AP-1反义寡聚脱氧核苷酸逆转人膀胱癌耐药细胞多药耐药的实验研究

AP-1 antisense oligodeoxynucleotides reverse the phenotype of multidrug resistance in human bladder cancer cells by the inhibition of glutathione synthesis

目的 观察以激活剂蛋白 1(AP 1)位点为靶序列的特异性反义寡聚脱氧核苷酸 (a sODNs)对人膀胱癌耐药细胞株多药耐药性的逆转作用。方法 设计针对谷胱甘肽 (GSH )生物合成的限速酶γ 谷氨酰半胱氨酸合成酶 (γ GCS)基因启动子区域AP 1序列的特异性asODNs ,用阳离子脂质体包被后转染人膀胱癌细胞株BIU87及其多药耐药 (MDR)亚株BIU87/A ,然后分别用定量逆转录 聚合酶链反应 (RT PCR)法 [以 β 肌动蛋白 (β actin)为内参照 ]、荧光分光光度法、MTT法等检测AP 1 asODNs对两株细胞γ GCSmRNA表达、细胞内GSH水平及其对 6种抗癌药物肿瘤细胞抑制率的变化。结果 AP 1 asODNs转染 72h后 ,BIU 87和BIU 87/A细胞的γ GCSmRNA的表达和细胞内GSH的水平均出现下降。同时 6种抗癌药物对反义转染后的两株细胞的抑制率大部分都有所提高。结论 γ GCS特异的AP 1 asODNs可通过降低细胞内的GSH水平增强膀胱癌细胞对化疗的敏感性 ,逆转肿瘤细胞的耐药。

Objective To study the reversal effect of specific antisenseoligodeoxynucleotides (asODNs) to the phenotype of multidrug resistance (MDR) in human bladder cancer cells.Methods Two 18-bp long phosphorothioate antisense and sense oligodeoxynucleotides (ODNs) targeting the promoter domain activator protein-1 (AP-1) sequence of γ-glutamylcysteine synthetase catalytic subunit (γ-GCSh) gene were designed and synthesized according to known 5'NCR sequence of human γ-GCSh gene,which were encapsulated in cationic lipid and transfected human bladder cancer cell line BIU87 and its durg resistance subline BIU87/A.The expression of γ-GCSh mRNA,intracellular glutathione (GSH) levels and inhibitory effect of 6 chemotherapy agents were measured respectively by the quantitative reverse transcription polymerase chain reaction (RT-PCR),fluoro-spectrophotography and MTT assay before and 72 h after transfection.Results In comparison with sense ODNs,antisense ODNs inhibited significantly the expression of γ-GCSh mRNA,decreased intracellular GSH content in BIU87 and BIU87/A at 72 h after transfections,and then increased two cell lines drug sensibitity to most of chemotherapy agents.Conclusion Ap-1-asODNs targeting at γ-GCSh particularly can reverse in vitro the phenotype of MDR in bladder cancer cells by reducing the levels of intracellular GSH.