Teaching & Research Section of Nuclear Medicine, An-hui Medical University, Hefei, China.
J Exp Clin Cancer Res. 2011 May 19;30(1):61. doi: 10.1186/1756-9966-30-61.
Hypoxia inducible factor-1 (HIF-1) has been considered as a critical transcriptional factor in response to hypoxia. It can increase P-glycoprotein (P-Gp) thus generating the resistant effect to chemotherapy. At present, the mechanism regulating HIF-1α is still not fully clear in hypoxic tumor cells. Intracellular redox status is closely correlated with hypoxic micro-environment, so we investigate whether alterations in the cellular redox status lead to the changes of HIF-1α expression. HepG2 cells were exposed to Buthionine sulphoximine (BSO) for 12 h prior to hypoxia treatment. The level of HIF-1α expression was measured by Western blot and immunocytochemistry assays. Reduce glutathione (GSH) concentrations in hypoxic cells were determined using glutathione reductase/5,5'-dithiobis-(2-nitrob-enzoic acid) (DTNB) recycling assay. To further confirm the effect of intracellular redox status on HIF-1α expression, N-acetylcysteine (NAC) was added to culture cells for 8 h before the hypoxia treatment. The levels of multidrug resistance gene-1 (MDR-1) and erythropoietin (EPO) mRNA targeted by HIF-1α in hypoxic cells were further determined with RT-PCR, and then the expression of P-Gp protein was observed by Western blotting. The results showed that BSO pretreatment down-regulated HIF-1α and the effect was concentration-dependent, on the other hand, the increases of intracellular GSH contents by NAC could partly elevate the levels of HIF-1α expression. The levels of P-Gp (MDR-1) and EPO were concomitant with the trend of HIF-1α expression. Therefore, our data indicate that the changes of redox status in hypoxic cells may regulate HIF-1α expression and provide valuable information on tumor chemotherapy.
缺氧诱导因子-1（HIF-1）被认为是应对缺氧的关键转录因子。它可以增加 P 糖蛋白（P-Gp），从而产生对化疗的耐药性。目前，缺氧肿瘤细胞中调节 HIF-1α 的机制尚不完全清楚。细胞内氧化还原状态与缺氧微环境密切相关，因此我们研究细胞内氧化还原状态的变化是否导致 HIF-1α 表达的变化。在缺氧处理前，HepG2 细胞用 Buthionine sulphoximine（BSO）孵育 12 小时。通过 Western blot 和免疫细胞化学检测来测量 HIF-1α 的表达水平。使用谷胱甘肽还原酶/5,5'-二硫代双（2-硝基苯甲酸）（DTNB）循环测定法测定缺氧细胞中还原型谷胱甘肽（GSH）的浓度。为了进一步证实细胞内氧化还原状态对 HIF-1α 表达的影响，在缺氧处理前 8 小时向培养细胞中加入 N-乙酰半胱氨酸（NAC）。通过 RT-PCR 进一步测定缺氧细胞中 HIF-1α 靶向的多药耐药基因-1（MDR-1）和促红细胞生成素（EPO）mRNA 的水平，然后通过 Western blot 观察 P-Gp 蛋白的表达。结果表明，BSO 预处理下调了 HIF-1α，且呈浓度依赖性，另一方面，NAC 增加细胞内 GSH 含量可部分提高 HIF-1α 的表达水平。P-Gp（MDR-1）和 EPO 的水平与 HIF-1α 表达的趋势一致。因此，我们的数据表明，缺氧细胞中氧化还原状态的变化可能调节 HIF-1α 的表达，并为肿瘤化疗提供有价值的信息。
