姜黄素逆转人结肠癌耐奥沙利铂细胞株HCT-116/L-OHP的耐药性

Effect of Curcumin in Reversing Resistance of Human Colon Carcinoma Against Oxaliplatin Cell Line HCT-116/L-OHP

目的:构建人结肠癌奥沙利铂(oxaliplatin,L-OHP)耐药细胞株HCT-116/L-OHP,观察姜黄素(curcumin,Cur)对其耐药的逆转作用,并探讨其可能的耐药机制。方法:采用浓度梯度递增法,逐步提高作用于亲代人结肠癌细胞HCT-116的L-OHP浓度,建立耐L-OHP的细胞株HCT-116/L-OHP;通过cell counting kit-8(CCK-8)法检测L-OHP和Cur对HCT-116以及HCT-116/L-OHP细胞的细胞毒性,观察Cur能否逆转耐药;蛋白免疫印迹法(Western blot)检测耐药相关蛋白的表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测相关mRNA的改变。结果:成功建立了耐L-OHP的人结肠癌耐药细胞株并命名为HCT-116/L-OHP,其耐药指数为12. 6。与HCT-116细胞株比较,HCT-116/L-OHP细胞株中切除修复交叉互补基因1(ERCC1)蛋白及mRNA表达水平显著上升(P <0. 01),B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2),谷胱甘肽-巯基-转移酶-π(glutathione-s-transferase-π,GST-π),多药耐药相关蛋白(multidrug resistance-associated protein, MRP),P-糖蛋白(P-glycoprotein,P-gp),凋亡抑制基因(Survivin)的表达亦显著上升(P <0. 01);不同浓度姜黄素(5,10,20,30,40μmol·L-1)作用后,ERCC1表达下降(P <0. 01),Bcl-2,GST-π,MRP,P-gp,Survivin表达也有不同程度下降(P <0. 05)。结论:HCT-116/L-OHP细胞株具有稳定的耐药性,其耐药机制可能为ERCC1的表达上调,而导致Bcl-2,GST-π,MRP,P-gp,Survivin等相关蛋白表达上调,使肿瘤细胞获得耐药性。姜黄素对HCT-116/L-OHP的耐药性具有逆转作用,其作用机制可能是通过降低ERCC1的表达,从而下调Bcl-2,GST-π,MRP,P-gp,Survivin等耐药相关mRNA及蛋白的表达,增加肿瘤对L-OHP的敏感性,从而逆转肿瘤细胞的耐药。

Objective:To construct oxaliplatin(L-OHP)drug-resistant cell line HCT-116/L-OHP in human colon cancer,in order to observe the reversal effect of curcumin(cur)on its drug resistance,and preliminarily explore the possible drug resistance mechanism.Method:The concentration gradient increasing method was used to gradually increase the L-OHP concentration of HCT-116 in parental colon cancer cells,and the cell line HCT-116/L-OHP resistant to L-OHP was established.The cytotoxicity of L-OHP and curcumin to HCT-116 and HCT-116/L-OHP cells was detected by cell counting kit-8(CCK-8)method to observe whether curative resistance could be reversed.Western blot was used to detect the expressions of drug-resistance-related proteins.Real-time PCR was used to detect changes in related genes.Result:Human colon cancer cell line resistant to L-OHP were successfully established and named as HCT-116/L-OHP,with a drug resistance index of12.6.Compared with HCT-116 cell lines,the expression levels of resected and repaired cross complementation gene1(ERCC1)protein and gene in HCT-116/L-OHP cell lines were significantly increased(P<0.01).The expressions of B-cell lymphoma-2(Bcl-2),glutathione-s-transferase-consciousness(GST-consciousness),multidrug resistance-associated protein(MRP),P-glycoprotein(P-gp)and apoptotic inhibitory gene(Survivin)also increased significantly(P<0.01).After the treatment with different concentrations of curcumin(5,10,20,30,40μmol·L-1),the expression of ERCC1 decreased(P<0.01),and the expressions of Bcl-2,GST-π,MRP,P-gp,Survivin also decreased to different degrees(P<0.05).Conclusion:HCT-116/L-OHP cell lines have a stable drug resistance,and its drug resistance mechanism may be up-regulated with the expression of ERCC1,which leads to the up-regulation of Bcl-2,GST-π,MRP,P-gp,Survivin and other related proteins,and enables tumor cells to acquire drug resistance.Curcumin can reverse the drug resistance of HCT-116/L-OHP,and its mechanism may be to reduce the expression of ERCC1,thereby down-regulating the expressions of Bcl-2,GST-π,MRP,P-gp,Survivin and other drug-resistant related genes and proteins,and increase the sensitivity of tumor to L-OHP,so as to reverse the drug resistance of tumor cells.