小G蛋白Cdc42对人结肠癌奥沙利铂耐药细胞多药耐药性的影响

The Effect of Small GTPase Cdc42 on the Multidrug Resistance of Oxaliplatin-resistant Colon Cancer Cell Lines

目的研究细胞分裂周期蛋白(cell division cycle 42,Cdc42)对人结肠癌奥沙利铂耐药细胞系多药耐药性的影响。方法 Western blot检测人结肠癌SW480、Colo320细胞与已成功构建的奥沙利铂耐药细胞系SW480/L-OHP、Colo320/L-OHP细胞中Cdc42的表达,用LipofectamineTM2000介导重组质粒pDEST26-His-Cdc42转染入Cdc42表达相对较低的亲代细胞系,介导Cdc42 siRNA转染入Cdc42表达相对较高的耐药细胞系。Western blot和RT-PCR检测转染是否成功。利用Cell Counting Kit-8(CCK-8)检测各细胞系5氟尿嘧啶、顺铂、奥沙利铂,紫杉醇,依托泊苷,长春新碱,阿霉素等结直肠癌常用化疗药物的药物敏感性,计算出各种药物的半数致死浓度(IC50)。行Western blot检测不同细胞系P-gp、MRP1蛋白表达水平。结果在耐药细胞系中Cdc42的表达高于亲代细胞系。成功转染外源Cdc42基因的亲代细胞CCK-8(CCK-8)法检测对多种化疗药物的IC50以及P-gp、MRP1蛋白表达水平高于未转染细胞系。成功转染Cdc42 siRNA的耐药细胞CCK-8法检测对多种化疗药物的IC50显著低于耐药细胞系。结论 Cdc42与人结肠癌细胞耐药性形成有关,Cdc42可增强人结肠癌细胞的多药耐药性的形成。提示Cdc42可能成为降低结肠癌细胞多药耐药性的分子靶点。

Objective To determine the effect of cell division cycle42（Cdc42） on the multidrug resistance of oxaliplatin-resistant human colon cancer cells.Methods The protein expression levels of Cdc42 in oxaliplatin-resistant colon cancer cells and parental cells were examined with Western blot.pDEST26-His-Cdc42 was transfected by lipofectamineTM 2000 into SW480 and Colo320 cells with low expression of Cdc42.Cdc42 siRNA was transfected by lipofectamineTM 2000 into SW480/L-OHP and Colo320/L-OHP cells with high expression of Cdc42.The expression of Cdc42 in these cell lines were examined by Western blot and RT-PCR 48 hours after transfection.The sensitivity of colon cancer cells to antitumor drugs was evaluated using CCK-8 assay.The concentration of each drug that caused a 50% reduction in the numbers of cells（IC50） was calculated.The expression of P-gp,MRP1 in SW480/ Cdc42 and Colo320/ Cdc42 cells were examined with Western blot.Results Cdc42 was over-expressed in the SW480/L-OHP and Colo320/L-OHP cell lines.Over-expression of Cdc42 significantly enhanced the resistance of colon cancer cells to multiple antitumor drugs and up-regulated the expression of P-gp and MRP1.Conclusion Cdc42 enhances the resistance of colon cancer cells to several antitumor drugs.It might become a potential target for reversing multidrug resistance of colon cancer.