The Efficacy Of ABT-199 On Proliferation And Apoptosis Of Acute Lymphoblastic Leukemia Cells And Its Mechanism

Background:Acute lymphoblastic leukemia(ALL)is a group of highly genetically heterogeneous diseases.Although the multi-drug combination of induction regimens significantly improved complete remission(CR)rate,there are still a considerable number of patients with ALL become resistant to chemotherapy or recurrence at some point in their course and succumb to their disease even after high-dose of chemotherapy and hematopoietic stem cell transplantation(HSCT)followed complete remission.Once recurrence means extremely poor prognosis.In recent years,researchers have found that the overexpression level of anti-apoptotic protein Bcl-2(B-cell lymphoma/leukemia 2)subfamily has responsibility for tumor resistance.ABT-199,which functions as a small-moleculemimetic of the BH3 domain BH3-only protein and efficient oral medication,maintains specificity for BCL-2.ABT-199 could induce cancer cells by directly stimulating the mitochondrial apoptotic pathway.To date,the efficacy of the ABT-199 for leukemia is still in basic research and clinical trials stagein foreign countries,and there still need more research about the role of ABT-199 in ALL cells.Objective:This study aims to investigate the anti-leukemia effects of ABT-199,a Bcl-2 selectively inhibitor,in acute lymphoblastic leukemia cells as well as its molecular mechanismsin vitro.Method:1.We detected the inhibition of cell proliferation of different concentrations of ABT-199 treatment with Molt4 and Nalm6 cells after 48h by CCK-8 kit.2.Apoptosis features were observed under fluorescence microscope with DAPI staining on Molt4 cells.3.Annexin V/PI double scaining were used to determine apoptotic rates after treatment with ABT-199 on Molt4 and Nalm6 cells by flow cytometry.4.Caspase suppressing assay:a pan-inhibitor of the caspase family,Z-VAD-fink pretreatment Molt4 cells 2 hours,then Molt4 cells were continuely treated with ABT-199 for 24 h and then the apoptotic rate was measured by flow cytometry.5.JC-1 staining was used to determine the mitochondrial membrane potential of Molt4 cells by JC-1 probe.6.The expression of Bcl-2,cleaved-Caspase-3,PARP and Bax protein in Molt4 cells treated with ABT-199 was detected by Western blotting.7.Statistical analysis:Statistical analysis was performed by using professional statistical software SPSS 20.0.The experimental datas are expressed as mean ±standard deviation(mean ± SD).Due to the small sample size,the comparison between multiple independent samples were performed by nonparametric test(Kruskal-Wallis H)statistical analysis,with a = 0.05 for the test level.All tests were bilateral test.Results:1.ABT-199 was effective against the proliferation of Molt4 and Nalm6 cells.The IC50 on Molt4 and Nalm6 cells 4.63±0.15?mol/L and 9.79±0.06?mol/L for 48 hrs,respectively.2.After ABT-199 treatment for 48hrs,Chromatin condensation and apoptosis body were found in nucleus with DAPI stainingunder fluorescence microscope.The changes were remarkably associated with the increase of ABT-199 concentration.3.Annexin-V-APC/PI double staining showed that ABT-199 induced apoptosis of Molt4 and Nalm6 cells in a concentration-dependent manner.There was a significant difference between the ABT-199 treatment group and the control group)through nonparametric teststastical analysis(?2=1 8286,v=4,P=0.001;x2=18286,v=4,P=0.001).4.We found that the pan-inhibitor of the caspase family,Z-VAD-fmk,could suppressed ABT-199 induced apoptosis.The apoptotic rate of ABT-199 compared with ABT-199 plus Z-VAD-fink(20?mol/L)was statistically significant on Molt4 cells(P<0.05).The results suggested that ABT-199 induced acute lymphoblastic leukemia cell apoptosis in Caspase-dependent manner.5.JC-1 results showed the mitochondrial membrane potential was decreased after treatment with ABT-199 on Molt4 cells with the increase of concentration.The results of nonparametric test showed that ABT-199 could significantly down-regulate the mitochondrial membrane potential after Molt4 cells and showed a concentration-dependent relationship(?2=17.3 86,v=4,P=0.002).6.Western blotting showed that the molecular mechanism of ABT-199-induced apoptosis of leukemia cells was related to the activation of mitochondrial apoptosis pathway.The expression of Bcl-2 was markedly decreased in the presence of ABT-199(?2=9.024,v=4,P=0.029),accompanied by PARP degradation(?2=7.619,v=3,P=0.045)and increased caspase-3 cleavage(activation).The expression of proapoptotic protein Bax did not change significantly,suggesting that ABT-199 migth promoting Bax translocation.Conclusion:1.ABT-199 markedly inhibited proliferation of Molt4 and nalm6 cells,in dose-dependent mode.2.ABT-199 significantly induced ALL cells apoptosis.3.ABT-199 induced acute lymphoblastic leukemia apoptosis is mediated by Caspase.4.The molecular mechanism of ABT-199-induced apoptosis of leukemia cells was related to the activation of mitochondrial apoptosis pathway.ABT-199 could down-regulate the expression of Bcl-2 protein in the mitochondrial signaling pathway,and promote the translocation of Bax,leading to the decrease of mitochondrial membrane potential(MMP)and activation of Caspase-3 and PARP.