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Cellular and in vitro transport of glutathione conjugates by MRP
H Shen1, S Paul, L M Breuninger
1Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Abstract:
MRP is a recently identified ATP-binding cassette transporter. We previously established that MRP confers resistance to a spectrum of natural product cytotoxic drugs [Kruh, G.D., (1994) Cancer Res. 54, 1649-1652], that expression of MRP is associated with enhanced drug efflux [Breuninger, L.M., (1995) Cancer Res. 55, 5342-5347], and that MRP transcript is widely expressed in human tissues and solid tumor cell lines [Kruh, G.D., (1995) J. Natl. Cancer Inst. 87, 1256-1258]. In the present study the relationship between MRP and drug glutathione S-conjugates was examined. We observed that MRP was labeled by azidophenacylglutathione (APA-SG), a photoaffinity analog of glutathione, and that inside-out membrane vesicles prepared from MRP-overexpressing HL60/ADR cells transported this compound. Transport into membrane vesicles was ATP-dependent, sensitive to osmolarity, and saturable with regard to APA-SG and ATP concentrations, with Km values of 15 and 61 microM, respectively. APA-SG transport was competitively inhibited by the natural product cytotoxic drugs daunorubicin, vincristine, and etoposide, with Ki values of 4.8, 3.8, and 5.5 microM, respectively. Oxidized glutathione, the drug-glutathione S-conjugate DNP-SG, the LTD4 antagonist MK571 and arsenate were also competitive inhibitors, with Ki values of 9.0, 23.4, 1.1, and 15.0 microM, respectively. Analysis of the fate of monochlorobimane in MRP transfectants revealed reduced intracellular concentrations of drug-glutathione S-conjugates associated with enhanced efflux and altered intracellular distribution. These results indicate that MRP can transport glutathione conjugates in vitro and in living cells and suggest the possibility that the transporter may represent a link between cellular resistance to some classes of cytotoxic drugs and glutathione-mediated mechanisms of resistance. In addition, the observation that both mildly cationic or neutral natural product cytotoxic drugs and anionic compounds such as DNP-SG, MK571, and arsenate are competitive inhibitors of MRP action suggests that the substrate specificity of the transporter is quite broad.
Insights
The multidrug resistance-associated protein (MRP) transporter can move glutathione conjugates, linking drug resistance to glutathione mechanisms. This broad-specificity transporter handles both cytotoxic drugs and glutathione conjugates, impacting cellular drug efflux and distribution.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The multidrug resistance-associated protein (MRP) is an ATP-binding cassette transporter.
- Previous studies established MRP's role in conferring resistance to cytotoxic drugs and its wide expression in human tissues and tumors.
- MRP expression is linked to enhanced drug efflux.
Purpose of the Study:
- To investigate the relationship between MRP and drug glutathione S-conjugates.
- To determine if MRP can transport glutathione conjugates.
Main Methods:
- Labeling MRP with azidophenacylglutathione (APA-SG), a photoaffinity analog of glutathione.
- Transport assays using inside-out membrane vesicles from MRP-overexpressing HL60/ADR cells.
- Analysis of monochlorobimane fate in MRP transfectants to assess intracellular drug-glutathione S-conjugate concentrations.
Main Results:
- MRP was labeled by APA-SG, and membrane vesicles transported APA-SG in an ATP-dependent, saturable manner.
- Transport of APA-SG was competitively inhibited by cytotoxic drugs, oxidized glutathione, DNP-SG, MK571, and arsenate, indicating broad substrate specificity.
- MRP transfectants showed reduced intracellular drug-glutathione S-conjugates with enhanced efflux and altered distribution.
Conclusions:
- MRP can transport glutathione conjugates both in vitro and in living cells.
- MRP may link cellular resistance to cytotoxic drugs with glutathione-mediated resistance mechanisms.
- The broad substrate specificity of MRP includes both cytotoxic drugs and anionic conjugates.