IGF-1R抑制剂AG1024对头颈鳞癌FaDu细胞放射敏感性的影响

Radiosensitization of IGF-1R inhibitor AG1024 on head and neck squamous FaDu cells

背景与目的:胰岛素样生长因子1受体(insulin-like growth factor 1 receptor,IGF-1R)在多数头颈鳞癌中高表达。本研究拟探讨IGF-1R抑制剂AG1024对头颈鳞癌FaDu细胞的放射增敏作用。以此探索IGF-1R联合放射治疗头颈鳞癌的疗效及可能机理,为临床寻找有效的以IGF-1R为靶点的肿瘤治疗方法提供实验依据。方法:以头颈鳞癌FaDu细胞为研究对象,克隆形成实验分析细胞放射敏感性,应用流式细胞术(flow cytometry,FCM)检测AG1024对FaDu细胞早期凋亡及周期的影响。进一步应用γ-H2AX形成实验检测DNA双键断裂(double-strand breaks,DSBs)的修复情况;蛋白质印迹法(Western blot)及免疫沉淀法测定IGF-1R下游蛋白激酶B(protein B,Akt)和细胞外调节蛋白激酶1/2(extracellular regulated protein kinases1/2,Erk1/2)的活化水平,动物实验观察对肿瘤生长的影响。结果:AG1024联合放疗使细胞存活曲线的肩区明显变窄,反映放射敏感性指标的D0降低;FCM检测结果表明,AG1024联合放疗可增加FaDu细胞的早期凋亡水平(P<0.05),使细胞的G0/G1期比例增高(P<0.05),S期比例降低(P<0.05);在放射后24 h进一步的γ-H2AX形成实验显示,Ag1024能抑制DSBs的修复(P<0.05);同时,经AGl024处理后,细胞中磷酸化Akt(p-Akt)和磷酸化Erk1/2(p-Erk1/2)的表达水平明显降低;动物实验的结果显示药物联合照射能明显抑制肿瘤的生长。结论:IGF-1R抑制剂AG1024在体内和体外对头颈鳞癌FaDu细胞均有放射增敏作用,其机制可能与改变细胞周期并诱导细胞凋亡、抑制DSBs的修复、下调P13K/Akt及Ras/Raf/MAPK信号通路有关。

Background and purpose： IGF-1R is often overexpressed in head and neck squamous cell. The aim of this study was to investigate the radiosensitivity effect of IGF-1R inhibitor AG1024 on HNSCC FaDu in vitro and in vivo. Methods： Human HNSCC FaDu was used in this study. The radiosensitivity effect was performed by clonogenic assay. The apoptosis and cell cycle were examined by flow cytometry. The repair of DSBs was examined by y-H2AX loci formation. The activation of Akt and Erkl/2 which were downstream of IGF-1R, were detected by Western blot. Results： Clone formation asssy showed that AG1024 enhance radiosensitivity on HNSCC FaDu cell. When treated with AG1024 and radiation, cells were significantly accumulated in the G0/G1 phase （P〈0.05） and decreased in S phase （P〈0.05）, and the apoptosis rates were increased （P〈0.05）. y-H2AX loci formation showed that, AG1024 could inhibit the repair of double-strand breaks （DSBs）. pAkt and pErkl/2 were down-regulated by the treatment with AG1024. In vivo radiosensitization was measured by subcutaneous tumor growth delay. Conclusion： IGF-1R inhibitor AG1024 could change the cell cycle and induce cell apoptosis, delay the repair of DSBs and down- regulate the downstream protein p-Akt （mainly pAktl and pAkt2） and p-MAPK, that might be the immediate cause to enhance the radiosensitizing effects on head and neck squamou FaDu cells in vitro and in vivo.