托瑞米芬逆转乳腺癌耐药蛋白介导的多药耐药机制  

Regulation mechanism of breast cancer resistance protein by toremifene to reverse BCRP-mediated multidrug resistance in breast cancer cells

作　　者：张玉华[1] 李光[1] 俞进[1] 徐妙生[1] 刘朝霞[1] 

机构地区：[1]首都医科大学附属北京天坛医院病理科,100050

出　　处：《中华肿瘤杂志》2011年第9期654-660,共7页Chinese Journal of Oncology

摘　　要：目的探讨托瑞米芬逆转乳腺癌耐药蛋白（BCRP）介导的多药耐药机制。方法通过基因扩增，构建分别由BCRP启动子和巨细胞病毒（CMV）启动子启动表达BCRP的重组质粒pcDNA3-Pmmoter-BCRP和作为对照的质粒pcDNA3-CMV-BCRP，将其分别转染雌激素受体Ⅸ（ERa）阳性的MCF-7和ERα阴性的MDA-MB-231乳腺癌细胞系，建立由BCRP启动子和CMV启动子启动表达BCRP的4种耐药细胞系MCF-7／Promoter-BCRP、MCF-7／CMV-BCRP、MDA-MB-231／Promoter-BCRP和MDA—MB-231／CMV-BCRP。在耐药细胞培养基中加入托瑞米芬，通过逆转录聚合酶链反应（RT-PCR）、Western blot、外排实验以及细胞毒性实验观察托瑞米芬对不同细胞系的耐药逆转效果。结果与空白对照组（未加药物）相比，托瑞米芬以剂量依赖方式抑制BCRP mRNA的表达，0．1、1和10μmol／L托瑞米芬处理组MCF-7／Promoter—BCRP细胞中BCRP mRNA的表达水平分别下调29．5％（P〈0．05）、68．1％（P〈0．01）和97．4％（P〈0．01）；MCF-7／Promoter—BCRP细胞经托瑞米芬和17β-雌二醇联合处理后，细胞中BCRPmRNA的相对表达水平为64．2％±1．3％，明显高于托瑞米芬单独处理组（3．8％±0．2％，P〈0．01）。托瑞米芬对各组细胞系中BCRP蛋白表达的调控作用与mRNA相似。经托瑞米芬处理后，MCF-7／Promoter-BCRP细胞内米托蒽醌的荧光强度显著增强，外排米托蒽醌的能力降低了47．3％（P〈0．05）；经托瑞米芬和17β-雌二醇联合处理后，MCF-7／Promoter—BCRP细胞内米托蒽醌的荧光强度明显低于托瑞米芬单独处理组，外排米托蒽醌的能力升高了61．5％。托瑞米芬可有效逆转MCF-7／Promoter—BCRP细胞对米托蒽醌的耐药性。上述作用在MCF-7／CMV-BCRP、MDA—MB-231／Promoter-BCRP和MDA—MB-231／CMV-BCRP细胞中未能体现。结论托瑞米芬可能通过ERot的介导与BCRP启动子上游调控序列中的ERE结合，负性调节BCRP的表达Objective To explore the regulation mechanism of the reversal of breast cancer resistance protein-mediated multidrug resistance by toremifene. Methods Two recombinant plasmids （pcDNA3-promoter-BCRP and pcDNA3-CMV-BCRP） were designed to express the wild-type full-length BCRP eDNA enforced driven by its endogenous promoter containing a functional ERE and a CMV promoter as control, respectively. Two recombinant plasmids were transfected into ERa-positive MCF-7 and ERa- negative MDA-MB-231 breast cancer cell lines. Four kinds of BCRP expressing cell lines of MCF-7/ Promoter-BCRP, MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP were established in which BCRP was promoted by the BCRP promoter and a CMV promoter as control, respectively. The drug resistant cells were treated with toremifene. Then RT-PCR, Western blot, mitoxantrone efflux assays and cytotoxicity assay were performed to detect the reversal function of BCRP by toremifene on the drug resistance cell lines. Results Toremifene significantly downregulated BCRP mRNA levels in a dose-dependent manner in ERa-positive MCF-7/Promoter-BCRP cells than that of untreated control cells. In MCF-7/Promoter-BCRP ceils, toremifene at the dose of 0.1, 1 and 10 μmol/L decreased BCRPmRNAexpression by 29.5% （P 〈0.05）, 68. 1% （P 〈0.01） and 97.4% （P 〈0. 01）, respectively. After being treated with toremifene and 17[3-estradiol, the BCRP mRNA level in MCF-7/ Promoter-BCRP cells was 64.2% ± 1.3% , significantly higher than that of toremifene treatment control cells （3.8% ±0.2% , P 〈 0.01 ）. Furthermore, the effect of toremifene on BCRP protein is similar in BCRP mRNA. Toremifene obviously increased the mitoxantrone fuorescence intensity and decreased the efflux activity by 47.3 % （P 〈 0. 05） in MCF-7/promoter-BCRP cells when compared with the untreated control, whereas intracellular accumulation of mitoxantrone obviously decreased and the efflux activity increased by 61.5% were observed in combination with 17β-estradiol when com

关 键 词：乳腺癌耐药蛋白 托瑞米芬 基因表达调控 启动区 抗药性 肿瘤 

分 类 号：R737.9[医药卫生—肿瘤]