【摘要】 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Molt4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,AnnexinⅤ/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Molt4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,48h的IC50值为(4.63±0.15)μmol/L。荧光显微镜观察显示,ABT-199处理Molt4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;AnnexinⅤ/PI检测细胞凋亡结果显示,0、1、2、4、8μmol/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,χ~2=18.286,ν=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,χ~2=17.386,ν=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(χ~2=9.024,ν=3,P=0.029),并出现Caspase-3下游底物多聚ADP核糖聚合酶(poly ADP ribose polymerase,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。更多还原

【Abstract】 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-2selectively 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 AnnexinⅤ/PI double scaining were used to determine IC50 and apoptotic status.JC-1assay 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 IC50of(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 0,1,2,4and 8μmol/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(χ~2=18.286,ν=4,P=0.001).In addition,the administration of ABT-199 resulted in the disruption of mitochondrial membrane potential in Molt4cells(χ~2=17.386,ν=4,P=0.002).Western blot analysis revealed that the expression of Bcl-2was markedly decreased in the presence of ABT-199(χ~2=9.024,ν=4,P=0.029),accompanied by PARP degradation and increased caspase-3cleavage(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 induction.Mechanistically,these events may attribute to the activation of mitochondrial signaling pathway.更多还原