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.2019 Apr;93(4):953-964.
doi: 10.1007/s00204-019-02417-6. Epub 2019 Mar 12.

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

Affiliations

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

Tímea Windt et al. Arch Toxicol.2019 Apr.

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.

Keywords: ATP-binding cassette transporters; Anticancer drugs; Cytotoxicity; High-throughput screen; Organic Anon Transporting Polypeptides.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Cell growth can be reliably monitored based on the fluorescence of differently tagged cell lines.a Cells were plated in a 384 well plate at a final cell density of 1875 cells per well in the triple co-culture system (625 cells from Mes-Sa mCh/Mes-Sa Dx5 eGFP/ Mes-Sa-B1 mOr cell lines; 625 cells from A431 eGFP/A431-B1 mCh/A431-G2 mOr cell lines) and at 2500 cells per well in monocultures. Fluorescence corresponding to the density of the individual cell lines was recorded at regular intervals using a plate reader.b Validation of the triple-co-cultured fluorescence-based cytotoxicity assay. IC50 values measured in triple coculture condition and in monocultures. Mes-Sa, Mes-Sa-B1, Mes-Sa/Dx5 and A431, A431-B1, A431-G2 cells were seeded in 384 well plates and were incubated in the presence of increasing concentrations of etoposide or the MDR-selective compound NSC57969 in monoculture (left) and triple co-culture conditions (middle) measured with fluorescence-based cytotoxic assay, and with the PrestoBlue-based cytotoxicity assay (right). The fluorescence-based assay measured in triple coculture condition and in monoculture, compared with PrestoBlue-based cytotoxic assay provided concordant IC50 results (see Table S1)
Fig. 2
Fig. 2
Screening results of the DTP oncology set IV measured againsta parental Mes-Sa mCh and Pgp-expressing MDR derivatives [Mes-Sa/Dx5 eGFP (gray dots) or Mes-Sa-B1 mOr (black dots)];b parental A431 and its ABCG2-expressing derivative (A431-G2). Data points represent average pIC50 values derived from at least two independent experiments. To evaluate the selective toxicity of the compounds, resistance ratio (RR) was calculated by dividing the IC50 values measured against the multidrug resistant, transporter-expressing derivative by the cytotoxicity measured in the parental cell line, while the selectivity ratio (SR) was calculated as the inverse of RR. Compounds displaying at least a threefold difference in toxicity (dashed line) between the control and transporter-expressing cells were considered as putative ABCB1 or ABCG2 substrates (RR > 3, see inset); or putative MDR-selective compounds (SR > 3, see Table S2)
Fig. 3
Fig. 3
Characterization of the fluorescence-based OATP2B1 uptake transporter-expressing A431 co-culture system.a Transport activity of OATP2B1 can be selectively measured in the co-cultured cells. Co-culture of parental A431 eGFP (grey) and transporter expressing A431-2B1 mCh (black) cells were stained by Cascade Blue in the absence (middle) or presence (right) of the OATP2B1 inhibitor estrone-3-sulfate (cells without Cascade Blue are shown in the left panel). CB is selectively taken up by OATP2B1-expressing cells.b Growth of A431 eGFP and A431-2B1 mCh cells can be reliably monitored based on the differential tagging of cells. Cells were seeded in 384-well plates in monoculture (2500 cells/well) and in co-culture conditions (1250 A431 eGFP + 1250 A431-2B1 cells/well). The cells were incubated for 144 h in a final volume of 60 µl, at 37 °C and 5% CO2, under humidified atmosphere. Cell growth corresponding to each cell line was recorded using a microplate reader
Fig. 4
Fig. 4
Screening results of the DTP Oncology Set IV measured against parental A431 and its OATP2B1-expressing derivative (A431-2B1). Compounds displaying at least a threefold difference in toxicity (dashed line) between the control and transporter-expressing cells were considered as putative substrates (SR > 3, see inset)
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