穿心莲内酯对食管癌细胞的放射增敏作用

Radiosensitivity of Andrographolide on esophageal cancer cell

目的研究穿心莲内酯对食管癌细胞的放射增敏作用。方法培养食管癌ECA 109细胞,通过四甲基偶氮唑盐微量酶反应比色法(MTT法)检测穿心莲内酯对细胞生长的抑制;克隆集落形成实验观察穿心莲内酯的放射增敏作用;通过免疫荧光实验观察促凋亡蛋白Bax的表达情况;流式细胞仪检测细胞的凋亡和周期情况;蛋白质印迹法(Western Blot)检测细胞内核因子κB(NF κB),半胱氨酸天冬氨酸蛋白酶3(caspase3),Bax,B淋巴细胞瘤 2基因(Bcl 2)的表达;磷酸化组蛋白H2AX(γ H2AX)焦点形成检测DNA分钟损伤情况。细胞划痕实验检测细胞增殖、迁移的情况。结果穿心莲内酯抑制食管癌ECA 109细胞的生长,并且有明显的时间和剂量依赖性;通过单击靶模型拟合曲线,可见低浓度穿心莲内酯预处理6 h,相对比单纯照射组可以增加ECA 109细胞的放射敏感性(t=4.59,P=0.02),放射增敏比为1.28;与单纯照射组(26.5±2.2)%相比,加入穿心莲内酯联合X射线照射组中的细胞凋亡率(32.5±0.9)%明显增加(P<0.05);穿心莲内酯作用于食管癌细胞,可以使凋亡相关蛋白caspase3,Bax的表达升高,并且呈明显的量效关系,NF κB表达趋势与Bcl 2一致;相对于单纯照射组,穿心莲内酯与X射线联合作用,能够增加双链DNA断裂(DSB)数目;药物联合放射可以明显影响食管癌细胞的增殖迁移能力。结论穿心莲内酯能够提高食管癌细胞的放射敏感性,机制可能与其增加食管癌细胞的凋亡率和调节细胞内的NF κB的表达有关。

Objective To explore the radiosensitivity of Andrographolide on esophageal cancer.Methods The inhibition effects of Andrographolide were measured by MTT assay.Clonogenic survival assay was used to evaluate the effect of Andrographolide on ra diosensitivity of esophageal cancer cell.Immunofluorescence was employed to examine the expression of Bax.The change of cell apoptosis was assayed by flow cytometry.The expression of NF κB/caspase3/Bax/Bcl 2 was measured by Western Blot.DNA damage was detected byγ H2AX foci counting.Results With a clear dose and time effect,Andrographolide could inhibit the proliferation of esophageal cell line ECA 109.Clonogenic survival assay showed that Andrographolide could markedly enhance the radiosensitivity(t=4.59,P=0.02)and the sensitizing enhancement ratio(SER)was 1.28.Compared with the irradiation group(26.5±2.2)%,the apoptosis rate in the Andrographolide combined with X ray irradiation group(32.5±0.9)%was significantly increased(P<0.05).Andrographolide caused a dose dependent increase in caspase3/Bax protein expressions,in contrast Bcl 2/NF κB;Compared with control,the apoptosis in ECA 109 treated with Andrographolide was increased significantly;Compared with radiation alone group,Andrographolide combined with the X ray can increase the number of DNA double chain break(DSB).Conclusion Androgra pholide can increase the radiosensitivity of esophageal cell line ECA 109,which may be associated with reducing the level of NF κB and inducing apoptosis of esophageal cancer cells.