TGF-β1抑制剂增加H460肺癌细胞的放射敏感性

TGF-β1 inhibitors increasing the radiosensitivity of H460 lung cancer cells

探索一种TGF-β1受体激酶的选择性抑制剂SB431542对H460非小细胞肺癌细胞的放射增敏作用及对其DNA损伤应答体系的干扰。采用克隆形成实验检测SB431542对H460细胞放射敏感性的影响;用移植瘤实验验证SB431542在体内对H460细胞的放射增敏作用;采用流式细胞术检测细胞周期分布和细胞凋亡;用免疫荧光检测53BP1 foci和磷酸化的DNA-PKcs foci。结果发现,SB431542可增加H460的放射敏感性。照射前用SB431542预处理H460细胞虽然快速启动了细胞的DNA损伤应答反应,但却抑制了DNA双链断裂的非同源末端链接修复,因此,导致细胞残留更多未修复的DNA双链断裂,从而产生更严重的细胞周期阻滞。研究还发现,这种增敏作用与细胞凋亡无关。体内移植瘤实验表明,与单独照射组相比,连续3次用SB431542联合5 Gy的X-射线处理明显在处理后第5～12 d之间降低了肿瘤的生长。这些结果显示,在照射前用SB431542抑制肿瘤细胞的TGF-β1通路,能降低其DNA损伤修复能力,改变细胞周期分布,降低细胞克隆形成能力,延缓肿瘤的生长,因此用SB431542抑制TGF-β1通路可作为一些类型肺癌病人放疗的有效辅助手段。

The purpose of this study was to investigate the radiosensitizing effect of SB431542, a selective inhibitor of TGF-β1 receptor, on H460 non-small-cell lung cancer ceils and relative interfering effect on DNA damage response （DDR）. Clonogenic assay was used to measure the radiosensitizing effects of SB431542 on H460 cells; xenograft mouse model was used to study the radiosensitization in vivo; flow cytometry was used to determine cell cycle distribution and apoptosis; immunofluorescence microscopy was used to assess 53BP1 foci and pospho-DNA-PKcs foci. The results showed that SB431542 could increase the radiosensitivitv of H460 cells.Pretreatment of H460 cells with SB431542 prior to irradiation initiated fast DDR but inhibited DNA repair via non-homologous end joining pathway and thus resulting in more unrepaired DNA double strand breaks, which caused more severe cell cycle delay. However, apoptosis did not contribute to the radiosensitization. The data from xenograft model showed that compared with irradiation alone, the combination of SB431542 and 5 Gy of X-rays on three consecutive days significantly delayed the tumor growth from 5-12 d post treatment. All the above indicate that inhibition of TGF-β1 pathway using SB431542 prior to irradiation can attenuate DNA damage repair and disturb cell cycle distribution, leading to decreased clonogenic cell survival and tumor growth delay, which can be an effective adjunct in radiotherapy for certain lung cancer subtypes.