5-氮杂胞苷和顺铂联合TRAIL诱导肺癌A549细胞凋亡及其机制探讨

Synergistic induction effect of 5-azacytidine and cisplatin combined with TRAIL on apoptosis of lung cancer A549 cells and its mechanism

目的 :探讨5-氮杂胞苷、顺铂联合肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)诱导人非小细胞肺癌A549细胞凋亡的作用及可能的分子机制。方法:用5-氮杂胞苷单药、TRAIL单药、5-氮杂胞苷+TRAIL以及5-氮杂胞苷+顺铂+TRAIL联合用药分别作用A549细胞后,采用MTT法和FCM法分别检测细胞增殖抑制率和细胞凋亡率。采用RT-PCR和蛋白质印迹法检测5-氮杂胞苷处理前后A549细胞中死亡受体(4death receptor 4,DR4)m RNA及蛋白表达量的变化。甲基化特异性PCR(methylation-specific PCR,MSP)法检测5-氮杂胞苷处理前后A549细胞中DR4基因的启动子甲基化状态。结果 :5-氮杂胞苷+TRAIL组A549细胞的增殖抑制率及凋亡率均明显高于TRAIL单药组(P值均<0.05),而5-氮杂胞苷+顺铂+TRAIL组A549细胞的增殖抑制率及凋亡率均明显高于5-氮杂胞苷+TRAIL组(P值均<0.05)。A549细胞在经5-氮杂胞苷处理前,其DR4基因启动子呈甲基化状态,DR4 m RNA及蛋白表达水平均较低;经5-氮杂胞苷处理后,DR4基因启动子呈非甲基化状态,DR4表达水平明显提高,并呈时间依赖性(P值均<0.05)。结论 :5-氮杂胞苷可以逆转DR4基因启动子的甲基化状态,使其表达上调,增强TRAIL诱导A549细胞凋亡的作用,逆转细胞对TRAIL的耐药性。同时,亚毒性剂量的顺铂可进一步增强TRAIL对A549细胞的凋亡诱导作用。提示5-氮杂胞苷、顺铂联合TRAIL可能成为治疗肺癌的一种新策略。

Objective： To study the effect of 5-azacytidine and cisplatin combined with tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） on apoptosis of non- small cell lung cancer A549 cells, and to investigate its possible molecular mechanism.Methods： A549 cells were treated with 5-azacytidine, TRAIL, 5-azacytidine combined with TRAIL, and 5-azacytidine and cisplatin combined with TRAIL, respectively. The proliferation inhibitory rate and apoptosis rate of A549 cells were determined by MTT method and flow cytometry, respectively. Before and after 5-azacytidine treatment, the mRNA and protein expression levels of death receptor 4 （DR4） gene in A549 cells were detected by reverse transcripton （RT）-PCR and Western blotting, respectively; the methylation of DR4 gene promoter was examined by methylation-specific PCR （MSP）.Results： The proliferation inhibitory rate and apoptosis rate of A549 cells in 5-azacytidine combined with TRAIL group were significantly higher than those in TRAIL group （both P 〈 0.05）. As compared with 5-azacytidine combined with TRAIL group, the rates of proliferation inhibition and apoptosis in 5-azacytidine and cisplatin combined with TRAIL group were significantly increased （both P 〈 0.05）. In A549 cells not treated with 5-azacytidine, the promoter of DR4 gene was in methylation status, with low expressions of DR4 mRNA and protein. After A549 cells were treated with 5-azacytidine, the promoter of DR4 gene was demethylated, while the expression levels of DR4 mRNA and protein were significantly increased in a time-dependent manner （both P 〈 0.05）.Conclusion： 5-Azacytidine can reverse the methylation of DR4 gene promoter and up- regulate the expression level of DR4 gene, resulting in promoting the apoptosis of A549 cells induced by TRAIL and reversing TRAIL resistance of A549 cells. Moreover, sub-toxic dose of cisplatin can enhance the induction effect of TRAIL on apoptosis of A549 cells. Therefore, the combination of 5-azacytidine, cisplatin and TRAIL may become a new strategy for treatment of lung cancer.