去甲斑蝥酸钠对耐顺铂肺腺癌A549/DDP细胞的逆转作用及机制

Reversal effect of sodium norcantharidate on human lung adenocarcinoma cell line A549/DDP in vitro

目的 探讨去甲斑蝥酸钠（SNCTD）对耐顺铂人肺腺癌细胞系A549/DDP的逆转作用及可能分子机制.方法 （1）采用CCK法筛选出SNCTD对A549/DDP的无毒浓度（即对细胞抑制率＜10%的药物浓度）,并检测出顺铂及与无毒浓度SNCTD联合对耐药细胞株的IC50;（2）采用流式细胞仪检测无毒浓度SNCTD对细胞内罗丹明123的蓄积情况;（3）采用反转录-聚合酶链反应（RT-PCR）检测1、2倍无毒浓度SNCTD处理细胞48h后耐药相关基因mdrlmRNA,MRP1 mRNA的表达.结果 （1）SNCTD对A549/DDP的无毒浓度为5μg/ml,与顺铂联合用药降低A549/DDP的耐药性,逆转倍数为1.97;（2）无毒浓度SNCTD处理耐药细胞48h后细胞内罗丹明123荧光强度明显增强（F=36.99,P＜0.05）;（3）1、2倍无毒浓度SNCTD处理耐药细胞后mdr1,MRP1mRNA表达明显减低,并具有浓度依赖性.结论 SNCTD对A549/DDP具有耐药逆转作用,其作用机制可能与下调耐药相关基因mdr1,MRP1的表达,影响膜蛋白外排泵功能有关.

Objective To investigate the reversal effect and the mechanism of sodium norcantharidate（SNCTD）on human lung adenocarcinoma cell line A549/DDP. Methods CKK assay was used to screen out non-toxic concentration （less than 10 percent of cell inhibition ratio） of SNCTD, and to measure the IC50 of cisplatin and IC50 of innoxious concentration SNCTD plus cisplatin in drug-resistant cell line. The accumulation effect of Rh123 was assayed by flow cytometry after treatment with non-toxic concentration of SNCTD. PT-PCR was used to detect the expression of mdr1, MRP1 gene for the drug-resistant cell line treated with non-toxic concentration of SNCTD for 48h. Results （1）The non-toxic concentration of SNCTD was 5μg/ml. SNCTD could decrease drug resistance to cisplatin. The reversal fold was 1.97. （2）The fluorescence intensity of Rh123 in the cells treated with 5μg/ml SNCTD was obviously increased （F=36.99, P＜0.05）. （3）The expressions of mdr1, MRP1 gene decreased significantly in a concentration-dependent manner.Conclusions SNCTD could reverse the resistance to cisplatin in A549/DDP cell line. It possibly downregulates the expression of mdr1, MRP1 gene, and inhibits the function of efflux pump of membrance protein.