Vanhoefer U, Yin M B, Harstrick A, Seeber S, Rustum Y M
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Biochem Pharmacol. 1997 Mar 21;53(6):801-9. doi: 10.1016/s0006-2952(97)00010-5.
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.
细胞内谷胱甘肽（GSH）浓度最近被认为是多药耐药蛋白（MRP）介导的药物外排的调节决定因素。N，N-双（2-氯乙基）-N-亚硝基脲（BCNU）对谷胱甘肽还原酶（GR）活性的抑制作用已被广泛用于研究GSH氧化还原循环在细胞功能中的作用。本研究检测了BCNU对多药耐药的人纤维肉瘤细胞系HT1080/DR4（该细胞系过表达MRP）中MRP介导的阿霉素外排的影响。未检测到HT1080（亲代）细胞和多药耐药的HT1080/DR4细胞之间GR活性有显著差异（分别为38.6±2.2和37.8±5.28 nmol/min/10⁶细胞）。将HT1080和HT1080/DR4细胞暴露于100 - 500μM BCNU会使GR活性浓度依赖性降低，随后两种细胞系中的细胞内GSH池均减少。γ-谷氨酰半胱氨酸合成酶的特异性抑制剂D，L-丁硫氨酸-（S，R）-亚砜亚胺（D，L-BSO）对GSH生物合成的抑制作用显著降低了MRP介导的药物外排，并增强了阿霉素在表达MRP的HT1080/DR4细胞中的细胞毒性（剂量修正因子为20.8）。虽然在亲代细胞和耐药细胞中观察到BCNU对GR活性的抑制效果相同，但仅在表达MRP的HT1080/DR4细胞中阿霉素的细胞内潴留量显著增加。此外，用BCNU或D，L-BSO处理后对MRP功能的抑制与表达MRP的肿瘤细胞中GSH消耗程度直接相关[相关系数分别为r = 0.94（P < 0.001）和0.99（P < 0.001）]。基于对MRP mRNA水平的Northern印迹分析，将HT1080/DR4细胞暴露于BCNU并未导致MRP基因表达下调。本文报道的结果表明，具有氨甲酰化特性的亚硝基脲衍生物是MRP功能的有效抑制剂。通过抑制GSH氧化还原循环或GSH从头生物合成来消耗细胞内GSH池可显著抑制MRP介导的阿霉素转运，并在体外恢复细胞内药物浓度。
