ABT-199体外对急性T淋巴细胞白血病细胞株Molt4细胞增殖凋亡影响及其机制研究

目的 大多数肿瘤细胞对化疗诱导细胞凋亡的耐受与Bcl-2家族蛋白有关,ABT-199是针对抗凋亡蛋白Bcl-2的选择性拮抗剂.本研究旨在探讨ABT-199体外对急性T淋巴细胞白血病Molt4细胞株增殖凋亡的影响及其机制.方法 体外培养Molt4细胞,CCK8法检测不同浓度ABT-199对Moh4的增殖抑制作用,DAPI细胞核染色后荧光显微镜观察细胞凋亡特征,Annexin Ⅴ/PI双染法检测不同浓度ABT-199对Molt4细胞的诱导凋亡作用,JC-1染色法检测不同浓度ABT-199作用于Moh4细胞后线粒体膜电位的变化,蛋白质印迹法检测经ABT-199处理后线粒体信号通路相关蛋白(Bcl-2、PARP和cleaved Caspase-3蛋白)表达水平的变化.结果 ABT-199对Molt4细胞具有抑制增殖的作用,48 h的IC50值为(4.63±0.15) μmol/L.荧光显微镜观察显示,ABT-199处理Moh4细胞后细胞核染色质染色浓聚,细胞核碎裂,出现凋亡小体,且随着药物浓度的增加上述形态变化更加明显;Annexin Ⅴ/PI检测细胞凋亡结果显示,0、1、2、4、8 μmol/L的ABT-199诱导细胞凋亡的百分比分别为(3.09±1.16)％、(11.59±5.58)％、(25.37±7.42)％、(46.38±7.05)％和(80.60±11.18)％,给药组与对照组的凋亡比例差异有统计学意义,x2=18.286,v=4,P=0.001.JC-1检测结果显示,ABT-199能促使Molt4细胞的线粒体膜电位下降,呈浓度依赖性,给药组与对照组的线粒体膜电位下降比例差异有统计学意义,x2 =17.386,v=4,P=0.002.蛋白质印迹法检测结果显示,给药组线粒体通路中Bcl-2表达水平显著下降(x2=9.024,v=3,P=0.029),并出现Caspase-3下游底物多聚ADP核糖聚合酶(poly ADP ribose poly-merase,PARP)的裂解,同时出现了Caspase-3裂解片段的累积.结论 ABT-199在体外能抑制急性T淋巴细胞白血病Molt4细胞的增殖及诱导其凋亡,其机制与激活线粒体信号通路相关.

Inhibition effect of ABT-199 on proliferation and apotosis of T-cell acute lymphoblastic leukemia cell line Molt4 cells and its mechanism in vitro

OBJECTIVE Accumulating evidence demonstrates that resistance to chemotherapy strategies in a wide range of cancers is closely associated with B cell lymphoma/leukemia 2 (Bcl-2) family proteins.This study aimed to investigate the anti-leukemia effects of ABT-199,a Bcl-2 selectively inhibitor,in T-cell acute lymphoblastic leukemia cell line Molt4 as well as its molecular mechanisms in vitro.METHODS Molt4 cells were subjected to different ABT-199 treatment.Apoptosis features were observed under fluorescence microscope with DAPI staining.CCK8 assay and Annexin Ⅴ /PI double scaining were used to determine IC50 and apoptotic status.JC-1 assay was performed to assess the changes of mitochondrial membrane potential.Meanwhile,the expression of mitochondrial signaling pathway-related proteins,including Bcl-2,PARP and cleaved Caspase-3,were also evaluated by western blot after exposure to ABT-199 treatment in Molt4 cells.RESULTS ABT-199 exerted a great anti-leukemia action in Molt4 cells with an IC50 of (4.63-±-0.15) μmol/L for 48 h ours.After ABT-199 treatment,Chromatin condensation and apoptosis body were found in nucleus.The changes were remarkably associated with the increase of ABT 199 concentration.After treated with 0,1,2,4 and 8 μmol/L ABT-199,apoptosis ratio was (3.09±1.16)％,(11.59± 5.58) ％,(25.37±7.42) ％,(46.38±7.05) ％ and (80.60-±-11.18) ％,respectively.ABT-199 treatment induced higher apoptosis compared with control group (x2 =18.286,v =4,P =0.001).In addition,the administration of ABT-199 resulted in the disruption of mitochondrial membrane potential in Molt4 cells (x2=17.386,v=4,P=0.002).Western blot analysis revealed that the expression of Bcl-2 was markedly decreased in the presence of ABT-199 (x2 =9.024,v =4,P =0.029),accompanied by PARP degradation and increased caspase-3 cleavage (activation).CONCLUSIONS The results demonstrate that ABT-199 exhibits a well-characterized anti-leukemia function in T-cell acute lymphoblastic leukemia cell line Molt4 cells in vitro,reflected by proliferation inhibition and apoptosis in duction.Mechanistically,these events may attribute to the activation of mitochondrial signaling pathway.