Ziemann C, Bürkle A, Kahl G F, Hirsch-Ernst K I
Department of Toxicology, Institute of Pharmacology and Toxicology, University of Göttingen, Germany.
Carcinogenesis. 1999 Mar;20(3):407-14. doi: 10.1093/carcin/20.3.407.
P-glycoproteins encoded by multidrug resistance type 1 (mdr1) genes mediate ATP-dependent efflux of numerous lipophilic xenobiotics, including several anticancer drugs, from cells. Overexpression of mdr1-type transporters in tumour cells contributes to a multidrug resistance phenotype. Several factors shown to induce mdr1 overexpression (UV irradiation, epidermal growth factor, tumour necrosis factor alpha, doxorubicin) have been associated with the generation of reactive oxygen species (ROS). In the present study, primary rat hepatocyte cultures that exhibit time-dependent overexpression of the mdr1b gene were used as a model system to investigate whether ROS might participate in the regulation of intrinsic mdr1b overexpression. Addition of H2O2 to the culture medium resulted in a significant increase in mdrlb mRNA and P-glycoprotein after 3 days of culture, with maximal (approximately 2-fold) induction being observed with 0.5-1 mM H2O2. Furthermore, H2O2 led to activation of poly(ADP-ribose) polymerase, a nuclear enzyme activated by DNA strand breaks, indicating that ROS reached the nuclear compartment. Thus, extracellularly applied H2O2 elicited intracellular effects. Treatment of rat hepatocytes with the catalase inhibitor 3-amino-1,2,4-triazole (2-4 mM for 72 h or 10 mM for 1 h following the hepatocyte attachment period) also led to an up-regulation of mdrlb mRNA and P-glycoprotein expression. Conversely, antioxidants (1 mM ascorbate, 10 mM mannitol, 2% dimethyl sulphoxide, 10 mM N-acetylcysteine) markedly suppressed intrinsic mdr1b mRNA and P-glycoprotein overexpression. Intracellular steady-state levels of the mdrl substrate rhodamine 123, determined as parameter of mdr1-type transport activity, indicated that mdr1-dependent efflux was increased in hepatocytes pretreated with H2O2 or aminotriazole and decreased in antioxidant-treated cells. The induction of mdr1b mRNA and of functionally active mdr1-type P-glycoproteins by elevation in intracellular ROS levels and the repression of intrinsic mdrlb mRNA and P-glycoprotein overexpression by antioxidant compounds support the conclusion that the expression of the mdr1b P-glycoprotein is regulated in a redox-sensitive manner.
多药耐药1型（mdr1）基因编码的P-糖蛋白介导包括多种抗癌药物在内的众多亲脂性外源性物质从细胞中进行ATP依赖性外排。肿瘤细胞中mdr1型转运蛋白的过表达导致多药耐药表型。已显示几种诱导mdr1过表达的因素（紫外线照射、表皮生长因子、肿瘤坏死因子α、阿霉素）与活性氧（ROS）的产生有关。在本研究中，表现出mdr1b基因时间依赖性过表达的原代大鼠肝细胞培养物被用作模型系统，以研究ROS是否可能参与内在mdr1b过表达的调节。向培养基中添加H2O2导致培养3天后mdrlb mRNA和P-糖蛋白显著增加，在0.5-1 mM H2O2时观察到最大（约2倍）诱导。此外，H2O2导致聚（ADP-核糖）聚合酶激活，这是一种由DNA链断裂激活的核酶，表明ROS到达了核区室。因此，细胞外施加的H2O2引发了细胞内效应。用过氧化氢酶抑制剂3-氨基-1,2,4-三唑处理大鼠肝细胞（在肝细胞贴壁期后2-4 mM处理72小时或10 mM处理1小时）也导致mdrlb mRNA和P-糖蛋白表达上调。相反，抗氧化剂（1 mM抗坏血酸、10 mM甘露醇、2%二甲基亚砜、10 mM N-乙酰半胱氨酸）显著抑制内在mdr1b mRNA和P-糖蛋白过表达。作为mdr1型转运活性参数测定的mdrl底物罗丹明123的细胞内稳态水平表明，在用H2O2或氨基三唑预处理的肝细胞中mdr1依赖性外排增加，而在抗氧化剂处理的细胞中减少。细胞内ROS水平升高诱导mdr1b mRNA和功能活性mdr1型P-糖蛋白，抗氧化剂化合物抑制内在mdrlb mRNA和P-糖蛋白过表达，支持了mdr1b P-糖蛋白的表达以氧化还原敏感方式调节的结论。
