替拉扎明调节乏氧诱导鼻咽癌细胞HIF-1α和OPN mRNA的表达及其对放射增敏的作用

Regulation of hypoxia-induced mRNA expressions of HIF-1α and OPN and in vitro radiosensitization by tirapazamine in human nasopharyngeal carcinoma HNE-1 and CNE-1 cells

背景与目的:乏氧细胞毒性药物联合肿瘤放化疗是肿瘤治疗的一种重要途径,替拉扎明(tirapazamine,TPZ)是最受瞩目的乏氧细胞毒性药物之一,研究证实替拉扎明联合放疗作用于肿瘤细胞具有协同效应,但在乏氧条件下是否能下调乏氧诱导相关基因未见报道,因此本实验研究人鼻咽癌细胞HNE-1EB+和CNE-1EB-乏氧相关基因乏氧诱导因子1α(HIF-1α)和骨桥蛋白(osteopontin,OPN)的表达差异以及TPZ的放射增敏作用。方法:在常氧和乏氧条件下,用MTT法测定TPZ对HNE-1和CNE-1细胞的生长抑制作用,计算出IC50;用RT-PCR测定TPZIC10作用24h后HNE-1和CNE-1细胞HIF-1α和OPN mRNAs的表达;以及用克隆形成法测定TPZ预作用6h和1～6Gy60Coγ射线照射后细胞的存活率,用单击多靶数学模型拟合剂量-生存曲线,计算出D0、Dq和放射增敏比(SER)。结果:TPZ对HNE-1和CNE-1细胞的IC50在常氧条件下分别为34.81μmol/L、35.02μmol/L,在乏氧条件下分别为30.20μmol/L和28.48μmol/L;TPZ能明显下调HNE-1细胞乏氧后HIF-1α和OPN的表达,对CNE-1HIF-1α和OPN的表达无明显作用;放射剂量-生存曲线符合单击多靶数学模型特征;HNE-1aero和HNE-1hypox的D0和Dq分别为0.89Gy、0.28Gy,1.47Gy和0.44Gy;HNE-1hypox/TPZ的D0和Dq分别为0.66Gy和0.21Gy;CNE-1aero和CNE-1hypox的D0和Dq分别为0.72Gy、0.68Gy,0.95Gy和0.56Gy;CNE-1hypox/TPZ的D0和Dq分别为0.85Gy和0.79Gy。结论:TPZ对HNE-1EB+细胞明显的放射增敏作用可能与其下调乏氧诱导的HIF-1α和OPN表达有关,以及具有乏氧修饰剂的作用。

Background and Objective： Combined hypoxic cytotoxic drugs and chemoradiotherapy is an important mean of oncotherapy, and tirapazamine （TPZ） is one of the most remarkable drugs. It has been shown that TPZ has a synergistic effect with radiotherapy on tumor cells, but whether TPZ would down-regulate the expression of the hypoxia-induced genes has not been reported. This study was to investigate the hypoxiainduced expression of hypoxia inducible factor-1α（HIF-1α） and osteopontin （OPN） mRNA in human nasopharyngeal carcinoma HNE-1 and CNE-1 cells and the radiosensitization of TPZ, a hypoxia-specific drug, on HNE-1 and CNE-1 cells in vitro. Methods. The IC50 values of TPZ for HNE-1 and CNE-1 cells were measured using MTT assay, and the expression of HIF-1α and OPN mRNA in HNE-1 and CNE-1 cells was determined using RT-PCR under aerobic and hypoxic conditions, respectively. The survival rates of HNE-1 and CNE-1 cells treated with or without TPZ at IC10 in the presence or absence of oxygen for 6 h were determined using colony forming assay following exposure to 1-6 Gy of ^60Co radiation, and the dose-survival curves were plotted and the values of Do, Dq and SER were calculated as a singlehit multitarget model. Results. The IC50 values of TPZ were 34.81 μmol/L and 35.02 μmol/L in HNE-1 and CNE-1 cells under aerobic condition, and 30.20 μmol/L and 28.48 μmol/L under hypoxic condition. The expressions of HIF-1α and OPN mRNA were reduced by TPZ in HNE-1 cells, but not in CNE-1 cells under hypoxic condition. The values of Do and Dq in HNE-1 and CNE-1 cells following irradiation under aerobic and hypoxic conditions were 0.89 Gy, 0.28 Gy and 0.72 Gy, 0.68 Gy, and 1.47 Gy, 0.44 Gy and 0.95 Gy, 0.56 Gy, respectively. The values of Do and Dq for HNE-1 and CNE-1 cells treated with TPZ Under hypoxic condition following irradiation were 0.66 Gy, 0.21 Gy and 0.85 Gy, 0.79 Gy, respectively. Conclusion： TPZ would downregulate hypoxia-induced expression of HIF-1α and OPN mRNA of HNE-1 cells and radiosensitize the HNE-1 cells but not CNE-1 cells, and act as a hypoxia modifier.