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

Abstract

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. Electronic supplementary material The online version of this article (10.1007/s00204-019-02417-6) contains supplementary material, which is available to authorized users.

A novel fluorescence‐based functional assay for human OATP1A2 and OATP1C1 identifies interaction between third‐generation P‐gp inhibitors and OATP1A2

“…Since both inhibitors and transported substrates can inhibit the uptake of the labeled test substrates, we investigated the toxicity of the P‐gp inhibitors in A431‐OATP1A2 cells. We hypothesized that transported substrates may be more toxic to A431‐OATP1A2 cells as compared to the parental cell lines . Figure B shows that indeed OATP1A2 expression sensitized the cells against elacridar.…”

Structural Similarities between Some Common Fluorophores Used in Biology, Marketed Drugs, Endogenous Metabolites, and Natural Products

“…The basis of the main idea presented and tested here is that the structures of common fluorophores are in fact sufficiently similar to those of many drugs (including natural products) as to provide suitable surrogates for assessing their uptake via solute carriers of the SLC (and, indeed, their efflux via ABC) families. While the latter transporters are well known to be rather promiscuous, and to transport a variety of fluorophores [ 40 , 42 , 128 , 129 , 130 ], considerably less attention has been paid to the former. As mentioned in the Introduction, some marketed pharmaceutical drugs that are transported into cells are in fact naturally fluorescent, including anthracyclines [ 56 , 57 , 58 ], mepacrine (atebrin, quinacrine) [ 59 ], obatoclax [ 60 , 61 ], tetracycline derivatives [ 57 , 62 ] and topotecan [ 63 ], while the same is true of certain vitamins riboflavin [ 64 , 65 ] and certain bioactive natural products (e.g., [ 66 , 67 , 68 ]).…”

Relation of Metal-Binding Property and Selective Toxicity of 8-Hydroxyquinoline Derived Mannich Bases Targeting Multidrug Resistant Cancer Cells

“…CRL-1977™). ABCB1 was expressed in A431 and MES-SA cells using lentiviral transduction [ 42 , 61 ]. The human cervix carcinoma cell line KB-3-1 and the vinblastine selected KB-v1were kind gifts from Dr. Michael M. Gottesman, National Institutes of Health.…”