title: Culture period-dependent change of function and expression of ATP-binding cassette transporters in Caco-2 cells.
abstract: The objective of this study was to determine an appropriate culture period to assess whether a compound of interest is transported by efflux transporters such as human multidrug resistance 1 (hMDR1), human multidrug resistance-associated protein 2 (hMRP2), and human breast cancer resistance protein (hBCRP) in Caco-2 cells. Caco-2 cells were cultured on a Transwell for 1 to 6 weeks. The expression of these transporters in the mRNA and protein levels was examined using a real-time polymerase chain reaction and Western blotting, respectively. Transcellular transport activities using digoxin, ochratoxin A, olmesartan, and estrone-3-sulfate were also examined. Except for digoxin, the permeability coefficient (P(app)) ratio of the three compounds at 2 weeks was the highest in the periods tested. The P(app) ratio of digoxin at 2 weeks was higher than that at 3 weeks. The temporal expression profile of each transporter in the mRNA level was similar to that in the protein level, and the functions of hMRP2 and hBCRP were roughly correlated with the expression in the mRNA and protein levels, but that of hMDR1 was not. These data suggest that among all the culture periods evaluated a 2-week culture is the best culture period for transport studies to identify whether a compound is a substrate for hMDR1, hMRP2, and hBCRP.
authors.affiliation: Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan. kamiyama.emi.gf@daiichisankyo.co.jp
authors.forename: Emi
authors.initials: E
authors.lastname: Kamiyama
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sugiyama
authors.forename: Daisuke
authors.initials: D
authors.lastname: Nakai
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Miura
authors.forename: Osamu
authors.initials: O
authors.lastname: Okazaki
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000378:metabolism; D015153:Blotting, Western; D018938:Caco-2 Cells; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004077:Digoxin / Q000378:metabolism; D004553:Electric Conductivity; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D020024:Leukotriene Antagonists / Q000494:pharmacology; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D009793:Ochratoxins / Q000378:metabolism; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013777:Tetrazoles / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism; D013997:Time Factors; D014700:Verapamil / Q000494:pharmacology
chemical_list: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D047228:Angiotensin II Type 1 Receptor Blockers; D002121:Calcium Channel Blockers; D004077:Digoxin; D004970:Estrone; D007093:Imidazoles; D020024:Leukotriene Antagonists; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D009793:Ochratoxins; D011422:Propionates; D011804:Quinolines; D012333:RNA, Messenger; D013777:Tetrazoles; D014700:Verapamil; C000715087:ABCC2 protein, human; C116614:ABCG2 protein, human; C025589:ochratoxin A; C059141:verlukast; C437965:olmesartan; C017296:estrone sulfate