吴茱萸碱增强Eca-109食管鳞癌细胞的放射敏感性

Evodiamine enhances the radiosensitivity of esophageal squamous cell cancer Eca-109 cells

目的探讨吴茱萸碱(Evo)对Eca-109食管鳞癌细胞放射敏感性的影响,探索其可能机制。方法采用MTT法检测(10、20、40、60、80、100、120)μg/m L吴茱萸碱对Eca-109细胞生长的抑制作用,确定最佳处理剂量。实验分为放射组(仅给予0、2、4、6、8 Gy X线照射处理)、联合组(给予80μg/m L吴茱萸碱和0、2、4、6、8 Gy X线照射处理),克隆形成实验检测吴茱萸碱对Eca-109细胞克隆形成能力的影响,流式细胞术检测细胞周期;Western blot法检测Eca-109细胞中Ku70、Ku80、DNA激活蛋白激酶催化亚基(DNA-PKcs)、DNA双链修复蛋白Rad51蛋白水平。结果吴茱萸碱呈剂量依赖性抑制Eca-109细胞的生长,80μg/m L吴茱萸碱的抑制作用最大。克隆形成实验结果显示80μg/m L吴茱萸碱联合放射治疗后,平均致死剂量(D0)、准域剂量(Dq)值显著小于单纯放射时的值,放射增敏比(SER)为1.86±0.06,拟合的生存曲线左移。吴茱萸碱可减少放射诱导的细胞G2/M期阻滞,可显著抑制放射诱导的Ku70、Ku80、DNA-PKcs、Rad51蛋白上调。结论吴茱萸碱具有增加食管鳞癌细胞株Eca-109放射敏感性的作用,可能与抑制细胞周期G2/M期阻滞和降低DNA损伤修复蛋白表达水平有关。

Objective To investigate the effect of evodiamine on the radiosensitivity of esophageal squamous cell cancer Eca-109 cells. Methods Eca-109 cells were treated with various concentrations of evodiamine [ （ 10, 20, 40, 60, 80, 100, 120） μg/mL], and then cell proliferation was examined by MTT assay. After the optimal evodiamine concentration was determined, the cells were divided into radiation group （0, 2, 4, 6, 8 Gy X-ray radiation） and radiation combined with evodiamine group （80 μg/mL evodiamine and 0, 2, 4, 6, 8 Gy X-ray radiation） . The radiosensitivity of Eca-109 cells was detected using colony formation assay. Flow cytometry was used to determine cell cycle of Eca-109 cells. The protein expressions of Kur/O, Ku80, DNA-PKcs and Rad51 were examined by Western blotting. Remits MTT assay showed that evodiamine decreased the proliferation of Eca-109 cells in a concentration-dependent manner. The inhibition reached the maximal level at 80 μg/mL. Compared with radiotherapy alone, the combination of 80 μg/mL evodiamine and radiotherapy improved survival curve and decreased the values of DO and Dq. Sensitizer enhancement ratio was 1. 86±0. 06. Furthermore, cell cycle analysis revealed that evodiamine suppressed radiotherapy-induced the G2/M arrest. Additionally, evodiamine treatment also significantly inhibited radiotherapy-induced increase in Ku70, Ku80, DNA-PKcs and Rad51 expressions. Conclusion Evodiamine enhances radiosensitivity of Eca-109 cells during radiotherapy. The effect may be associated with the inhibition of G2/M arrest and the attenuation of Ku70, KuSO, DNA-PKcs and Rad51 expressions.