Role of multidrug resistance protein 1 (MRP1) and glutathione S-transferase A1-1 in alkylating agent resistance. Kinetics of glutathione conjugate formation and efflux govern differential cellular sensitivity to chlorambucil versus melphalan toxicity.
We investigated the role of phase II (conjugation) and phase III (efflux) detoxification of the anticancer drugs melphalan (MLP) and chlorambucil (CHB). Although both drugs are substrates of Alpha-class glutathione S-transferases (GST) and the monoglutathionyl conjugates formed in these enzymatic reactions are transported by MRP1, we found that GSTA1-1 and MRP1 acted in synergy to confer resistance to CHB but not to MLP (Morrow, C. S., Smitherman, P. K., Diah, S. K., Schneider, E., and Townsend, A. J. (1998) J. Biol. Chem. 273, 20114-20120). To explain this selectivity of MRP1/GST-mediated resistance, we report results of side-by-side experiments comparing the kinetics of MLP- versus CHB-glutathione conjugate: formation, product inhibition of GSTA1-1 catalysis, and transport by MRP1. The monoglutathionyl conjugate of CHB, CHB-SG, is a very strong competitive inhibitor of GSTA1-1 (K(i) 0.14 microM) that is >30-fold more potent than that of the corresponding conjugate of MLP, MLP-SG (K(i) 4.7 microM). The efficiency of GSTA1-1-mediated monoglutathionyl conjugate formation is more than 4-fold higher for CHB than MLP. Lastly, both CHB-SG and MLP-SG are efficiently transported by MRP1 with similar V(max) although the K(m) for CHB-SG (0.37 microm) is significantly lower than for MLP-SG (1.1 microM). These results indicate that MRP1 is required for GSTA1-1-mediated resistance to CHB in order to relieve potent product inhibition of the enzyme by intracellular CHB-SG formed. The kinetic properties of MRP1 are well suited to eliminate CHB-SG at pharmacologically relevant concentrations. For MLP detoxification, where product inhibition of GSTA1-1 is less important, GSTA1-1 does not confer resistance because of the relatively poorer catalytic efficiency of MLP-SG formation. Similar analyses can be useful for predicting the pharmacological and toxicological consequences of MRP and GST expression on cellular sensitivity to various other electrophilic xenobiotics.
Paumi
C M
CM
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
Ledford
B G
BG
Smitherman
P K
PK
Townsend
A J
AJ
Morrow
C S
CS
D018528:ATP-Binding Cassette Transporters / Q000502:physiology*; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D002699:Chlorambucil / Q000378:metabolism / Q000494:pharmacology*; D004351:Drug Resistance; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D008558:Melphalan / Q000378:metabolism / Q000494:pharmacology*; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014407:Tumor Cells, Cultured
D018528:ATP-Binding Cassette Transporters; D018906:Antineoplastic Agents, Alkylating; D002699:Chlorambucil; D005978:Glutathione; D005982:Glutathione Transferase; D007527:Isoenzymes; D008558:Melphalan; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C119995:glutathione S-transferase alpha