Transcriptional Modulation Of Breast Cancer Resistance Protein By Estrogen And Antiestrogen In Breast Carcinoma Cells

【Background and objective】Human cancer cells can exhibit a cross-resistant phenotype against several unrelated drugs that differ widely with respect to molecular structure and target specificity.This phenomenon has been termed multidrug resistance(MDR).It is the major cause of the failure of chemotherapy-based treatment modalities of breast cancer.As is known to all,endocrinotherapy and chemotherapy of breast cancer are the main principles and new trends besides of surgery.MDR,a common phenomenon in cancer patients,is the major obstacle in the successful treatment of cancer.Although the mechanistic basis for this phenomenon is complex,the overexpression of ABC transporters is often associated with this phenotype.Various members of the protein superfamily of ABC transporters have been associated with MDR of human cancers when overexpressed,including P-gp,the MRP and its homologues MRP2 and MRP3.BCRP is a relatively new member of the ABC-transporter family and overexpressed in a variety of human MDR cancer cell lines.Elevated expression of BCRP results in resistance to antitumor drugs,including mitoxantrone,topotecan,daunorubicin,doxorubicin and bisantrene,etc.The BCRP gene is located on chromosome 4q22,which spans over 66kb in length and consists of 16 exons and 15 introns.The BCRP promoter is the region from -266 to -36.BCRP is a 72 kDa half-transporter consisting of 655 amino acid residues and is located on cell membrane.Recently,some estrogen and agonists were found to reverse BCRP-mediated multidrug resistance as a substrate of BCRP.As is known to all,antiestrogens are now a first-line drug used in endocrinotherapy for breast cancer.Antiestrogens can interdict the biological effect of estrogen on breast cancer cells,which involves binding of ER through the classical pathway to inactivate transcription of gene.The 5' region upstream of the basal promoter is characterized by both positive and negative regulatory domains.A putative estrogen response element(ERE)revealed exists between positions -243 and -115 in the human BCRP promoter,which is associated with the expression of BCRP in breast cancer cells.Therefore, antiestrogens downregulate BCRP expression to reverse BCRP-mediated multidrug resistance via a novel transcriptionally mechanism,which might be involved in the ERE of BCRP promoter to repress transcription of BCRP gene.The present study aimed to investigate whether the human BCRP expression is regulated transcriptionally by 17β-estradiol and antiestrogens toremifene.Two recombinant plasmids were designed to express the full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a control constitutive cytomegalovirus(CMV)promoter,respectively,which were transfected into estrogen receptorα(ERα)-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cell lines.These results may therefore further shed light on the control of BCRP gene expression.In addition,the modulation of BCRP expression in breast adenocarcinoma cells by antiestrogens may be a useful strategy for sensitizing such cells to anticancer agents.【Methods】1.Plasmids and cloning(1)Extract the total DNA of MCF-7.Lookup the whole length of BCRP promoter, design the forward and reverse primer of BCRP gene using OLIGO(Version 6.0).The BCRP promoter in full-length was amplified by PCR. (2)Extract the total RNA of MCF-7,design forward and reverse primer to clone the whole length of BCRP gene by RT-PCR.The full-length BCRP cDNA was amplified by reverse transcription-PCR(RT-PCR).(3)The full-length BCRP promoter and BCRP cDNA was sublconed into expression vector pcDNA3.Two recombinant plasmids (pcDNA3-Promoter-BCRP and pcDNA3-CMV-BCRP)have been 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.Double enzyme digestion analysis was used to identify the targeting plasmid.In addition,all constructs were further verified by DNA sequencing.2.Two recombinant plasmids were transfected into ERα-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cell lines.Four 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 new cell line was identified by flow cytometry,cytotoxicity assay,efflux assays,RT-PCR,western blot analysis,etc.3.These drug resistant cell lines were cultured in medium containing 17β-estradiol or antiestrogen toremifene.Expression of BCRP mRNA and protein were detected by RT-PCR and Western blot.The function of BCRP was assessed by mitoxantrone efflux assay.MTT method was used to evaluate the 50%inhibition concentration of antineoplastic drugs mitoxantrone on drug-resistant breast cancer cells.The present study aimed to investigate whether the human BCRP expression is regulated transcriptionally by 17β-estradiol or toremifene in ERα-positive and ERα-negative breast cancer cell lines.4.Electrophoretic mobility shift assay(EMSA)further confirmed wheather the putative ERE in the promoter region of BCRP gene and ERαare essential for transcriptional activation of BCRP by 17β-estradiol or toremifene.The present study is to explore the regulation mechanism of BCRP by 17β-estradiol or toremifene.