桔梗皂苷D对肿瘤坏死因子相关凋亡诱导配体抗肺癌细胞活性的影响

Platycodin-DPromotes TRAIL-induced Apoptosis in Lung Cancer

目的探讨中药活性成分桔梗皂苷D对肿瘤坏死因子相关凋亡诱导配体(TRAIL)抗肺癌细胞活性的影响及其机制。方法将人肺癌细胞系A549分为对照组、桔梗皂苷D组、TRAIL组及桔梗皂苷D联合TRAIL组,MTT法检测A549细胞活力,Annexin V/PI染色检测A549细胞凋亡,免疫共沉淀法检测A549细胞RIP1-FADD-caspase-8复合物的形成,Western blot法检测A549细胞caspase-8的活化。结果对照组、桔梗皂苷D组、TRAIL组、桔梗皂苷D+TRAIL组对A549细胞活力的抑制率分别为0、(7.6±0.7)%、(13.4±1.0)%、(60.3±4.2)%,桔梗皂苷D组、TRAIL组、桔梗皂苷D+TRAIL组的细胞活力抑制率与对照组比较差异均有统计学意义(P<0.05)。对照组、桔梗皂苷D组、TRAIL组、桔梗皂苷D+TRAIL组对A549细胞凋亡的诱导率分别为(1.6±0.2)%、(4.5±0.3)%、(7.2±0.5)%、(40.2±2.8)%,TRAIL组、桔梗皂苷D+TRAIL组的细胞凋亡率较对照组有明显提高(P<0.05)。桔梗皂苷D+TRAIL组A549细胞内的RIP1-FADD-caspase-8复合物水平及caspase-8的活化程度显著提高。RIP1 si RNA及z-IETD-fmk能显著抑制桔梗皂苷D对TRAIL的协同作用。对照组、桔梗皂苷D+TRAIL组、桔梗皂苷D+TRAIL+RIP1 si RNA组、桔梗皂苷D+TRAIL+z-IETD-fmk组对A549细胞活力的抑制率分别为0、(61.2±5.0)%、(24.5±1.9)%、(17.5±1.7)%,与桔梗皂苷D+TRAIL组比较,桔梗皂苷D+TRAIL+RIP1 si RNA组、桔梗皂苷D+TRAIL+z-IETD-fmk组的细胞活力抑制率明显降低(P<0.05)。对照组、桔梗皂苷D+TRAIL组、桔梗皂苷D+TRAIL+RIP1 si RNA组、桔梗皂苷D+TRAIL+z-IETD-fmk组对A549细胞的凋亡诱导率分别为(1.8±0.2)%、(39.5±2.6)%、(12.3±1.1)%、(9.4±0.8)%,与桔梗皂苷D+TRAIL组比较,桔梗皂苷D+TRAIL+RIP1 si RNA组、桔梗皂苷D+TRAIL+z-IETD-fmk组的细胞凋亡率明显降低(P<0.05)。结论桔梗皂苷D可能通过促进死亡受体复合物的形成,增强TRAIL对肺癌细胞的凋亡诱导效应。

Objective To investigate the role of platycodin-Din TNF-related apoptosis-inducing ligand（TRAIL）-induced cell death in lung cancer. Methods The A549 cells were divided into control group, platycodin-D group,TRAIL group, and platycodin-D ＋TRAIL group, then the cell death, cell apoptosis, and the formation of RIP1-FADD-caspase-8 complex were detected by MTT assay, Annexin Ⅴ/PI staining, and co-immunoprecipitation, respectively. The activation of caspase-8 in A549 cells was evaluated by western blot assay. Results The cell viability inhibitory rate of A549 cells in control group, platycodin-D group, TRAIL group, and platycodin-D ＋TRAIL group was as follows： 0,（7.6±0.7）%,（13.4±1.0）%, and（60.3±4.2）%; significant differences in the cell viability inhibitory ratewas found between control group and platycodin-D group, TRAIL group, and platycodin-D ＋TRAIL group（all P〈0.05）. The apoptotic ratio of A549 cells in control group, platycodin-D group, TRAIL group, and platycodin-D＋TRAIL group was as follows：（1.6±0.2）%,（4.5±0.3）%,（7.2±0.5）%, and（40.2±2.8）%, with significant differences between control group and TRAIL group and platycodin-D＋TRAIL group（all P〈0.05）. The formation of RIP1-FADD-caspase-8 complex and the activation of caspase-8 in TRAIL-treated A549 cells were significantly increased in platycodin-D＋TRAIL group. Treatment of RIP1 siRNA and z-IETD-fmk significantly impaired the synergistic effect of platycodin-D on TRAIL-induced cell death. The cell viability inhibitory rate of A549 cells in control group, platycodin-D＋TRAIL group, platycodin-D＋TRAIL＋RIP1 siRNA group, and platycodin-D＋TRAIL＋zIETD-fmk group was as follows： 0,（61.2±5.0）%,（24.5±1.9）%,（17.5±1.7）%; significant differences in the cell viability inhibitory ratewas noted between platycodin-D＋TRAIL groupandplatycodin-D＋TRAIL＋RIP1 siRNA groupand platycodin-D＋TRAIL＋z-IETD-fmk group（all P〈0.05）. The apoptotic rate of A549 cells in control group, platycodin-D＋TRAIL group, platycodin-D＋TRAIL＋RIP1 siRNA group, and platycodin-D＋TRAIL＋z-IETD-fmk group was as follows：（1.8 ±0.2）%,（39.5 ±2.6）%,（12.3 ±1.1）%, and（9.4 ±0.8）%, with significant differences betweenplatycodin-D＋TRAIL groupand platycodin-D＋TRAIL＋RIP1 siRNA groupand platycodin-D＋TRAIL＋z-IETD-fmk group（all P〈0.05）. Conclusion platycodin-D promotes TRAIL-induced apoptosis by the formation of RIP1-FADD-caspase-8 complex in lung cancer.