香兰素衍生物BVAN08对人脑胶质瘤细胞的放射增敏作用及机制研究

Radiosensitization and relative mechanisms of vanillin derivative BVAN08 on human glioma U-251 cells

目的 研究香兰素衍生物6-溴异香兰素（BVAN08）对人脑胶质瘤细胞的增殖影响、放射增敏作用和相关机制,为开发新的放射增敏抗癌药物提供实验依据.方法 采用MTT法、克隆形成率法检测BVAN08对U-251细胞增殖活性和60Co γ射线敏感性的影响（照射剂量率为2.3 Gy/min）;光学显微镜观察BVAN08作用后细胞形态学变化;透射电镜检测细胞自吞噬死亡;流式细胞术检测细胞周期和凋亡的变化;Western blot检测DNA修复蛋白DNA-PKcs的表达变化.结果 6-溴异香兰素BVAN08对U-251细胞增殖有显著抑制作用（t=1.83～3.07,P＜0.05）,在10～100 μmol/L浓度范围内呈剂量和作用时间依赖性,药物作用48和72 h的IC50分别为55.3和52.7 μmol/L.60 μmol/L BVAN08作用4 h后,U-251细胞产生明显的G2/M期阻滞,12 h达到峰值,G2/M阻滞达到63.3%,并开始同时出现细胞凋亡和自吞噬死亡.BVAN08对U-251细胞有明显的放射增敏作用,而且在照射前12 h给药的增敏效果最好,20 μmol/L浓度对2 Gy照射杀伤U-251细胞的增强比为3.14.Western blot检测表明BVAN08能显著抑制DNA-PKcs的表达.结论 香兰素衍生物BVAN08具有明显抑制人脑胶质瘤U-251细胞增殖和辐射增敏作用、并同时诱发凋亡和自吞噬死亡.BVAN08对U-251细胞的辐射增敏作用可能与G2/M期阻滞和DNA双链断裂修复关键蛋白DNA-PKcs表达的抑制敏感性有关.

Objective To provide more convincing evidences and experimental data for exploring vanillin derivative BVAN08,6-bromine-5-hydroxy-4-methoxy-benzaldehyde,as a new anticancer drug,and to investigate the effect on the growth,radiosensitization of human glioma cell line U-251 and the relative mechanism.Methods The effect of BVAN08 on cell proliferation of U-251 and radiosensitivity to 60Co γ-rays （irradiation dose rate 2.3 Gy/min） were analyzed with MTT and colony-forming ability assay.Change in cellular morphology was observed by using light microscope.Change in cell cycle and apoptosis was detected with flow cytometry.The autophagy was observed by using TEM （irradiation dose rate is transmission electron microscope）.DNA-PKcs protein level was detected through Western blot analysis.Results BVAN08 exhibited a dose- and time-dependent inhibition on the proliferation of U-251 cells during the concentration range of 10-100 mol/L （t = 1.83-3.07,P ＜ 0.05）.IC50 at 48 h and 72 h after administration with BVAN08 were 55.3 and 52.7 mol/L,respectively.Obvious G2/M arrest was induced in U-251 cells after 4 h administration with BVAN08,and reached peak at 12 h.The G2/M population reached 63.3% in U-251 cells after 12 h administration of 60 μmol/L BVAN08 and kept increasing with the time,while both apoptosis and autophagic cell death were induced.The most effective radiosensitization time for BVAN08 treatment was 12 h before irradiation.The enhancement ratio of radiosensitivity was 3.14 for 20 μmol/L of BVAN08 12 h before 2 Gy irradiation.Conclusions BVAN08 can nduce apoptosis as well as autophygic cell death of U-251 cells,and sensitize U-251 cells.The mechanism of its radiosensitizing effect might be associated with the induction of G2/M arrest and inhibition of DNA-PKcs expression.BVAN08 seemed to be a romising radiosensitizing anticancer drug.