【Results】1.Constructed BCRP expressing vector which was identified by enzymy digestion, the positive clones can be cut into two segments after being digested with HindⅢ/BamH I.Double enzyme digestion analysis and DNA sequencing confirmed that the recombinant plasmids were successfully constructed.2.MCF-7/Promoter-BCRP,MCF-7/CMV-BCRP,MDA-MB-231/Promoter-BCRP, and MDA-MB-231/CMV-BCRP cells were established by transfection of MCF-7 and MDA-MB-231 cells,respectively,with recombinant plasmids pcDNA3-Promoter-BCRP and pcDNA3-CMV-BCRP.RT-PCR was performed to observe the expression of BCRP mRNA and Western blot was used to measure BCRP protein levels.The capacity of effluxing mitoxantrone increased,the BCRP mRNA and protein increased obviously too.3.In MCF-7/Promoter-BCRP cells,17β-estradiol at 0.03 nM,0.3 nM and 3 nM significantly increased BCRP mRNA expression to 2.69-fold,4.46-fold and 8.03-fold,respectively.BCRP mRNA expression driven by the endogenous BCRP promoter in MCF-7/Promoter-BCRP cells,could be upregulated by 17β-estradiol in a dose-dependent manner and the induction effect was effectively abolished by antiestrogen tamoxifen.Moreover,in MCF-7/Promoter-BCRP cells after being treated with 17β-estradiol and tamoxifen for 72 hours,relative BCRP mRNA levels was 8.5%±0.1%,significantly lower than that of untreated control cells(61.3%±1.2%,P＜0.01).However,in MCF-7/CMV-BCRP cells, 17β-estradiol treatment did not affect the expression of BCRP mRNA driven by a CMV promoter.As expected,no change was observed in BCRP-transduced ERα-negative MDA-MB-231 cells after 17β-estradiol treatment.4.In MCF-7/Promoter-BCRP cells,toremifene at 0.3μM,3μM and 10μM decreased BCRP mRNA expression by 27.1%,72.3%and 94.0%,respectively. Moreover,estrone induction of BCRP mRNA expression was effectively banished by toremifene in a dose-dependent manner.In MCF-7/Promoter-BCRP cells after being treated with toremifene(0.3μM,3μM,and 10μM)and estrone (5 nM)for 48 hours,relative BCRP mRNA levels were 194.2%±6.5%,61.7%±2.6%and 25.4%±1.9%respectively,significantly lower than that of estrone treatment control cells(251.3%±7.2%,P＜0.05).However,in MCF-7/CMV-BCRP cells,toremifene treatment did not affect the expression of BCRP mRNA driven by a CMV promoter.Not unexpectedly,no significant changes of the BCRP mRNA levels were found in BCRP-transfected ERα-negative MDA-MB-231 cells after toremifene treatment,indicating that ERαis indispensable for the transcriptional repression of BCRP.5.In MCF-7/Promoter-BCRP cells,toremifene decreased mitoxantrone efflux activity by 41.7%in MCF-7/Promoter-BCRP cells when compared with the untreated control(P＜0.05).In MCF-7/P-BCRP cells,toremifene decreased the degree of mitoxantrone resistance to 48%compared with the untreated control cells,equivalent to 52%reversal(P＜0.05).6.Electrophoretic mobility shift assays further demonstrated that the putative ERE in the promoter region of the BCRP gene and ERαare essential for transcriptional activation of BCRP by 17β-estradiol or toremifene.Our findings indicate that expression of BCRP is regulated by 17β-estradiol or toremifene via a novel transcriptional mechanism which might be involved in 17β-estradiol- or toremifene- ER complexes binding to the ERE of BCRP promoter through the classical pathway to activate or inactivate transcription of BCRP gene.【Conclusion】1.Two plasmid vectors have been successfully constructed 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. MCF-7/Promoter-BCRP,MCF-7/CMV-BCRP,MDA-MB-231/Promoter-BCRP, and MDA-MB-231/CMV-BCRP cells were established,which express BCRP highly. 2.17β-Estradiol significantly upregulated BCRP mRNA and protein expression in a dose-dependent manner and the effect was abolished by antiestrogen tamoxifen.3.Toremifene significantly downregulated BCRP mRNA and protein levels in ERα-positive MCF-7 cells in a dose-dependent manner to reverse BCRP-mediated multidrug resistance.It will therefore be of interest that clinical treatment of hormone-responsive breast cancer with antiestrogen toremifene, conversely,may help to reduce the incidence of drug resistance.4.Taken together,our findings indicate that expression of BCRP is upregulated or downregulated by 17β-estradiol or antiestrogen toremifene,via a novel transcriptional mechanism which might be involved in the ERE of BCRP promoter through the classical pathway to activate or inactivate transcription of BCRP gene.