ATP7A对结肠癌细胞耐奥沙利铂的机制初探  

Preliminary exploration on the mechanism of ATP7A on oxaliplatin-resistant colon cancer cells

作　　者：余文燕[1] 王国娟[2] 许建华[3] 王桦影 范忠泽[3] YU Wenyan;WANG Guojuan;XU Jianhua;WANG Huaying;FAN Zhongze(Research Center for Differentiation and Development of Basic theory of Traditional Chinese Medicine(TCM),Jiangxi University of TCM(Key Laboratory of Etiology and Biology of TCM in Jiangxi Province,No.JA009500),Nanchang 330004,China)

机构地区：[1]江西中医药大学中医基础理论分化发展研究中心江西省中医病因生物学重点实验室No.JA009500 [2]江西中医药大学附属医院肿瘤科 [3]上海中医药大学附属普陀医院肿瘤科

出　　处：《实用医学杂志》2019年第2期186-190,共5页The Journal of Practical Medicine

基　　金：江西省自然科学基金项目(编号:20161BAB215208);江西省卫生计生委中医药科研课题(编号:2015A047);江西省教育厅科学技术项目(编号:GJJ150838);江西中医药大学校级课题(编号:2014BS002)

摘　　要：目的探讨铜转运蛋白ATP7A对人结肠癌耐药细胞HCT116/L-OHP耐奥沙利铂的作用机制。方法选用RNA沉默(siRNA)ATP7A稳转结肠癌耐药细胞siATP7A-HCT116/L-OHP,同时设立空转染组negATP7A-HCT116/L-OHP,以化疗药奥沙利铂(oxaliplatin,L-OHP)作为用药组,分别采用四甲基偶氮唑盐比色法(MTT)、Annexin V-FITC/PI双染法及蛋白免疫印迹法检测ATP7A蛋白沉默前后对HCT116/L-OHP细胞抑制率、凋亡率及ATP7A蛋白表达的影响。结果 (1)随着L-OHP浓度的增加对细胞抑制率增强,IC50(μg/mL)由85.81±2.76降至41.30±3.31,逆转耐药2.07倍。(2)negATP7A-HCT116/L-OHP和siATP7A-HCT116/L-OHP细胞空白组48 h凋亡率分别为(1.4±0.1)%和(4.47±0.15)%,L-OHP组细胞凋亡率分别为(4.97±0.06)%和(25.87±0.21)%。与negATP7A-HCT116/L-OHP细胞相比,同一组别siATP7A-HCT116/L-OHP细胞凋亡率均显著升高(P <0.001)。(3)与negATP7A-HCT116/L-OHP细胞L-OHP组相比,siATP7A-HCT116/L-OHP细胞L-OHP组内除CTR1蛋白上调外,ATP7A、ATP7B、ATOX1蛋白均呈不同程度的下调(P <0.001)。结论 ATP7A降低结肠癌耐药细胞HCT116/L-OHP对L-OHP的敏感性及细胞凋亡,沉默后能上调铜转运相关蛋白CTR1的表达、下调ATP7B、ATOX1表达,可能是人结肠癌细胞耐奥沙利铂的重要机制之一。Objective To investigate the mechanism of copper transporter ATP7A on oxaliplatin resistance in human colon cancer cell line HCT116/L-OHP.Methods RNAi(siRNA)ATP7A was used to stably transfect the colon cancer cell line siATP7A-HCT116/L-OHP.At the same time,empty-transfected group negATP7A-HCT116/L-OHP was established.Chemotherapy drug oxaliplatin(L-OHP)was used as the medical group.The in hibitory rates,apoptosis rates and the expression of ATP7A protein in HCT116/L-OHP cells before and after the RNA silence of it were detected by MTT assay,Annexin V-FITC/PI double staining and Western blot(WB)re spectively.Results The inhibitory rates(%)of L-OHP at different concentrations were-8.51±58.52,25.37±6.19,32.72±2.92,53.81±6.96,72.23±3.32,93.37±2.82(μg/mL),which decreased from 85.81±2.76 to 41.30±3.31,reversing the resistance by 2.07 times.In addition,the apoptotic rates of blank groups in negATP7A-HCT116/L-OHP and siATP7A-HCT116/L-OHP were(1.4±0.1)%and(4.47±0.15)%respectively after 48h,while the ones of L-OHP groups in them were(4.97±0.06)%and(25.87±0.21)%.Compared with the apoptosis rates in negATP7A-HCT116/L-OHP cells,the ones in the same group of siATP7A-HCT116/L-OHP increased significantly(P<0.001).Moreover,the expressions of ATP7A,ATP7B and ATOX1 protein were down-regulated differently except the up-regulation of CTR1 protein in siATP7A-HCT116/L-OHP cells compared with negATP7A-HCT116/L-OHP group(P<0.001).Conclusion ATP7A can reduce the sensitivity and apoptosis of L-OHP in duced by HCT116/L-OHP in colon cancer cells.After silencing,ATP7A can up-regulate the expression of CTR1 and down-regulate the expression of ATP7B and ATOX1,which may be one of the important mechanisms of oxalipl atin resistance in human colon cancer cells

关 键 词：ATP7A 结肠癌 奥沙利铂 增殖 凋亡 

分 类 号：R735.35[医药卫生—肿瘤]