Carbamoylation of glutathione reductase by N,N-bis(2-chloroethyl)-N- nitrosourea associated with inhibition of multidrug resistance protein (MRP) function.

Intracellular glutathione (GSH) concentrations have been implicated recently as a regulatory determinant of multidrug resistance protein (MRP)-mediated drug efflux. Inhibition of glutathione reductase (GR) activity of N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU) has been employed extensively to investigate the role of GSH redox cycle in cellular function. The present study examined the effect of BCNU on the MRP-mediated efflux of doxorubicin in the multidrug-resistant human fibrosarcoma cell line HT1080/DR4 overexpressing MRP. No significant difference in GR activity between HT1080 (parental) and multidrug-resistant HT1080/DR4 cells was detected (38.6 +/- 2.2 and 37.8 +/- 5.28 nmol/min/10(6) cells, respectively). Exposure of HT1080 and HT1080/DR4 cells to 100-500 microM BCNU decreased GR activity concentration dependently with subsequent reduction in cellular GSH pools in both cell lines. Inhibition of GSH biosynthesis by D,L-buthionine-(S,R)-sulfoximine (D,L-BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced MRP-mediated drug efflux and potentiated the cytotoxicity of doxorubicin in MRP-expressing HT1080/DR4 cells (dose modifying factor 20.8). While equally effective inhibition of GR activity by BCNU was observed in parental and resistant cells, a significant increase in intracellular retention of doxorubicin was only achieved in MRP-expressing HT1080/DR4 cells. Furthermore, inhibition of MRP function following treatment with BCNU or D,L-BSO was directly related to the degree of GSH depletion in MRP-expressing tumor cells [r = 0.94 (P < 0.001) and 0.99 (P < 0.001), respectively]. Based on northern blot analysis of MRP mRNA levels, exposure of HT1080/DR4 cells to BCNU did not produce down-regulation of MRP gene expression. The results reported herein indicate that derivatives of nitrosourea with carbamoylating properties are potent inhibitors of MRP function. Depletion of intracellular GSH pools by inhibition of the GSH redox cycle or GSH de novo biosynthesis significantly inhibited MRP-mediated doxorubicin transport and restored intracellular drug concentrations in vitro.