CAJ | 학술논문

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-Promoter-BCRP和作为对照的质粒pcDNA3-CMV-BCRP，将其分别转染雌激素受体α（ERα）阳性的MCF-7和ERα阴性的MDA-MB-231乳腺癌细胞系，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/PromoterBCRP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10 μmol/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BCRP mRNA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，抑制BCRP蛋白的功能，在体外有效逆转BCRP介导的多药耐药。目的探討託瑞米芬逆轉乳腺癌耐藥蛋白(BCRP)介導的多藥耐藥機製。方法通過基因擴增，構建分彆由BCRP啟動子和巨細胞病毒(CMV)啟動子啟動錶達BCRP的重組質粒pcDNA3-Promoter-BCRP和作為對照的質粒pcDNA3-CMV-BCRP，將其分彆轉染雌激素受體α（ERα）暘性的MCF-7和ERα陰性的MDA-MB-231乳腺癌細胞繫，建立由BCRP啟動子和CMV啟動子啟動錶達BCRP的4種耐藥細胞繫MCF-7/Promoter-BCRP、MCF-7/CMV-BCRP、MDA-MB-231/PromoterBCRP和MDA-MB-231/CMV-BCRP。在耐藥細胞培養基中加入託瑞米芬，通過逆轉錄聚閤酶鏈反應(RT-PCR)、Western blot、外排實驗以及細胞毒性實驗觀察託瑞米芬對不同細胞繫的耐藥逆轉效果。結果與空白對照組（未加藥物）相比，託瑞米芬以劑量依賴方式抑製BCRP mRNA的錶達，0.1、1和10 μmol/L託瑞米芬處理組MCF-7/Promoter-BCRP細胞中BCRP mRNA的錶達水平分彆下調29.5％(P＜0.05)、68.1％ (P＜0.01)和97.4％(P＜0.01)；MCF-7/Promoter-BCRP細胞經託瑞米芬和17β-雌二醇聯閤處理後，細胞中BCRP mRNA的相對錶達水平為64.2％±1.3％，明顯高于託瑞米芬單獨處理組(3.8％±0.2％，P＜0.01)。託瑞米芬對各組細胞繫中BCRP蛋白錶達的調控作用與mRNA相似。經託瑞米芬處理後，MCF-7/Promoter-BCRP細胞內米託蒽醌的熒光彊度顯著增彊，外排米託蒽醌的能力降低瞭 47.3％ (P ＜0.05)；經託瑞米芬和17β-雌二醇聯閤處理後，MCF-7/Promoter-BCRP細胞內米託蒽醌的熒光彊度明顯低于託瑞米芬單獨處理組，外排米託蒽醌的能力升高瞭61.5％。託瑞米芬可有效逆轉MCF-7/Promoter-BCRP細胞對米託蒽醌的耐藥性。上述作用在MCF-7/CMV-BCRP、MDA-MB-231/Promoter-BCRP和MDA-MB-231/CMV-BCRP細胞中未能體現。結論託瑞米芬可能通過ERot的介導與BCRP啟動子上遊調控序列中的ERE結閤，負性調節BCRP的錶達，抑製BCRP蛋白的功能，在體外有效逆轉BCRP介導的多藥耐藥。
목적 탐토탁서미분역전유선암내약단백(BCRP)개도적다약내약궤제。방법 통과기인확증，구건분별유BCRP계동자화거세포병독(CMV)계동자계동표체BCRP적중조질립pcDNA3-Promoter-BCRP화작위대조적질립pcDNA3-CMV-BCRP，장기분별전염자격소수체α（ERα）양성적MCF-7화ERα음성적MDA-MB-231유선암세포계，건립유BCRP계동자화CMV계동자계동표체BCRP적4충내약세포계MCF-7/Promoter-BCRP、MCF-7/CMV-BCRP、MDA-MB-231/PromoterBCRP화MDA-MB-231/CMV-BCRP。재내약세포배양기중가입탁서미분，통과역전록취합매련반응(RT-PCR)、Western blot、외배실험이급세포독성실험관찰탁서미분대불동세포계적내약역전효과。결과여공백대조조（미가약물）상비，탁서미분이제량의뢰방식억제BCRP mRNA적표체，0.1、1화10 μmol/L탁서미분처리조MCF-7/Promoter-BCRP세포중BCRP mRNA적표체수평분별하조29.5％(P＜0.05)、68.1％ (P＜0.01)화97.4％(P＜0.01)；MCF-7/Promoter-BCRP세포경탁서미분화17β-자이순연합처리후，세포중BCRP mRNA적상대표체수평위64.2％±1.3％，명현고우탁서미분단독처리조(3.8％±0.2％，P＜0.01)。탁서미분대각조세포계중BCRP단백표체적조공작용여mRNA상사。경탁서미분처리후，MCF-7/Promoter-BCRP세포내미탁은곤적형광강도현저증강，외배미탁은곤적능력강저료 47.3％ (P ＜0.05)；경탁서미분화17β-자이순연합처리후，MCF-7/Promoter-BCRP세포내미탁은곤적형광강도명현저우탁서미분단독처리조，외배미탁은곤적능력승고료61.5％。탁서미분가유효역전MCF-7/Promoter-BCRP세포대미탁은곤적내약성。상술작용재MCF-7/CMV-BCRP、MDA-MB-231/Promoter-BCRP화MDA-MB-231/CMV-BCRP세포중미능체현。결론 탁서미분가능통과ERot적개도여BCRP계동자상유조공서렬중적ERE결합，부성조절BCRP적표체，억제BCRP단백적공능，재체외유효역전BCRP개도적다약내약。
Objective To explore the regulation mechanism of the reversal of breast cancer resistance protein-mediated multidrug resistance by toremifene. MethodsTwo recombinant plasmids (pcDNA3-promoter-BCRP and PODNA3-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 Erαnegative 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 Erαt-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 μ mol/L decreased BCRPmRNA 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.Conclusion 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 Ermediated to inactivate the transcription of BCRP gene.