treatment.Method:(1)In the experiment,we selected the chronic myelogenous leukemia cell lines K562 as the object of our research.To use the concentration gradient method induce the imatinib resistantance cell line K562 / G.(2)When different concentrations of imatinib act on K562 and K562 / G,we adopt CCK 8 method to detect cell proliferation inhibition rate of two kinds of cell lines,test the IC50 value about the two cell lines for imatinib and calculate of multiple drug resistance.(3)With different concentration of imatinib acting on the two cell lines for a certain period of time,we use the trypan blue count number of viable cells and draw the growth curve.(4)To determine the imatinib,ROS inhibitors NAC working concentration and action time by preliminary experiments,Through flow cytometry test to observe the effects of cell apoptosis and ROS level after different concentrations of inhibitors acting on K562 and K562/G cell lines.(5)By real-time fluorescent quantitative PCR(RT-PCR)to detect the m RNA level of STAT5 A,STAT5B and internal reference GAPDH in the CML cell line K562 and imatinib resistantance cell line K562/G.(6)To detect the expression of STAT5 and p-STAT5 protein level by western blot method to analyze the related pathways signal molecule protein levels.(7)To collect of 40 cases of different treatment period for chronic myelogenous leukemia patients and 20 cases of normal control group in peripheral blood,the extraction of RNA,detection the STAT5 and BCR-ABL m RNA level by real-time fluorescent quantitative PCR.(8)To detect the expression of JAK2 protein level by western blot method and the cytokine IL3,IL4,IL6,GM-CSF m RNA level by real-time fluorescent quantitative PCR between K562 and K562/G.(9)To detect the expression of P53,Bax,Bcl-2 m RNA level by real-time fluorescent quantitative PCR between K562 and K562/G.Result:(1)In the experiment,we established the stable imatinib resistant cell line K562/G and The IC50 of K562/G was eighty times higher than K562 by CCK-8.(2)By cell count the results showed that the concentration gradient of imatinib inhibit the growth of K562 cells through different concentration gradient of imatinib act on K562 cell a stitch.We found cell vitality reduced obviously after 24 h,even down to zero.The same method,K562/G cells grew well in imatinib concentration below 10umol/L.The viable cell number was no difference between 96 h with 0h.With imatinib concentration increasing,viable count decrease progressively since more than 10umol/L concentration.Therefore K562 / G cells have obvious resistance for imatinib.(3)Compared with K562 cells,The apoptosis level of K562/G cells decreased significantly with different concentrations of imatinib;However,The expression of ROS in K562/G cells were significantly increased(P < 0.05).(4)The expression of STAT5 A and STAT5 B m RNA in K562/G cells were obviously higher than that of K562 cells(P < 0.05).(5)The level of STAT5 and STAT5 phosphorylation protein increased significantly in K562/G cells compared with K562 cells(P < 0.05).(6)The expression of STAT5 A and STAT5 B in CML patients was significantly higher than normal control group(P < 0.05),patients with CML resistant group was obviously higher than that of CML non resistant group(P < 0.05).(7)The level of JAK2 protein was no significantly in K562/G cells compared with K562 cells,but the the cytokine IL3,IL4,IL6,GM-CSF m RNA level by real-time fluorescent quantitative PCR had significantly in K562/G cells compared with K562cells(P < 0.05).(8)The expression of P53 and Bcl-2 had obvious difference in K562/G cells compared with K562 cells(P < 0.05).Conclusion:STAT5 is usually in a high active with CML patients,when BCR/ABL act on STAT5,the increasing of ROS lead to DNA oxidative damage and increase the cell mutation,then cause CML imatinib resistant.THe research of upstream and downstream markers about STAT5-ROS cell signaling pathway provide new ideas for chronic myelogenous leukemia imatinib resistant,which will provide a scientific basis for exploring multiple targets for treatment.