The Role Of DBO In Inducing Apoptosis And Autophagy Of K562 Cells

Background:Chronic myeloid leukemia(CML)is one of myeloid malignant proliferative tumors originating from pluripotent stem cells.t(9;22)(q34;q11)is a characteristic chromosomal change of CML and leads to the formation of BCR-ABL Fusion gene at the molecular level.The fusion protein formed by this BCR-ABL fusion gene is a variety of signal protein-related tyrosine kinases that can lead to cell transformation.At present,tyrosine kinase inhibitors(TKI)(including imatinib,Gleevec,etc.)have been widely used in CML treatment,so that the survival of most patients with CML extended to achieve cytogenetic remission or molecular biology remission.With the prolongation of TKI clinical application,TKI resistance is becoming more and more obvious,and actively explore new drugs is an urgent problem to be solved.DBO(6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine)is a benzoxazine derivative,studies have confirmed that it can be used as a mammalian rapamycin target protein(m TOR)inhibitor to induce human umbilical vein endothelial cells to produce cell reactive oxygen species(ROS),which induced human umbilical vein endothelial cells producing autophagy and promoted its apoptosis.However,at present,its anti-tumor effect has not been extensively studied,and the mechanism of anti-tumor effect is not yet conclusive.Research on whether DBO is causing autophagy or apoptosis in leukemia cells has not been reported.In this study,human K562 cells were used to study the effect of DBO on the proliferation of K562 cells and their effects on induction of apoptosis and autophagy.Objective:This study was to investigate the effect of DBO on proliferation,autophagy and apoptosis of human chronic myeloid leukemia K562 cells.Method:K562 cells were revived and cultured by conventional method.The control group(DMSO)and DBO treatment group were set up.Induced by DBO for 24 h,48 h and 72 h,the protein expression of LC3 was detected by Western blotting.The expression of LC3 spots was detected by cell immunofluorescence assay.Autophagy was observed by transmission electron microscopy.Cell viability and proliferation were analyzed using CCK-8 colorimetry.The ability of K562 cell division was detected by cell cycle assay.The cell apoptosis was tested by Annexin V-FITC / PI double staining flow cytometry.Results:Western blot showed that after treated with rapamycin,the protein expression of LC3-II in K562 cells increased and that of p62 reduced.After treated with 50 ?mol / L DBO for 24 h,48 h and 72 h,the protein expression of LC3-II and p62 in K562 cells is time-dependently similar to that of rapamycin treatment.Treated with different concentrations of DBO(10 ?mol / L,25 ?mol / L,50 ?mol / L),the protein expression of LC3-II in K562 cells increased dose-dependently,and that of p62 decreased dose-dependently.While the autophagy inhibitor 3–MA is used,LC3-II protein expression is reduced and p62 expression is increased.Cell immunofluorescence experiments showed that compared with the control group,the expression of LC3 spots in the DBO-treated group was significantly higher after treated with DBO for 72 h.Transmission electron microscopy showed that many autophagosome and autolysosome,irregular nucleus,marginalized chromatin appeared in the DBO-treated cells cytoplasm.CCK8 results showed that DBO inhibited the proliferation of K562 cells in a dose-and time-dependent manner.K562 cells were treated with DBO in different concentrations(10 ?mol / L,25 ?mol / L,50 ?mol / L,100 ?mol / L)for 24 h,the proliferation inhibition rates were(0.68 ± 0.05)%,(2.76 ± 0.35)%,(12.64 ± 3.90)% and(22.58 ± 2.41)% respectively,F = 67.389,P < 0.001;While after 48 h,the proliferation inhibition rates were(3.83 ± 1.06)%,(6.23 ± 1.27)%,(15.90 ± 1.10)% and(24.50 ± 2.51)respectively,F= 145.738,P< 0.001;and after 72 h,the proliferation inhibition rates were(8.78 ± 1.28)%,(21.38 ± 1.47)%,(32.11 ± 2.01)% and(34.27 ± 2.59)% respectively,F= 225.820,P< 0.001.There were significant differences between the control group and the treated groups.Flow cytometry analysis showed,compared with the control group,the ratio of G2 / M phase of K562 cells treated with DBO for 72 h was risen,?2= 276.706,P< 0.001.Compared with the control group,flow cytometry analysis showed that the apoptotic rate of K562 cells that treated with DBO for 72 h was risen,?2= 227.384,P< 0.001.Conclusion:DBO can inhibit the growth of K562 cells obviously,which was related to autophagy and apoptosis of K562 cells.