蛋白激酶C抑制剂对人非小细胞肺癌细胞株药物敏感性的影响

Effects of protein kinase C inhibitor,chelerythrine chloride,on drug-sensitivity of NSCLC cell lines

背景与目的蛋白激酶C(PKC)在癌变过程中的作用使其成为肿瘤治疗的潜在重要靶点。非小细胞肺癌(NSCLC)中存在PKC-α的异常表达和活性增高,PKC抑制剂能通过诱导肿瘤细胞凋亡、增强细胞毒作用及下调多药耐药基因的表达而发挥其抗肿瘤作用。通过观察PKC抑制剂白屈菜红碱(CH)对四种人NSCLC细胞株药物敏感性的影响,初步探讨其作用机制。方法以PKC抑制剂CH分别处理四种NSCLC细胞株H1299、H460、A549及耐顺铂A549细胞株,逆转录聚合酶链式反应法(RT-PCR)及蛋白印迹法(Westernblot)检测PKC-α的mRNA及蛋白表达水平,流式细胞仪检测细胞凋亡率,四甲基偶氮唑蓝(MTT)法检测细胞株对顺铂药物敏感性。结果用药前A549/DDP细胞株中PKC-αmRNA及蛋白的表达水平高于NSCLC细胞株H1299、H460及亲本A549细胞株(P<0.05),CH处理后四种NSCLC细胞株中PKC-αmRNA及蛋白的表达水平均有不同程度的下降,CH处理4h及24h后H1299、H460、A549细胞株的凋亡率无明显增加,仅A549/DDP细胞株的凋亡率明显增加,用药后NSCLC细胞株对顺铂的药物敏感性即IC50值有不同程度的下降,以A549/DDP细胞株降低更为明显(P<0.05)。结论四种NSCLC细胞株中存在PKC-αmRNA及蛋白的高表达。通过抑制NSCLC细胞株中PKC-αmRNA及蛋白的表达,PKC抑制剂CH能增加其对顺铂的药物敏感性。与亲本A549细胞株相比,PKC抑制剂CH能通过抑制耐顺铂A549细胞株中PKC-α蛋白的表达及增加细胞凋亡率,而更有效地增加其对顺铂的药物敏感性。

Background and objective Protein kinase C(PKC) is a potentially important target for can-cer therapeutics due to its potential role in carcinogenesis.Abnormal expression and increasing activity of PKC-α are present in non-small cell lung cancer(NSCLC).PKC inhibitor can show anti-tumor effects through inducing tumor cell apoptosis,enhancing cytotoxic effects and down-regulating expressions of multidrug resistance gene.By observing the effects of PKC inhibitor chelerythrine chloride(CH) on drug-sensitivity to cisplatin of four NSCLC cell lines its mechanism of effect initially is explored.Methods NSCLC cell lines(H1299,H460,A549 and cisplatin-resistant A549) were dealed with PKC inhibitor CH respectively.The expressions of PKC-α mRNA and protein in NSCLC cell lines were examined by reverse transcription polymerase chain reaction(RT-PCR) and Western blot.The apoptosis rates of NSCLC cells lines were detected by flow cytometry.The drug-sensitivity to cisplatin of NSCLC cells lines was measured by methabenzthiazuron(MTT) assay.Results The expression levels of PKC-α mRNA and protein in cisplatin-resistant A549 cell lines were significantly higher than H1299,H460 and parent A549 cell lines(P<0.05).The expression levels of PKC-α mRNA and protein in four NSCLC cell lines decreased at different extent.The apoptosis rates of cisplatin-resistant A549 cell lines increased obviously after treating with CH for 4 and 24 hours,but it was not seen in H1299,H460 and parent A549 cell lines.The IC50 value of cisplatin of NSCLC cell lines decreased at different degree after treating with CH and it was more obvious in cisplatin-resistant A549 cell lines(P<0.05).Conclusion High expressions of PKC-α mRNA and protein exist in all four NSCLC cell lines.PKC inhibitor CH can enhance the drug-sensitivity of NSCLC cell lines to cisplatin by inhibiting their expression of PKC-α mRNA and protein.When compared with parent A549 cell lines,cisplatin-resistant A549 cell line's drug-sensitivity to cisplatin is increasing more efficiently by PKC inhibitor CH through inhibition of PKC-α protein's expression and elevation of tumor cell apoptosis rates.