基因工程腺病毒H101逆转A549/DDP细胞对顺铂耐药性的实验研究

背景及目的:耐药性产生是化疗失败的主要原因之一,克服耐药性的方法之一是使用逆转剂。目前尚无一种理想的、可应用于临床的顺铂(DDP)耐药逆转剂。本研究探讨基因工程腺病毒H101对DDP耐药性的逆转作用及其机制。方法:体外利用人肺腺癌细胞株A549的DDP耐药模型A549/DDP,采用MTT法检测H101对A549/DDP细胞耐药性的逆转作用。用流式细胞仪(FCM)测定细胞多药耐药蛋白1(multidrugresistantprotein1,MRP1)、谷胱甘肽鄄S鄄转移酶(GST)及TOPO鄄Ⅱ蛋白表达量,荧光鄄考马斯亮蓝法检测细胞内GSH含量,原子吸收法测定细胞内铂浓度。结果:A549/DDP细胞对DDP的耐药指数为14.3。加入H101后,DDP对A549/DDP的半数抑制浓度(IC50)由352.4μmol/L降至61.6μmol/L,逆转倍数为5.7倍。FCM检测结果显示,A549/DDP细胞的GST和TOPO鄄Ⅱ蛋白水平均较A549细胞高,而两种细胞内均未检测到MRP1的表达,经H101处理后的A549/DDP细胞胞内GST蛋白和TOPO鄄Ⅱ蛋白水平均明显下降。A549/DDP胞内GSH含量比A549高2倍多;H101感染A549/DDP细胞后,胞内的GSH含量下降,随着H101剂量的增加,胞内GSH含量逐渐下降,达A549细胞内GSH水平。原子吸收法测定细胞内铂含量的结果显示,A549细胞内铂含量是A549/DDP细胞内铂含量的3倍,加入H101后,A549细胞内铂的含量无明显变化,但A549/DDP细胞内铂的含量明显增加。结论:H101能逆转A549/DDP对DDP的耐药性,H101逆转DDP耐药性的机制可能是减少A549/DDP细胞内GSH和TOPOⅡ蛋白的水平,增加了A549/DDP细胞内铂的蓄积。

Reverse effect of genetically modified adenovirus H101 on drug-resistance of A549/DDP cells to cisplatin

BACKGROUND & OBJECTIVE: Multidrug resistance is one of the main causes of treatment failure of chemotherapy. Reversal agents may be used to reverse drug-resistance. To date, no appropriate reversal agent is used clinically. This study was to evaluate reverse effect of genetically modified adenovirus H101 on drug-resistance of A549/DDP cells to cisplatin (DDP), and to explore its mechanisms. METHODS: Lung adenocarcinoma cell line A549 and its DDP-resistant cell line A549/DDP were treated with DDP and H101. Reverse effect of H101 on drug-resistance of A549/DDP to DDP was tested by MTT assay. The levels of cellular multidrug resistant protein 1 (MRP1), glutathione-S-transferase (GST), and topoisomerase-II (TOPO-II) were detected by flow cytometry (FCM). The level of intracellular glutathione (GSH) was measured by fluorescence-Coomassie light blue method. The intracellular concentration of platinum was measured by atomic absorption spectrometry. RESULTS: The resistance index of A549/DDP cells to DDP was 14.3. After infection of H101, the 50% inhibitory concentration (IC(50)) of DDP to A549/DDP cells reduced from 352.4 micromol/L to 61.6 micromol/L by 5.7 folds. Protein levels of GST and TOPO-II were higher in A549/DDP cells than in A549 cells, which were markedly decreased in A549/DDP cells after infection of H101. No MRP1 protein was detected in both A549 cells and A549/DDP cells. Intracellular amount of GSH in A549/DDP cells was 2 times higher than that in A549 cells, which was significantly decreased after infection of H101. The concentration of platinum in A549 cells was 3 times higher than that in A549/DDP cells. After infection of H101, the concentration of platinum in A549 cells had no significant change, but that in A549/DDP cells was significantly increased. CONCLUSION: H101 may reverse drug-resistance of A549/DDP cells to DDP through down-regulating GST and TOPO-IIand increasing intracellular cumulation of platinum.