title: A new intestinal cell culture model to discriminate the relative contribution of P-gp and BCRP on transport of substrates such as imatinib.
abstract: P-glycoprotein (P-gp/MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) play an important role in transport of a wide variety of endogenous compounds, drugs and toxins. Transport of some drugs, for example the tyrosine kinase inhibitor imatinib, is influenced by both P-gp and BCRP. Establishing an intestinal Caco-2 cell culture model with specific knock-downs of P-gp and BCRP and double knock-down of both proteins, we aimed to elucidate the impact of each transporter on transport of imatinib. Stable single and double knock-downs of P-gp and BCRP were obtained by RNA interference (RNAi). Transporter expression was measured on RNA and protein level using real-time RT-PCR and Western blot, respectively. Functional activity was quantified by transport of specific substrates across Caco-2 cells. MDR1 and BCRP mRNA expression was reduced to 75% and 90% compared to wild-type control in single MDR1- and BCRP-knock-down clones, respectively. In double knock-down clones, MDR1 expression decreased to 95% and BCRP expression to 80%. Functional activity of P-gp and BCRP was diminished as transport of the P-gp-specific substrate (3)H-digoxin and the BCRP-specific substrate (14)C-PhIP was augmented in the opposite direction, when the respective transporter was knocked down. Similar effects were observed by chemical inhibition of the respective transporter. Bidirectional transport studies with (14)C-imatinib revealed an abrogation of asymmetric transport when P-gp was knocked down, either in single or double knock-down clones compared to wild-type cells. This was not observed in single BCRP-knock-down clones. In conclusion, this newly established cell system with single and concomitant knock-down of P-gp and BCRP can be used to quantify the specific partial impact of the transporters on transport of substrates that are transported by both proteins. For imatinib transport, the contribution of P-gp seems to be more important compared to BCRP in this Caco-2 cell system.
authors.affiliation: Division of Gastroenterology and Hepatology, University Hospital of Basel, CH-4031, Basel, Switzerland.
authors.forename: Angelika
authors.initials: A
authors.lastname: Graber-Maier
authors.forename: Heike
authors.initials: H
authors.lastname: Gutmann
authors.forename: Juergen
authors.initials: J
authors.lastname: Drewe
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001549:Benzamides / Q000493:pharmacokinetics*; D001693:Biological Transport, Active; D018938:Caco-2 Cells; D055785:Gene Knockdown Techniques; D006801:Humans; D000068877:Imatinib Mesylate; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010879:Piperazines / Q000493:pharmacokinetics*; D047428:Protein Kinase Inhibitors / Q000493:pharmacokinetics; D011743:Pyrimidines / Q000493:pharmacokinetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C513055:ABCB1 protein, human; C116614:ABCG2 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001549:Benzamides; D009363:Neoplasm Proteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D012333:RNA, Messenger; D000068877:Imatinib Mesylate