Identification of anticancer OATP2B1 substrates by an in vitro triple-fluorescence-based cytotoxicity screen

Membrane transporters play an important role in the absorption, distribution, metabolism and excretion of drugs. The cellular accumulation of many drugs is the result of the net function of efflux and influx transporters. Efflux transporters such as P-glycoprotein/ABCB1 have been shown to confer multidrug resistance in cancer. Although expression of uptake transporters has been confirmed in cancer cells, their role in chemotherapy response has not been systematically investigated. In the present study we have adapted a fluorescence-based cytotoxic assay to characterize the influence of key drug-transporters on the toxicity of approved anticancer drugs. Co-cultures of fluorescently labeled parental and transporter-expressing cells (expressing ABCB1, ABCG2 or OATP2B1) were screened against 101 FDA-approved anticancer drugs, using a novel, automated, triple fluorescence-based cytotoxicity assay. By measuring the survival of parental and transporter-expressing cells in co-cultures, we identify those FDA-approved anticancer drugs, whose toxicity is influenced by ABCB1, ABCG2 or OATP2B1. In addition to confirming known substrates of ABCB1 and ABCG2, the fluorescence-based cytotoxicity assays identified anticancer agents whose toxicity was increased in OATP2B1 expressing cells. Interaction of these compounds with OATP2B1 was verified in dedicated transport assays using cell-impermeant fluorescent substrates. Understanding drug-transporter interactions is needed to increase the efficacy of chemotherapeutic agents. Our results highlight the potential of the fluorescence-based HT screening system for identifying transporter substrates, opening the way for the design of therapeutic approaches based on the inhibition or even the exploitation of transporters in cancer cells.