香加皮杠柳苷对肺癌A549细胞凋亡及Survivin表达的影响

Effects of Periplocin from Cortex Periplocae on Apoptosis of Human Lung Cancer A549 Cells and Expression of Survivin

目的研究香加皮杠柳苷(CPP)对人肺癌A549细胞凋亡及survivin表达的影响,探讨其抗肿瘤作用及作用机制。方法采用噻唑蓝(MTT)法检测1.25,2.50,5.00,10.00,20.00 ng·m L-1CPP处理24,48,72 h对人肺癌A549细胞的抑制作用;应用流式细胞术分析不同浓度CPP(2.50,5.00,10.00 ng·m L-1)分别作用于A549细胞6,12,24,48,72 h对细胞凋亡和凋亡率的影响;应用吖啶橙/溴乙啶(AO/EB)荧光染色法和透射电镜观察CPP处理前后A549细胞凋亡的形态学及超微结构变化;采用反转录聚合酶链反应(RT-PCR)法检测CPP作用后A549细胞中凋亡抑制基因survivin的mRNA表达情况;应用Western blot检测CPP对A549细胞survivin蛋白表达的影响。结果 CPP能显著抑制人肺癌A549细胞的生长,最大抑制率(93.46±2.35)%。流式细胞术结果显示,CPP处理组可见典型的凋亡峰,与对照组比较,A549细胞的凋亡率明显升高(P<0.05)。荧光显微镜下可见CPP处理组A549细胞呈橘红色的凋亡细胞形态。透射电镜下可见经CPP处理的A549细胞体积变小,出现细胞质凝缩,核内染色质凝聚边集于核膜,内质网扩张,细胞质空泡化等凋亡细胞的特征性超微结构改变。RT-PCR结果显示,经CPP处理的A549细胞中survivin mRNA表达降低(P<0.05),10.0 ng·m L-1CPP组survivin mRNA表达由对照组的(0.928±0.016)降至(0.251±0.012);Western blot结果显示CPP组细胞中survivin蛋白表达明显减弱。结论 CPP可诱导人肺癌A549细胞发生凋亡,可通过下调survivin基因的mRNA和蛋白表达而发挥诱导细胞凋亡作用。

Objective To investigate the effects of periplocin from Cortex Periplocae （CPP） on apoptosis of human lung cancer A549 cells and expression of survivin, and demonstrate its anti-tumor effect and the possible mechanism. Methods Inhibitory effect of CPP at different concentrations （1. 25, 2. 50, 5. 00, 10. 00, 20. 00 ng·mL-1 ） and for different time length （24, 48, 72 h） on A549 cell proliferation was tested by MTT method. Apoptosis rate of A549 cells treated with CPP at different concentrations （2. 50, 5. 00, 10. 00 ng·mL-1 ） were measured using flow cytometry （FCM） for 6, 12, 24, 48, 72 h, respectively. The morphological and ultrastructural changes of the apoptosis cells were observed by acridine orange/ ethidium bromide （AO/ EB） staining and transmission electron microscopy （TEM）. The effects of CPP on mRNA and protein expression of apoptosis associated gene survivin were assessed by RT-PCR and Western blotting. Results CPP could significantly inhibit the growth of A549, and the inhibition rate reached （93. 46±2. 35）% . The results of FCM showed that the apoptosis rate of A549 cells treated with CPP was increased significantly as compared to the control group （ P〈0. 05）. Meanwhile, typical apoptotic peaks were detected. The characteristic morphological changes of apoptosis were observed in A549 exposed to CPP, including cell shrinkage, the nuclei became yellow-red by AO/ EB staining, and typical ultrastructural changes, including nuclear chromatin condensation along the nuclear membrane, vacuolar degeneration of cytoplasm observed by TEM. The result of RT-PCR indicated that survivin mRNA expression decreased obviously in A549 cells exposed to CPP. The protein expression of survivin in A549 cells treated with 10. 0 ng·mL-1 CPP（0. 251±0. 012）was weaker than that in control group（0. 928±0. 016）. Conclusion CPP can induce apoptosis in human lung cancer cell lines A549, and the probable mechanism is related to the down-regulation of survivin mRNA and protein.