Suppression of Canine ATP Binding Cassette ABCB1 in Madin-Darby Canine Kidney Type II Cells Unmasks Human ABCG2-Mediated Efflux of Olaparib.

Endogenous canine ATP binding cassette B1 (cABCB1) is expressed abundantly in Madin-Darby canine kidney type II (MDCKII) cells, and its presence often complicates phenotyping of the transport process. Errors in estimating the corrected efflux ratio (cER), as a result of the variable expression of cABCB1, were examined for the dual substrates of ABCB1 and ABCG2 in MDCKII cells expressing human ABCG2 (hABCG2). cABCB1 mRNA and protein expression was 60% and 55% lower, respectively, in MDCKII cells expressing hABCG2 compared with the wild type, suggesting that the expression of endogenous cABCB1 became variable after the expression of hABCG2. To minimize the contribution of endogenous efflux, cABCB1 was suppressed kinetically (using verapamil as a selective inhibitor) or biochemically (transfecting short-hairpin RNA against cABCB1). Under these suppression conditions, cER values for irinotecan and topotecan (dual substrates of ABCB1 and ABCG2) were elevated by more than 4-fold and 2-fold, respectively, compared with cER values without the suppression. The cER of olaparib was similarly increased to 3- and 5-fold in MDCKII cells under the kinetic and biochemical suppression of cABCB1, respectively, suggesting that hABCG2-mediated efflux cannot be ruled out for olaparib. Since the substrate selectivity for ABCB1 and ABCG2 overlapped considerably, the possibility of an inaccurate estimation of cER must be considered for dual substrates in the case of the variable expression of cABCB1 in MDCKII cells.
(Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)