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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。
Purpose The tolerance of most tumor cells to chemotherapy-induced apoptosis is related to the Bcl-2 family of proteins, and ABT-199 is a selective antagonist of the anti-apoptotic protein Bcl-2. The purpose of this study was to investigate the effects and mechanisms of ABT-199 on proliferation and apoptosis of human acute lymphoblastic leukemia Molt4 cells in vitro. Methods Molt4 cells were cultured in vitro. The proliferation of Molt4 cells was detected by CCK8 assay. The apoptosis of Molt4 cells was observed by DAPI staining. The apoptosis of Molt4 cells was detected by AnnexinⅤ / PI double staining. The changes of mitochondrial membrane potential in Molt4 cells treated with different concentrations of ABT-199 were detected by JC-1 staining. The expressions of Bcl-2, PARP and cleaved Caspase -3 protein) expression level changes. Results ABT-199 had the inhibitory effect on Molt4 cells proliferation, and the IC50 value at 48h was (4.63 ± 0.15) μmol / L. Fluorescence microscopy showed that apoptotic bodies were found in the nuclei of Molt4 cells treated with ABT-199, and the morphological changes were more obvious with the increase of drug concentration. The apoptosis results of AnnexinⅤ / PI The percentages of apoptotic cells induced by ABT-199 at 0,1,2,4 and 8μmol / L were (3.09 ± 1.16)%, (11.59 ± 5.58)%, (25.37 ± 7.42)% and (46.38 ± 7.05) )% And (80.60 ± 11.18)%, respectively. There was a significant difference in the percentage of apoptotic cells between the treated group and the control group (χ ~ 2 = 18.286, ν = 4, P = 0.001). The results of JC-1 showed that ABT-199 could decrease the mitochondrial membrane potential of Molt4 cells in a concentration-dependent manner, and there was a significant difference in the mitochondrial membrane potential decrease between the treated group and the control group (χ ~ 2 = 17.386, ν = 4, P = 0.002. The results of Western blotting showed that the expression of Bcl-2 in the mitochondrial pathway was significantly decreased (χ ~ 2 = 9.024, ν = 3, P = 0.029) (poly ADP ribose polymerase, PARP) cleavage, while the accumulation of Caspase-3 cleavage fragments. Conclusion ABT-199 can inhibit the proliferation and induce the apoptosis of Molt4 cells in vitro, which is related to the activation of mitochondria signaling pathway.