Zhang Yu-hua, Li Guang, Yu Jin, Xu Miao-sheng, Liu Zhao-xia
Department of Pathology, Capital Medical University, Beijing, China.
Zhonghua Zhong Liu Za Zhi. 2011 Sep;33(9):654-60.
To explore the regulation mechanism of the reversal of breast cancer resistance protein-mediated multidrug resistance by toremifene.
Two recombinant plasmids (pcDNA3-promoter-BCRP and pcDNA3-CMV-BCRP) were designed to express the wild-type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a CMV promoter as control, respectively. Two recombinant plasmids were transfected into ERα-positive MCF-7 and ERα-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.
Toremifene significantly downregulated BCRP mRNA levels in a dose-dependent manner in ERα-positive MCF-7/Promoter-BCRP cells than that of untreated control cells. In MCF-7/Promoter-BCRP cells, toremifene at the dose of 0.1, 1 and 10 µmol/L decreased BCRP mRNA expression by 29.5% (P < 0.05), 68.1% (P < 0.01) and 97.4% (P < 0.01), respectively. After being treated with toremifene and 17β-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 fluorescence 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 compared with toremifene treatment alone. The results therefore suggested that toremifene reversed mitoxantrone resistance in MCF-7/Promoter-BCRP cells. However, in MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP cells, toremifene or in combination with 17β-estradiol did not affect intracellular mitoxantrone uptake.
Taken together, our findings indicate that expression of BCRP is downregulated by toremifene, via a novel transcriptional mechanism which might be involved in the ERE of BCRP promoter through ER-mediated to inactivate the transcription of BCRP gene.
探讨托瑞米芬逆转乳腺癌耐药蛋白介导的多药耐药的调控机制。
设计两种重组质粒（pcDNA3-启动子-BCRP和pcDNA3-CMV-BCRP），分别表达由含功能性雌激素反应元件（ERE）的内源性启动子强制驱动的野生型全长BCRP cDNA以及作为对照的CMV启动子。将两种重组质粒转染至雌激素受体α（ERα）阳性的MCF-7和ERα阴性的MDA-MB-231乳腺癌细胞系中。建立了四种BCRP表达细胞系，即MCF-7/启动子-BCRP、MCF-7/CMV-BCRP、MDA-MB-231/启动子-BCRP和MDA-MB-231/CMV-BCRP，其中BCRP分别由BCRP启动子和作为对照的CMV启动子促进表达。用托瑞米芬处理耐药细胞。然后进行逆转录-聚合酶链反应（RT-PCR）、蛋白质免疫印迹法（Western blot）、米托蒽醌外排试验和细胞毒性试验，以检测托瑞米芬对耐药细胞系中BCRP的逆转作用。
与未处理的对照细胞相比，托瑞米芬在ERα阳性的MCF-7/启动子-BCRP细胞中以剂量依赖性方式显著下调BCRP mRNA水平。在MCF-7/启动子-BCRP细胞中，0.1、1和10 μmol/L剂量的托瑞米芬分别使BCRP mRNA表达降低了29.5%（P<0.05）、68.1%（P<0.01）和97.4%（P<0.01）。用托瑞米芬和17β-雌二醇处理后，MCF-7/启动子-BCRP细胞中的BCRP mRNA水平为64.2%±1.3%，显著高于托瑞米芬处理的对照细胞（3.8%±0.2%，P<0.01）。此外，托瑞米芬对BCRP蛋白的作用与对BCRP mRNA的作用相似。与未处理的对照相比，托瑞米芬在MCF-7/启动子-BCRP细胞中明显增加了米托蒽醌荧光强度，并使外排活性降低了47.3%（P<0.05），而与单独托瑞米芬处理相比，与17β-雌二醇联合使用时，米托蒽醌的细胞内蓄积明显减少，外排活性增加了61.5%。因此，结果表明托瑞米芬逆转了MCF-7/启动子-BCRP细胞中的米托蒽醌耐药性。然而，在MCF-7/CMV-BCRP、MDA-MB-231/启动子-BCRP和MDA-MB-231/CMV-BCRP细胞中，托瑞米芬或与17β-雌二醇联合使用均不影响细胞内米托蒽醌的摄取。
综上所述，我们的研究结果表明，托瑞米芬通过一种新的转录机制下调BCRP的表达，该机制可能通过ER介导参与BCRP启动子的ERE，从而使BCRP基因的转录失活。
