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。
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 prol
出处
《中华肿瘤防治杂志》
CAS
北大核心
2016年第22期1470-1476,1482,共8页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(81570156)
广东省重大研究专项(2015B020227003)
