摘要
目的通过构建结肠癌耐奥沙利铂细胞株(HCT116/L-OHP),探讨5-氮杂-2′-脱氧胞苷(5-Aza-CdR)逆转奥沙利铂耐药的可能机制及其与切除修复交叉互补基因1(ERCC1)、人类错配修复基因1(hMLH1)的关系。方法采用奥沙利铂浓度梯度递增法,建立耐奥沙利铂的细胞株HCT116/L-OHP,CCK法筛选出奥沙利铂、5-Aza-CdR对各组细胞的半数抑制浓度(IC50),流式细胞仪检测细胞凋亡水平,实时荧光定量PCR(qRT-PCR)和蛋白印迹法分别检测使用5-Aza-CdR处理细胞前后ERCC1、hMLH1基因和蛋白表达水平。结果成功构建了稳定耐奥沙利铂细胞株HCT116/L-OHP,选取5μmol/L 5-Aza-CdR和100 mg/L奥沙利铂行体外敏感性实验,随着时间延长,各组对HCT116/L-OHP细胞株增殖抑制率呈上升趋势,5-Aza-CdR联合奥沙利铂组增殖抑制率较高,药物和时间的作用效应存在交互作用,F分组=89.743,P<0.001;F时间=28.249,P<0.001;F分组×时间=42,785,P<0.001。经5-Aza-CdR联合奥沙利铂处理HCT116/L-OHP 24 h后细胞凋亡结果显示,联合用药组相对于奥沙利铂单药组的细胞凋亡率由(17.94±1.58)%上升至(34.88±2.15)%,两药存在协同性的交互作用,F=48.232,P<0.001。RT-PCR结果显示,HCT116对照组、HCT116/L-OHP组和5-Aza-CdR处理组ERCC1基因水平分别为1.54±0.08、1.85±0.07和1.72±0.06,F=254.126,P<0.001;hMLH1水平分别为1.14±0.07、0.81±0.05和1.02±0.06,F=156.827,P<0.001。蛋白质印迹结果显示,HCT116对照组、HCT116/L-OHP组和5-Aza-CdR处理组ERCC1水平分别为0.29±0.03、0.80±0.05和0.54±0.02,F=251.274,P<0.001;hMLH1水平分别为0.56±0.08、0.46±0.05和0.76±0.09,F=153.683,P<0.001。结论 5-Aza-CdR可明显抑制奥沙利铂作用下人结肠癌耐药细胞株HCT116/L-OHP的增殖作用,诱导凋亡,其作用可能与影响ERCC1和hMLH1表达有关。
Objective To evaluate the effect of 5-Aza-CdR on the oxaliplatin sensitivity of HCT116/L-OHP cells and explore whether the effects of 5-Aza-CdR involve ERCC1 and hMLH1.Methods Human colon carcinoma oxaliplatin resistant cells line HCT116/L-OHP were cultured in vitro by the concentration gradient increasing method.The cytotoxicities in HCT116 and HCT116/L-OHP cells induced by L-OHP and 5-Aza-CdR were detected by CCK assay.Cell apoptosis was determined by flow cytometry.Real-time fluorescent quantitative PCR was used to detect the expression of ERCC1 mRNA and hMLH1 mRNA in different groups,and Western blot was used to detect the expressions of drug-resistance-related proteins.Results The HCT116/L-OHP cells with stable oxaliplatin resistance were successfully established.5 μmol/L 5-Aza-CdR and 100 mg/L of oxaliplatin were selected for in vitro sensitivity experiments.With the prolongation of time,the inhibition rate of proliferation of HCT116/L-OHP cell lines in each group showed an upward trend,and the inhibition rate of 5-Aza-CdR combined with oxaliplatin group was higher,and the interactive effects of time and drug on the HCT116/L-OHP cells proliferation was existed(Fdrug=89.743,P<0.001;Ftime=28.249,P<0.001;Fdrug×time=42,785,P<0.001).The apoptosis results of 5-Aza-CdR combined with oxaliplat in HCT116/L-OHP showed that the apoptosis rate of the combined group increased from(17.94±1.58)% to(34.88±2.15)%,and there was synergistic interaction between the two drugs(F=48.232,P<0.001).Fluorescence quantitative PCR showed that the expressions of ERCC1 in the control group,oxaliplatin group and 5-Aza-CdR group were 1.54±0.08,1.85±0.07,1.72±0.06,respectively(F=254.126,P<0.001),and the hMLH1 expressions were 1.14±0.07,0.81±0.05,1.02±0.06,respectively(F=156.827,P<0.001),Western blot analysis showed that the expressions of ERCC1 in the control group,oxaliplatin group and5-Aza-CdR group were 0.29±0.03,0.80±0.05,0.54±0.02,respectively(F=251.274,P<0.001),and the hMLH1 expressions were 0.56±0.08,0.46±0.05,0.76±0.09,respectively(F=153.683,P<0.001).Conclusion Results of the investigation suggest that a combination of 5-Aza-CdR and cisplatin has a significant inhibitory effect on the prolifertion of human colon cancer cell line HCT116/L-OHP cells under oxaliplatin,and its regulation may be resulted from the expressions of ERCC1 and hMLH1.
作者
张修振
闫燕
杨勇进
陈成武
亓玉琴
刘吉勇
ZHANG Xiu-zhen;YAN Yan;YANG Yong-jin;CHEN Cheng-wu;QI Yu-qin;LIU Ji-yong(Department of Gastroenterology,Affiliated Lai Gang Hospital of Tai Shan Medical College,Jinan 271126,China;Department of Gastroenterology,Qingdao City Hospital,Qingdao 266011,China;Department of Gastroenterology,Shandong Provincial Hospital,Jinan 250000,China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2021年第13期988-993,共6页
Chinese Journal of Cancer Prevention and Treatment
基金
济南市卫计委科技计划(2019-2-67)。
