Abstract
Accumulating evidence suggests a critical role of intracellular glutathione in tumor cell resistance to alkylating agents. The present study provides evidence for the direct interaction between cis-diamminedichloroplatinum(II) (cisplatin) and glutathione (GSH) both in a cell-free system, as well as in L1210 murine leukemia cells. We have isolated the reaction product and identified it by a combination of high performance liquid chromatography and atomic absorption spectroscopy. Stoichiometric analysis showed a 2:1 molar ratio of GSH/cisplatin for the reaction. The molecular mass assessed by mass spectroscopy was 809 Da, corresponding to a GS-platinum chelate complex, bis-(glutathionato)-platinum. The GS-platinum complex was detected in L1210 leukemia cells incubated with 20 microM cisplatin. The intracellular content of the GS-platinum complex reached a maximal level after 12 h, corresponding to about 60% of the intracellular platinum content. Thus, formation of the GS-platinum complex is considered a significant part of the cellular metabolism of cisplatin. The GS-platinum was found to inhibit cell-free protein synthesis in a rabbit reticulocyte lysate system using both chloramphenicol acetyltransferase mRNA and poly(A) mRNA from HL-60 human promyelocytic leukemia cells (IC50 = 190 microM the GS-platinum complex). Elimination of the GS-platinum complex from tumor cells may represent an important mechanism which reduces the intracellular accumulation of the platinum complex. Using plasma membrane vesicles prepared from L1210 cells, the transport of the GS-platinum complex across the plasma membrane was found to be an ATP-dependent process (apparent Km values: 49 microM, ATP; 110 microM, GS-platinum complex). The ATP-dependent transport of the GS-platinum complex was inhibited by vanadate (IC50 = 35 microM) as well as by S-(2,4-dinitrophenyl)-glutathione, leukotriene C4, and GSSG, but not by doxorubicin, daunorubicin, or verapamil. The ATP-dependent glutathione S-conjugate export pump, "GS-X pump" (Ishikawa, T. (1992) Trends Biochem. Sci. 17, 463-468), is suggested to play a role in the elimination of the GS-platinum complex from tumor cells.
Highlights
From the DeDartment o,f EmIerimental Pediatrics. the University of Texas, M
The present study provides evidencmeodulation of thiol-disulfide status of cellular proteins, profor the direct interaction betwceise-ndiamminedichlo- tection of cells from oxidative stress, detoxication of electroroplatinum(I1) and glutathione (GSH) both philic compounds, and synthesis and transpoorftbiologically in a cell-free system,as well as in L1210 murine leu- active,endogenous substances [1,2,3,4,5]
The GS-platinum was found to inhibit hitherto been identified, which include decreaseidntracellular cell-free protein synthesis in a rabbit reticulocyte ly- accumulation of cisplatin [17, 18],elevated cellular GSH [19]
Summary
From the DeDartment o,f EmIerimental Pediatrics. the University of Texas, M. ATP-dependent Transport of GS-Platinum Complex across cisplatin-treated cells (Fig. 8B, Coomassie staining), the au- Plasma Membrane of L1210 Cells-Active elimination of the GSH-associated Cisplatin Metabolism and Export
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