Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.

Abstract

Multidrug-resistance-associated protein [6][7][8] and is able to decrease cellular drug levels against a concentration gradient (4, 6). However, recent work has also indicated interesting differences between MRP and Pgp. Increased cellular MRP levels are associated with increased reduced glutathione (GSH) S-conjugate carrier (GS-X pump) activity in isolated plasma membrane vesicles (9-11). This suggests that MRP is a GS-X pump (12) present in many, if not all, mammalian cells (9-13). These pumps transport substrates containing a large hydrophobic moiety and at least two negative charges (12, 13), as present in drug GSH Sconjugates. Moreover, recent studies indicate that GS-X pumps are also involved in the export of cisplatin (14-16) and arsenite (11). Indeed, some cell lines overexpressing MRP are moderately resistant to arsenite (4, 11). These results link MRP to older experiments in which resistance to anthracyclines was found to correlate with increased levels of cellular GSH, GSH synthesis, or GSH Stransferases (17)(18)(19). This link is supported by the strong decrease in drug resistance in two MDR lung carcinoma cell lines that overexpress MRP by DL-buthionine (S,R)-sulfoximine (BSO) (20)(21)(22), an inhibitor of y-glutamylcysteine synthetase, the enzyme that catalyzes the first step in GSH synthesis (23). The interpretation of these inhibitor experiments is not unambiguous, however. In both cell lines, other resistance mechanisms (e.g., alterations in topoisomerase II) contribute to resistance, and it is not clear whether the GSH depletion in these cells does not result in membrane damagee.g., by lipid peroxidation. Damage of the plasma membrane could increase drug influx and, hence, decrease resistance. To test whether GSH is specifically required for MDR caused by MRP but not by Pgp, we have analyzed the effects of BSO treatment on lung cancer cells transfected with an expression vector containing either MRP cDNA or MDR1 cDNA. MATERIALS AND METHODSCell Lines. S1(MRP) was obtained after transfection of non-small cell lung cancer SW-1573/S1 cells with an expression vector containing MRP cDNA and a neomycin-resistance marker gene (pRc/RSV-MRP), followed by selection with geneticin (G418) (6). Sl(MDR1) was previously named S1(1.1) (24) and was obtained after transfection of S1 cells with the expression vector pJ3fl (25) containing MDR1 cDNA, followed by selection with 10 nM vincristine. GLC4/ADR is a MRP-overexpressing subline of the non-small cell lung cancer cell line GLC4 and was obtained by selection with doxorubicin (20,21).Clonogenic Survival Assay. In six-well dishes, 400 cells per well were seeded and incubated in medium with or without 25 ,uM BSO for 24 hr prior to incubation with increasing concentrations of drug. After 1 hr, drug was removed, the wells were rinsed with phosphate-buffered saline, and drug-free medium without BSO was added. Seven days after the start of the experiment, the percentage of cells that were able to produce a colony of >50 cells was used as a measure of cell sur...

Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export

“…The evidence available suggests that such conjugates do not exist for anthracyclines or vinca alkaloids (Tew, 1994). Zaman et al (1995) analysed the medium from MRP1-overexpressing cells incubated in the presence of vincristine or daunorubicin and found that all drug recovered was in the unmodified form. The simplest interpretation of our data is that vinblastine is transported as free drug.…”

“…Besides organic anions MRP1 can also transport neutral and basic cytotoxic drugs not known to be conjugated to GSH or other negatively charged compounds Zaman et al, 1994). Nevertheless, MRP1 requires the presence of intracellular glutathione for the transport of these drugs (Versantvoort et al, 1995;Zaman et al, 1995). The data available are most consistent with a model in which MRP1 co-transports cytotoxic drugs with GSH (Rappa et al, 1997;Loe et al, 1998).…”

Transport of the glutathione conjugate of ethacrynic acid by the human multidrug resistance protein MRP

“…SI(MRP) is a subline of the human non-small cell lung cancer cell line SW-1573/S1 that overexpresses MRP 15-fold [3,8]. This cell line was obtained by transfection of S1 ceils with an expression vector containing MRP cDNA and a neomycin-resistance gene and selection with Geneticin (G418) [3].…”

“…(ii) MRP requires GSH to export anti-cancer drugs and arsenite out of the cell [8]. (iii) Drug transport by MRP is relatively insensitive to classic reversal agents of Pgp-mediated multidrug resistance (MDR), such as verapamil and cyclosporin (analogues) [2,3], but is modulated by inhibitors of organic anion transport, such as probenecid and sulfinpyrazone [6,7,9,10].…”

“…(iii) Drug transport by MRP is relatively insensitive to classic reversal agents of Pgp-mediated multidrug resistance (MDR), such as verapamil and cyclosporin (analogues) [2,3], but is modulated by inhibitors of organic anion transport, such as probenecid and sulfinpyrazone [6,7,9,10]. These data suggest that MRP is a GS-X pump [I1] and link MRP-mediated MDR to drug resistance associated with GSH and GSH S-transferases (GSTs) [5,8,12].…”