Effect Of Tyrosine Kinase Inhibitor On Autophagy Of Chronic Myeloid Leukemia And Its Mechanism

Objective:To explore the changes of autophagy level and its specific mechanism in vitro and in vivo by tyrosine kinase inhibitor in CML cell lines and CML patients,and to lay a foundation for the basic research and clinical application of CML.Methods: 1.In vitro studies 1.1 Cell culture: chronic myeloid leukemia cell lines K562 and KCL22.1.2 CCK-8 assay was used to detect the proliferation inhibition rate of different concentrations of tyrosine kinase inhibitor(0.1,0.2,0.5,1.0,2.0ug/ml)in K562 and KCL22 cell lines at different times(6h,12 h,18h),and to study the change of proliferation inhibition rate of different concentrations of tyrosine kinase inhibitor(0.5ug/ml,1.0ug/ml)on chronic myeloid leukemia cell lines at 12 h.1.3 Acridine orange staining was used to observe the changes of autophagy levels in K562 and KCL22 cells in the blank group and different concentrations of TKI(0.5ug/ml,1.0ug/ml)for 12 hours.1.4 Real-time quantitative PCR(RT-qPCR)and Western Blot was used to detect the autophagy related factors LC3 B,mTOR and Beclin-1's mRNA and protein expression differently in the blank group and the 2 groups with different concentrations of tyrosine kinase inhibitor(0.5ug/ml,1.0ug/ml)for 12 h after the treatment of chronic myeloid leukemia cell lines.2.In vivo studies 2.1 Patients collection: patients were collected from the Department of Hematology,Affiliated Hospital of Chengde Medical College from December 2017 to March 2019.Ten patients who met the CML chronic phase diagnostic criteria and were regularly followed up with TKI for 3 months were included in the study.2.2 Bone marrow lymphocytes extracted and cryopreserved: Lymphocytes were extracted from the bone marrow of 10 patients with chronic CML who were diagnosed and regularly treated with TKI for 3 months,and frozen at-80 °C.2.3 Blood routine tests of 10 patients before and after medication were detected by Sysmex xe-2100 automatic blood analyzer.2.4 Real-time quantitative polymerase chain reaction(rq-pcr)was used to detect the transcription level of BCR-ABL fusion gene in bone marrow of 10 patients.2.5 Western Blot was used to detect the the autophagy related factors LC3 B,mTOR and Beclin-1's protein expression in patients with chronic myeloid leukemia at the first diagnosis and 3 months after the application of tyrosine kinase inhibitor.Results: 1.In vitro studies 1.1 Proliferation inhibition rates of chronic myeloid leukemia cell lines K562 and KCL22 were detected by CCK-8 method.The proliferation inhibition rate of KCL22 and K562 cell lines treated with different concentrations of tyrosine kinase inhibitors(0.1,0.2,0.5,1.0,2.0ug/ml)for different time(6,12,18h).With the increase of concentration of tyrosine kinase inhibitor and the prolongation of treatment time,the proliferation inhibition rates increased gradually(P<0.05).In order to better observe autophagy,tyrosine kinase inhibitor with concentrations of 0.5ug/ml and 1.0ug/ml were selected in our subsequent experiments,and the action time was 12 h.1.2 Acridine orange staining was used to observe the autophagy levels of K562 and KCL22 cells in the blank group and treated with different concentrations of TKI(0.5ug/ml and 1.0ug/ml)for 12 hours.1.3 RT-qPCR and Western Blot were used to analyze the autophagy related factors' mRNA and protein expression in K562 and KCL22 cell lines.Tyrosine kinase inhibitor(0.5ug/ml and 1.0ug/ml)treated chronic myeloid leukemia cell lines after 12 h,compared with the control group,the expression of mRNA and protein of autophagy related factors LC3 B,Beclin-1increaced,mTOR decreased,and the differences are statistically significant(P < 0.05).2.0 In vivo studies 2.1 According to the Chinese guidelines for the diagnosis and treatment of chronic myeloid leukemia,all the 10 patients treated with TKI for 3 months achieved complete hematological remission,and the level of BCR-ABL of the 10 patients treated with TKI decreased compared with that initial diagnosis,but none of them achieved complete molecular biological response.2.2 The protein expression level of autophagy-related factors LC3 B,mTOR and Beclin-1 were analyzed by Western Blot in 10 patients with chronic CML who were diagnosed and regularly treated with TKI for 3 months.Compared with the same newly diagnosed patients,the expression of autophagy-related factors LC3 B and Beclin-1 was increased and the expression of mTOR protein was decreased in CML patients who were treated with TKI for 3 months(P < 0.05).Conclusion:1.TKI inhibited proliferation of K562 and KCL22 cell lines in a time-dependent and concentration-dependent manner.2.Low level of autophagy was observed in CML cell lines and in CML patients.3.TKI can increase the level of autophagy and autophagy-related proteins in CML cell lines,and increase the level of autophagy-related proteins in CML patients.