β-catenin介导乳腺癌细胞多柔比星耐药实验研究  

Experimental study of β-catenin-mediated resistance to adriamycin in human breast cancer cells

作　　者：张为家 李爽[1] 苏小岩[1] 刘峰[1] 曾海 ZHANG Wei-jia;LI Shuang;SU Xiao-yan;LIU Feng;ZENG Hai(Department of Oncology , Clinical Medical College of Yangtze University, Jingzhou 434000, P. R. China;Department of Chemoradiotherapy , Second Clinical College of Wuhan University ,Wuhan 430071, P. R. China)

机构地区：[1]长江大学附属临床医院肿瘤科,湖北荆州434000 [2]武汉大学第二临床学院肿瘤放化疗科,湖北武汉430071

出　　处：《中华肿瘤防治杂志》2018年第4期232-237,共6页Chinese Journal of Cancer Prevention and Treatment

摘　　要：目的多柔比星是乳腺癌最常用的一线化疗药物之一,其耐药机制至今尚未完全阐明。本研究探讨Wnt/β-catenin信号分子在乳腺癌多柔比星耐药中的作用及机制。方法以多柔比星低剂量诱导结合间歇大剂量冲击的方法建立MCF7多柔比星耐药细胞株MCF7/ADR。MTS实验比较MCF7与MCF7/ADR细胞对多柔比星的敏感性。蛋白质印迹检测MCF7和MCF7/ADR细胞间Wnt/β-catenin蛋白的表达改变;并以双荧光素酶报告基因系统检测β-catenin的转录活性。采用小分子干扰RNA(small interfering RNA,siRNA)干扰MCF7/ADR细胞中β-catenin的表达,MTS检测多柔比星药物敏感性改变。实时定量PCR及蛋白质印迹检测β-catenin靶分子sp5、Axin2、P-gp的表达。另外,以同样的方法诱导建立乳腺癌多柔比星耐药细胞株MDA-MB231/ADR。蛋白质印迹检测β-catenin及其靶分子sp5、Axin2、P-gp的表达。siRNA干扰MDA-MB231/ADR细胞中β-catenin的表达,MTS检测MDA-MB231/ADR细胞对多柔比星药物敏感性改变。结果诱导建立了乳腺癌多柔比星耐药细胞株MCF7/ADR。MTS细胞毒实验结果显示,MCF7/ADR细胞对多柔比星耐药132倍。蛋白质印迹结果显示,MCF7/ADR细胞中β-catenin活化,报告基因显示耐药细胞中β-catenin转录活性由6.891±0.876上调至25.873±2.054,t=13.526,P<0.01。多柔比星对β-catenin干扰组IC50值显著低于干扰组对照组和未干扰组,F=120.529,P<0.05。在MCF7/ADR细胞中P-gp mRNA及蛋白均上调,t=13.091,P<0.05;而sp5、Axin2表达差异无统计学意义,t值分别为1.792和0.836,均P>0.05。干扰β-catenin后P-gp表达被抑制(0.883±0.127 vs 0.312±0.035),t=11.351,P<0.05。同样的,诱导建立的乳腺癌多柔比星药细胞系MDA-MB231/ADR,耐药指数为68.450倍。β-catenin及其调控分子P-gp明显上调。干扰β-catenin后MDA-MB231/ADR中P-gp表达被抑制,对多柔比星的敏感性增加,F=97.445,P<0.05。结论β-catenin通过上调P-gp介导乳腺癌细胞对多柔比星耐药。OBJECTIVE Adriamycin is one of several commonly used chemotherapeutic agents in breast cancer,and the chemoresistance of adriamycin has not been fully elucidated yet.This research was to investgate the role of Wnt/β-catenin signaling pathway in the adriamycin resistance of human breast cancer.METHODS The human cancer MCF7 cells were exposed in gradually increasing or interval high dose of adriamycin.MTS assay was used to detect the cytotoxic activity of adriamycin against MCF7 and MCF7/ADR cells.The protein expressions of actived-β-catenin in MCF7 and MCF7/ADR cells were analyzed by western blot.Reporter assays were used for Wnt/β-catenin activity.Knockdownβ-catenin in MCF7/ADR cells and assess the chemosensitivity to adriamycin by MTS.Real-time PCR and Western blot analysis were used to detected the expressions of sp5,Axin2,P-gp in MCF7,MCF7/ADR and the si-MCF7/ADR cells.Addtionly,the same method was used to induce another of adriamycin resistant breast cancer cell line MDA-MB231/ADR.The expressions of actived-β-catenin and its target molecules SP5,Axin2 and P-gp in MDA-MB-231,MDA-MB-231/ADR cells were analyzed by western blot.Interfer with the expression ofβ-catenin in MDA-MB231/ADR cells and assess the chemosensitivity to adriamycin by MTS.RESULTS The adriamycin resistance cell line MCF7/ADR was established.The resistance index（RI）of MCF7/ADR cell to the adriamycin was 132.Western blotting analysis showed that compared to the MCF7 cells,active-β-catenin in the nucleus was upregulated in MCF7/ADR cells[（6.891±0.876）vs（25.873±2.054）,t=13.526,P〈0.01].When knockdown ofβ-catenin in MCF7/ADR cell（si-β-catenin）,the IC50 of adriamycin was significantly lower than that of the control（si-Con）and Mock groups（Mock;F=120.529,P〈0.05）.The expression levels of P-gp mRNA and protein were significantly higher in MCF7/ADR cells than in the MCF7（t=13.091,P〈0.05）.The expressions of SP5 and Axin2 were not significantly different（t=1.792,P〉0.05;t=0.836,P〉0.05）.The expression of P-

关 键 词：乳腺癌 多柔比星耐药 Β-CATENIN P-GP 

分 类 号：R737.9[医药卫生—肿瘤]