title: [Regulation mechanism of breast cancer resistance protein by toremifene to reverse BCRP-mediated multidrug resistance in breast cancer cells].
abstract: 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 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathology, Capital Medical University, Beijing, China. yuhuazhang111@yahoo.com.cn
authors.forename: Yu-hua
authors.initials: YH
authors.lastname: Zhang
authors.forename: Guang
authors.initials: G
authors.lastname: Li
authors.forename: Jin
authors.initials: J
authors.lastname: Yu
authors.forename: Miao-sheng
authors.initials: MS
authors.lastname: Xu
authors.forename: Zhao-xia
authors.initials: ZX
authors.lastname: Liu
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D018931:Antineoplastic Agents, Hormonal / Q000008:administration & dosage / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D003587:Cytomegalovirus / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D018432:Drug Resistance, Multiple / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008942:Mitoxantrone / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D017312:Toremifene / Q000008:administration & dosage / Q000494:pharmacology*
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D018931:Antineoplastic Agents, Hormonal; D047628:Estrogen Receptor alpha; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004958:Estradiol; D017312:Toremifene; D008942:Mitoxantrone