Applications of BCRP-silenced Caco-2 cells and murine Bcrp-transfected MDCK cells in identifying BCRP substrates in vitro

The objective of this study was to evaluate the abilities of a human Caco-2 subclone, in which BCRP was silenced by RNA interference, and MDCK cells, transfected with the murine Bcrp1 gene, to identify BCRP substrates. The human BCRP-silenced Caco-2 subclone (CPT-B1) was created by transduction with a lentiviral vector containing BCRP-targeting shRNA, while murine Bcrp1-expressing MDCK cells (Bcrp1-MDCK) were generated by transfection with the murine Bcrp1 gene to create Bcrp1-overexpressing cells. Bidirectional transport experiments were conducted with commercial compounds in the CPT-B1 and Bcrp1-MDCK assay systems. Compound susceptibilities to efflux by BCRP or Bcrp1 were assessed by their vectorial transport relative to parental Caco-2 cells or GF120918-inhibited Bcrp1-MDCK cells, respectively. BCRP expression in parental Caco-2 cells was confirmed by molecular biology and functional assays. In addition, full-length cDNA sequencing showed that the BCRP gene allele expressed by this Caco-2 subclone does not contain any mutation. In the CPT-B1 assay system, 14 of 22 test compounds were identified as BCRP substrates and 8 as BCRP nonsubstrates; in the Bcrp1-MDCK assay system, 11 were classified as Bcrp1 substrates and 11 as non-substrates. Similarities as well as discrepancies were observed between the two assay systems. Of the 22 compounds tested, 7 compounds were identified as substrates of both BCRP and Bcrp1, 4 as non-substrates of both transporters, 7 as BCRP but not Bcrp1 substrates, and 4 as Bcrp1 but not BCRP substrates. Species difference in BCRP/Bcrp1 substrate specificity may be partially responsible for the discrepancies, but could not account for the fact that some well-known BCRP/Bcrp1 substrates (e.g., sulfasalazine and mitoxantrone) were identified as BCRP substrates in the CPT-B1 system but as Bcrp1 non-substrates in the Bcrp1-MDCK system. An alternative hypothesis is that known and/or as-yet unidentified uptake transporters in the basolateral membrane of Caco-2 cells facilitate cellular uptake of the compounds, whereas MDCK cells may lack such basal uptake systems. The current study demonstrates the utility of the CPT-B1 model for the identification of BCRP substrates, as well as the differences observed between well-characterized in vitro models, suggesting that appropriate control compounds should be benchmarked. Applications of BCRP-silenced Caco-2 cells and murine Bcrp-transfected MDCK cells in identifying BCRP substrates in vitro Jibin Li1, Erin D. Hugger2, Dung Nguyen2, Samantha M. Allen1, Wei Zhang1, Chris Bode1, Albert Owen1 and Ismael J. Hidalgo1 1Absorption Systems L.P., Exton, Pennsylvania; 2GlaxoSmithKline, King of Prussia, Pennsylvania METHODS Establishment of stable BCRP knockdown Caco-2 cells: Lentiviral particles containing human BCRPtargeting and non-targeting shRNAs were used to transduce Caco-2 cells to generate BCRP knockdown (CPTB1) cells and vector control (VC) cells (1). Stable transduced cells were cultured in DMEM containing 10% FBS with puromycin as the selecting agent.