N-ethylmaleimide increases P-glycoprotein photoaffinity labeling with iodoaryl-azidoprazosin in multidrug resistant cells

Abstract

P-glycoprotein (P-gp) mediates a multidrug resistance (MDR) phenotype in tumor cell lines selected with lipophilic cytotoxic drugs. Transport studies using purified P-glycoprotein reconstituted into defined liposomes have shown energy-dependent drug efflux of structurally dissimilar drugs. In this report, we have examined the effects of N-ethylmaleimide, a potent inhibitor of the P-gp ATPase, on P-gp drug binding in intact MDR cells and in plasma membranes. Our results show that short term treatment of MDR cells with 1-50 microM N-ethylmaleimide led to a concentration dependent increase in P-gp photoaffinity labeling with iodoaryl-azidoparazosin (IAAP). In addition, N-ethylmaleimide increases [3H] vinblastine accumu-lation in drug-resistant but not in sensitive cells. Comparison of IAAP photolabeled P-gp from intact cells with or without N-ethylmaleimide treatment did not show differences in the pattern of IAAP photolabeled peptides. Thus, the observed increase in P-gp photolabeling with IAAP in N-ethylmaleimide treated cells is not due to photolabeling at different sites. Incubation of MDR cells with [14C] N-ethylmaleimide showed that P-gp is directly modified at several Cysteine residues, as found from a complete proteolytic digestion of [14C] Nethylmaleimide labeled P-gp. The comparison of V8 staphylococcus aureas peptides from [14C] Nethylmaleimide or IAAP modified P-gp showed some peptides to co-migrate on SDS PAGE. However, modification of plasma membranes from drug resistant cells treated with N-ethylmaleimide did not show a dose-dependent increase in P-gp photolabeling with IAAP as seen with intact MDR cells. Interestingly, N-ethylmaleimide increases P-gp phosphorylation by inhibiting the turnover of Pgp phosphates. However, inhibition of P-gp phosphorylation with calyculin A did not show an increase in P-gp photolabeling in MDR cells. Taken together, the results of this study suggest that N-ethylmaleimide potentiates P-gp photolabeling with IAAP by inhibiting P-gp ATPase thereby increasing the local concentration of IAAP in intact MDR cells. Furthermore, inhibition of P-gp ATPase by N-ethylmaleimide does not lead to conformational changes that affects P-gp drug binding.