title: Breast Cancer Resistance Protein Abundance, but Not mRNA Expression, Correlates With Estrone-3-Sulfate Transport in Caco-2.
abstract: Transporter mRNA and protein expression data are used to extrapolate in vitro transporter kinetics to in vivo drug disposition predictions. Breast cancer resistance protein (BCRP) possesses broad substrate specificity; therefore, understanding BCRP expression-activity relationships are necessary for the translation to in vivo. Bidirectional transport of estrone-3-sulfate (E-3-S), a BCRP probe, was evaluated with respect to relative BCRP mRNA expression and absolute protein abundance in 10- and 29-day cultured Caco-2 cells. BCRP mRNA expression was quantified by real-time PCR against a housekeeper gene, Cyclophilin A. The BCRP protein abundance in total membrane fractions was quantified by targeted proteomics, and [(3)H]-E-3-S bidirectional transport was determined in the presence or absence of Ko143, a potent BCRP inhibitor. BCRP mRNA expression was 1.5-fold higher in 29- versus 10-day cultured cells (n = 3), whereas a 2.4-fold lower (p < 0.001) BCRP protein abundance was observed in 29- versus 10-day cultured cells (1.28 ± 0.33 and 3.06 ± 0.22 fmol/μg protein, n = 6, respectively). This correlated to a 2.45-fold lower (p < 0.01) efflux ratio for E-3-S in 29- versus 10-day cultured cells (8.97 ± 2.51 and 3.32 ± 0.66, n = 6, respectively). Caco-2 cell BCRP protein abundance, but not mRNA levels, correlates with BCRP activity, suggesting that extrapolation strategies incorporating BCRP protein abundance-activity relationships may be more successful.
authors.affiliation: Gut Barrier Group, Inflammation and Repair, University of Manchester, Salford Royal NHS Trust, Salford M6 8HD, UK; Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield S2 4SU, UK. Electronic address: matthew.harwood@certara.com.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Harwood
authors.affiliation: Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield S2 4SU, UK.
authors.forename: Sibylle
authors.initials: S
authors.lastname: Neuhoff
authors.affiliation: Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield S2 4SU, UK; Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester M13 9PT, UK.
authors.forename: Amin
authors.initials: A
authors.lastname: Rostami-Hodjegan
authors.affiliation: Gut Barrier Group, Inflammation and Repair, University of Manchester, Salford Royal NHS Trust, Salford M6 8HD, UK.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Warhurst
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2 / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D018938:Caco-2 Cells; D004970:Estrone / Q000031:analogs & derivatives* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D004970:Estrone; C017296:estrone sulfate
keywords: ABC transporters; Caco-2 cells; efflux pumps; intestinal absorption; liquid chromatography; mass spectrometry; proteomics