木犀草素对人胃癌细胞DNA双链断裂及同源重组修复的影响

Effects of luteolin on DNA double-strand breaks and homologous recombination repair in human gastric cancer cells

目的探讨木犀草素对人胃癌细胞DNA双链断裂(DNA double-strand breaks,DSBs)及同源重组(homologous recombination,HR)修复的影响。方法采用流式细胞仪检测作用木犀草素后各组细胞内ROS水平变化及GFP阳性率;彗星实验检测木犀草素对DSBs的影响;Western blotting检测DNA损伤标志性蛋白γH2AX和HR修复蛋白Rad51的表达;免疫荧光检测DNA损伤修复相关蛋白表达的募集情况。结果木犀草素以剂量依赖性方式增加胃癌细胞内ROS含量;用I-Scel质粒转染DR-GFP后木犀草素处理组GFP阳性细胞比例明显少于未加木犀草素组;但彗星实验表明,木犀草素处理后人胃癌AGS细胞后增加彗星尾炬;经木犀草素处理后,胃癌细胞中DNA的γH2AX上调,修复关键蛋白Rad51的表达下调;免疫荧光结果表明,在木犀草素处理人胃癌AGS细胞后HR修复蛋白Rad51在DNA损伤位点的募集减少。结论木犀草素能够促进人胃癌细胞DSBs,并抑制其HR修复。

Objective To investigate the effects of luteolin on DNA double-strand breaks(DSBs)and homologous recombination(HR)repair in human gastric cancer cells.Methods The changes of ROS level and the number of positive GFP cells in each group after luteolin treatment were detected by flow cytometry.Comet assay was used to detect the effect of luteolin on DSBs in cells.Western blotting was used to detect the expression of DNA damage marker proteinγH2AX and HR key protein Rad51.The recruitment of DNA damage marker protein and HR repair protein were detected by immunofluorescence.Results With luteolin treatment,the level of DCF-reactive ROS was significantly increased in human gastric cancer cells in a dose-dependent manner.The number of positive GFP cells in luteolin treated DR-GFP cells was lower than that in the group without luteotin.Comet assay showed that comet tailing increased after luteolin treatment in human gastric cancer AGS cells.In the gastric cancer cells(AGS,NCI-N87,MKN45),DNA damage markerγH2AX was increased upon luteolin treatment,whereas the key HR repair protein Rad51 was suppressed in luteolin treatement groups.Immunofluorescence results showed that after luteolin treating AGS cells,the recruitment level of the key HR repair protein Rad51 was decreased.Conclusion Luteolin induces DSBs and impairs the HR repair in human gastric cancer cells.