5-氮-2＇-脱氧胞苷逆转人非小细胞肺癌A549/DDP细胞对顺铂的耐药性

Reversal effect of 5-Aza-dc on cisplatin-resistance in human NSCLC cells in vitro

目的 探讨5-氮-2＇-脱氧胞苷（5-Aza-dc）逆转耐顺铂（DDP）的A549/DDP细胞对DDP的耐药性及其可能的机制.方法 以耐DDP的人非小细胞肺癌（NSCLC）细胞系A549/DDP为研究对象,采用四甲基偶氮唑蓝（MTT）法检测5-Aza-de对A549/DDP细胞的毒性作用和对A549/DDP细胞的耐药逆转指数（RI）;采用甲基化特异性聚合酶链反应（MSP）、逆转录聚合酶链反应（RT-PCR）和Western blot法检测5-Aza-de干预前后的A549/DDP细胞hMLH1基因启动子甲基化状态和基因表达情况.结果 20 μmol/L 5-Aza-dc（无毒剂量组）和40 μmol/L5-Aza-dc（低毒剂量组）干预A549/DDP细胞48 h后,其R1分别为1.82和4.42.MSP结果显示,A549/DDP细胞中,hMLH1基因启动子区呈部分甲基化状态;经无毒和低毒剂量5-Aza-dc处理A549/DDP细胞48 h后,hMLH1基因启动子区发生DNA去甲基化.无毒剂量组和低毒剂量组的hMLH1 mRNA相对表达量分别为2.05±0.08和2.00±0.03,hMLHl蛋白相对表达鼍分别为1.74±0.14和1.65±0.11.结论 5-Aza-dc能部分逆转A549/DDP细胞的DDP耐药,其机制可能与去除hMLH1基因启动子甲基化、上调其基因表达有关.

Objective The aim of this study was to investigate the effect of 5-aza-2＇-deoxycytidine （5-Aza-dc）, a methylation inhibitor, on cisplatin-resistance in non-small cell lung cancer cell line A549/ DDP and to explore its possible mechanism. Methods MTT assay was used to test the cytotoxicity of 5-Aza-dc on A549/DDP cells, and the IC50, and cisplatin resistance index of A540/DDP cells at 48 hours after 5-Aza-dc （0 μmol/L, 20 μmol/L, 40 μmol/L） treatment at different concentrations. MSP, fluorescence quantitative RT-PCR （real-time RT-PCR） and Western blot were used to detect the hMLH1 methylation status,mRNA and protein expressions, espectively. Results The IC50 value of cisplatin in AS49/DDP cells was 30.15 ±0.76 μmol/L. The MTT assay results demonstrated that during the 5-Aza-dc treatment for 48 hours, the dose of 20 μmol/L was non-toxic and 40 μmol/L was low-toxic. 5-Aza-dc at those two doses reduced IC50 value of cisplatin to 16. 54 ±0. 35 μmol/L （RI = 1. 82） and 6. 82 ±0. 16 μmol/L （ RI = 4.42） , respectively. MSP, real-time RT-PCR and Western blot showed that 5-Aza-dc at non-toxic and low-toxic doses removed the partial hMLH1-hypermethylation, and up-regulated hMLH1 mRNA and protein expressions. Conclusions Low dose 5-Aza-dc can partially reverse the cisplatin-resistance in A549/DDP cells, which may be achived through removal of hMLH1 hypermethylation and increased expression of hMLH1 gene. 5-Aza-dc may have a role in increasing the efficacy of chemotherapy for patients whose tumors are lack of hMLHl expression because of hMLHl promoter hypermethylation.