经典Wnt通路与食管癌细胞放射抵抗相关关系探讨

Correlation between classic Wnt signaling pathway and radioresistance of esophageal cancer cells

目的明确经典Wnt通路在食管癌细胞放射抵抗中的作用,探讨经典Wnt通路介导食管癌细胞放射抵抗的机制,为临床上增强食管癌放射敏感性提供重要的分子靶点。方法应用克隆形成实验检测人食管癌细胞EC9706、ECA109、KYSE70、KYSE150的放射敏感性。通过蛋白质印迹和RT-PCR检测照射后经典Wnt通路的活化情况。通过添加经典Wnt通路激活剂(AZD2858)和抑制剂(XAV-939)综合评价经典Wnt通路对食管癌细胞放射敏感性的影响。通过细胞免疫荧光技术检测照射后细胞DNA双链断裂(DSB)的产生、修复和DNA双链断裂修复蛋白焦点的形成。结果4种食管癌细胞的放射敏感性由高到低分别为EC9706、ECA109、KYSE70、KYSE150细胞。KYSE150细胞照射后细胞核内β联蛋白增加,c-Myc基因转录上调(均P<0.05);而EC9706细胞照射后细胞核内β联蛋白、c-Myc基因转录与照射前相近(均P>0.05)。EC9706细胞经AZD2858处理后放射抗性增加(P<0.05),而KYSE150细胞经XAV-939处理后放射抗性降低(P<0.05)。AZD2858使EC9706细胞DNA双链断裂修复加快(P<0.05),而XAV-939使KYSE150细胞DNA双链断裂修复减慢(P<0.05);XAV-939通过抑制同源重组修复相关蛋白(BRCA1和RAD51),而不是非同源末端连接修复相关蛋白(Ku80和XRCC4)降低DNA双链断裂修复能力。结论经典Wnt通路通过调控照射后DNA双链断裂的同源重组修复参与对食管癌细胞放射敏感性的调控,抑制经典Wnt通路可以克服食管癌细胞的放射抵抗,增强放射对食管癌细胞的杀伤作用。

Objective To clarify the role of classic Wnt signaling pathway in the radioresistance of esophageal cancer cells(ECC),and investigate the underlying mechanism,aiming to identify critical molecular targets for clinically enhancing the radiosensitivity of esophageal cancer.Methods The radiosensitivity of four types of ECCs(EC9706,ECA109,KYSE70 and KYSE150)were assessed by colony formation assay.Western blot and RT-PCR were used to detect the activation of classical Wnt signaling pathway after irradiation.Classic Wnt signaling pathway activator(AZD2858)and inhibitor(XAV-939)were utilized to comprehensively evaluate the effect of classic Wnt signaling pathway on the radiosensitivity of ECCs.Cellular immunofluorescence staining was performed to detect the production and repair of DNA double-strand breaks(DSB),as well as the foci formation of DSB repair proteins after irradiation.Results The results of colony formation assay showed that the radiosensitivity of four types of ECCs from high to low was EC9706,ECA109,KYSE70 and KYSE150.In KYSE150,a radioresistant cell type,the level of nuclear β-catenin and the transcription of c-Myc gene were significantly increased after irradiation(both P<0.05).However,in EC9706,a radiosensitive cell type,the level of nuclearβ-catenin and c-Myc gene transcription were not affected by irradiation(both P>0.05).Moreover,EC9706 cells showed enhanced radioresistance in the presence of AZD2858(P<0.05),whereas XAV-939 treatment decreased the radioresistance in KYSE150 cells(P<0.05).AZD2858 accelerated the DSB repair in EC9706 cells(P<0.05),whereas XAV-939 delayed the DSB repair in KYSE150 cells(P<0.05).Furthermore,the results of immunofluorescence staining showed that XAV-939 reduced the DSB repair capacity by inhibiting homologous recombination repair-related proteins(BRCA1 and RAD51)rather than non-homologous end junction repair-related proteins(Ku80 and XRCC4).Conclusions The classic Wnt signaling pathway participates in the regulation of radiosensitivity in ECCs by regulating the homologous recombination repair of DSB after irradiation.Inhibition of the classic Wnt signaling pathway can counteract the radioresistance of ECCs and enhance the killing effect of irradiation on ECCs.