ATP-Dependent Transport of Lipophilic Cytotoxic Drugs by Membrane Vesicles Prepared from MRP-Overexpressing HL60/ADR Cells

Abstract

MRP is an ATP-binding cassette family transporter that confers cellular resistance to a variety of natural product cytotoxic agents. However, the biochemical mechanism by which MRP confers resistance has not been established. To gain insight into its mechanism of action, the in vitro substrate specificity of MRP was examined by analyzing drug uptake into membrane vesicles prepared from MRP-overexpressing HL60/ADR cells. Compared to control HL60 membrane vesicles, HL60/ADR membrane vesicles exhibited markedly enhanced ATP-dependent transport of daunorubicin, etoposide, and vincristine. In contrast, little or no increased uptake was observed for vinblastine and Taxol. This pattern of in vitro substrate specificity was consistent with the analysis of the HL60/ADR drug resistance phenotype, which revealed substantial levels of resistance to anthracyclines, etoposide, and vincristine, but only slightly increased resistance to vinblastine and Taxol. Drug transport into HL60/ADR membrane vesicles was osmotically sensitive and dependent on ATP concentration, with a K_m value of 45 μM for ATP. Lineweaver−Burk analysis indicated that substrate transport was concentration-dependent, with apparent K_m values of 6.1, 5.7, and 5.5 μM for daunorubicin, etoposide, and vincristine, respectively. The P-glycoprotein-modulating agents cyclosporin A, PSC833, and verapamil, which have modest reversing effects on MRP-overexpressing cell lines, were weak competitive inhibitors of daunorubicin transport, with K_i values of 35, 84, and 95 μM, respectively. In addition, the glutathione analog azidophenacylglutathione, oxidized glutathione, and the LTD₄ antagonist MK571 were competitive inhibitors of daunorubicin transport, with K_i values of 69, 31, and 3.0 μM, respectively. Genistein, an MRP-specific modulating agent, and arsenate, a compound for which MRP has previously been reported to confer resistance, were also competitive inhibitors, with K_i values of 17 and 29 μM, respectively. These results are consistent with a previous report in which we demonstrated that HL60/ADR membrane vesicles transport azidophenacylglutathione and that transport of this agent is competitively inhibited by daunorubicin, vincristine, and etoposide [Shen et al., (1966) Biochemistry 35, 5719−5725]. Together, these uptake studies performed with HL60/ADR membrane vesicles constitute a consistent body of evidence that indicates that MRP transports both glutathione S conjugates and unaltered natural product drugs and support the idea that the direct transport of unaltered lipophilic cytotoxic drugs is the predominant biochemical mechanism whereby MRP confers multidrug resistance.