论文部分内容阅读
目的探讨2-甲氧基雌二醇(2-methoxyestradiol,2-Me OE2)对人鼻咽癌CNE-2干细胞(nasopharyngeal cancer stem cells,NPCSCs)增殖、迁移及放疗敏感性的影响及其可能机制。方法收集本课题组前期富集并已鉴定的鼻咽癌CNE-2干细胞,Western blot检测亲本CNE-2细胞及NPCSCs乏氧诱导因子-1α(HIF-1α)和核转录因子(NF-κB p65)的蛋白表达,克隆形成实验检测两组细胞的放射敏感性。按2-Me OE2浓度分为3组,分别用0、4、8μmol/L 2-Me OE2处理鼻咽癌干细胞,倒置显微镜观察细胞形态变化,CCK-8及Transwell检测细胞增殖及迁移能力,再联合X射线(0、2、4、8 Gy)照射,克隆形成实验检测3组细胞放疗敏感性;同时用4μmol/L 2-Me OE2处理鼻咽癌干细胞后不同时间(0、24、48 h)提取蛋白,Western blot检测不同浓度2-Me OE2处理以及4μmol/L 2-Me OE2处理后0、24、48 h提取的干细胞HIF-1α和NF-κB p65蛋白表达。结果相对于亲本CNE-2细胞,干细胞HIF-1α和NF-κB p65蛋白表达明显增高(P均<0.01),X射线辐射后干细胞各放射学参数D0、Dq、N及SF2均高于亲本CNE-2细胞。0、4、8μmol/L 2-Me OE2处理干细胞后,随浓度增加,致密球状生长的干细胞微球体逐渐变得离散,干细胞增殖活力降低(P<0.01);迁移能力降低(P<0.01);放疗敏感性增高,4μmol/L2-Me OE2组放射增敏比(sensitivity enhancement ratio,SER)为1.19,8μmol/L 2-Me OE2组SER为1.39;HIF-1α、NF-κB p65蛋白表达降低(P<0.01),呈浓度依赖性。2-Me OE2(4μmol/L)处理干细胞后随时间延长HIF-1α、NF-κB p65蛋白表达明显降低(P<0.01),呈时间依赖性。结论相对于亲本CNE-2细胞,干细胞高表达HIF-1α和NF-κB p65,并且对放疗具有较低的敏感性。2-Me OE2能有效抑制鼻咽癌CNE-2干细胞增殖、迁移并增加其放疗敏感性,其机制可能为2-Me OE2抑制HIF-1α和NF-κB p65蛋白表达。
Objective To investigate the effects of 2-methoxyestradiol (2-Me OE2) on the proliferation, migration and radiosensitivity of human nasopharyngeal cancer stem cells (NPCSCs) and their possible mechanisms . Methods Nasopharyngeal carcinoma CNE-2 stem cells enriched and identified in our group were collected. CNE-2 cells and hypoxia inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF- ) Protein expression, clone formation assay to test the radiosensitivity of two groups of cells. The cells were divided into 3 groups according to 2-Me OE2 concentration. Nasopharyngeal carcinoma stem cells were treated with 0, 4, and 8μmol / L 2-Me OE2 respectively. The morphological changes of the cells were observed by inverted microscope. Cell proliferation and migration were detected by CCK-8 and Transwell The radiosensitivity of the three groups of cells was detected by X-ray irradiation (0, 2, 4, 8 Gy) and clonogenic assay. The cells were treated with 4μmol / L 2-Me OE2 for different times Western blot was used to detect the expression of HIF-1αand NF-κB p65 protein in stem cells extracted with different concentrations of 2-Me OE2 and 0, 24 and 48 h after treated with 4μmol / L 2-Me OE2. Results Compared with parental CNE-2 cells, the expression of HIF-1α and NF-κB p65 protein in stem cells were significantly increased (all P <0.01). The radiological parameters D0, Dq, N and SF2 of stem cells after X-ray irradiation were higher than those of CNE -2 cells. After treated with 0, 4 and 8μmol / L 2-Me OE2, the stem cells microspheres gradually became discrete with increasing concentration, the proliferation activity of stem cells decreased (P <0.01) and migration ability decreased (P <0.01) The sensitivity of radiotherapy increased, the SER of 1. 4μmol / L2-Me OE2 group was 1.19, SER of 1.39 in 8μmol / L 2-Me OE2 group, and the expression of HIF-1α and NF- P <0.01), in a concentration-dependent manner. After treated with 2-Me OE2 (4μmol / L), the expression of NF-κB p65 protein decreased significantly (P <0.01) in a time-dependent manner. Conclusions Stem cells overexpress HIF-1α and NF-κB p65 relative to their parental CNE-2 cells and are less sensitive to radiotherapy. 2-Me OE2 can effectively inhibit CNE-2 stem cell proliferation, migration and radiosensitivity, and its mechanism may be that 2-Me OE2 inhibits the expression of HIF-1α and NF-κB p65 protein.