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 re cently as a regulatory determinant of multidrug resistance protein (MR P)-mediated drug efflux. Inhibition of glutathione reductase (GR) acti vity of N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU) has been employed extensively to investigate the role of GSH redox cycle in cellular fun ction. The present study examined the effect of BCNU on the MRP-mediat ed 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 100-5 00 mu M BCNU decreased GR activity concentration dependently with subs equent reduction in cellular GSH pools in both cell lines. Inhibition of GSH biosynthesis by D,L-buthionine-(S,R)-sulfoximine (D,L-BSO), a s pecific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced MRP-mediated drug efflux and potentiated the cytotoxicity of d oxorubicin in MRP-expressing HT1080/DR4 cells (dose modifying factor 2 0.8). While equally effective inhibition of GR activity by BCNU was ob served in parental and resistant cells, a significant increase in intr acellular retention of doxorubicin was only achieved in MRP-expressing HT1080/DR4 cells. Furthermore, inhibition of MRP function following t reatment with BCNU or D,L-BSO was directly related to the degree of GS H 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 indica te chat derivatives of nitrosourea with carbamoylating properties are potent inhibitors of MRP function. Depletion of intracellular GSH pool s by inhibition of the GSH redox cycle or GSH de novo biosynthesis sig nificantly inhibited MRP-mediated doxorubicin transport and restored i ntracellular drug concentrations in vitro. (C) 1997 Elsevier Science I nc.