硼替佐米对喉鳞状细胞癌细胞的放射增敏作用的体内外研究

目的 探讨蛋白酶抑制剂硼替佐米对喉鳞状细胞癌(简称鳞癌)Hep-2细胞的放射增敏作用及其机制.方法 体外培养的Hep-2细胞在100 nmol/L的硼替佐米作用2 h后,经过0、2、46、8及10 Gy照射后,检测其放射敏感性的变化,并通过建立的裸鼠移植瘤模型验证其体内放射增敏作用.利用Trans AMTM NF-κB P65活性测定试剂盒检测硼替佐米对Hep-2细胞辐射诱导NF-κB活化的影响;用流式细胞仪分析细胞周期及照射前后的细胞凋亡;Hoechst 33342荧光染色法观察细胞凋亡状况.结果 克隆形成实验显示硼替佐米可增加Hep-2细胞的放射敏感性34%.裸鼠移植瘤模型的肿瘤生长延迟放射增敏值为1.46.经2 Gy和8 Gy照射后2 h可诱导NF-κB活化,且存在剂量相关性(r=0.989,P<0.05).硼替佐米在0、2、8 Gy不同的照射剂量均能降低Hep-2细胞NF-κB的活性(t值分别为20.02、14.20、17.46,P<0.01);经硼替佐米干预的细胞有明显的G2-M期阻滞作用(t=22.31,P=0.000),并在照射后明显的细胞凋亡率增加(P值均<0.01).Hoecht 33342染色可见细胞核均匀染色,凋亡细胞的核凝集,呈强蓝色荧光,细胞核碎裂呈花瓣状.硼替佐米照射组凋亡比例高于单纯照射组.结论 硼替佐米通过可影响Hep-2细胞的细胞周期分布、诱导细胞凋亡及抑制NF-κB活化的途径,从而增加其对放射的敏感性.

Proteasome inhibitor bortezomib sensitizes Hep-2 human laryngeal squamous cell carcinoma cells to ionizing radiation in vitro and in vivo

OBJECTIVE: To determine if proteasome inhibitior bortezomib leads to enhanced radiation sensitivity of Hep-2 human laryngeal cancer cells and the relative mechanisms. METHODS: Hep-2 cells with or without bortezomib were irradiated at 0, 2, 4, 6, 8, and 10 Gy. Growth and clonogenic survival data were obtained to assess effects of treatment on radiosensitization. In vitro results were tested in vivo using a Hep-2 xenograft model. Nuclear factor-kappaB (NF-kappaB) activation was determined by Trans AM NF-kappaB P65 kit. The distribution of cell cycle and apoptosis were evaluated by flow cytometry. Morphological evidence of apoptotic cells were observed with Hochest 33342. RESULTS: It decreased cell growth and clonogenic survival. A 34% increase in radiosensitivity was observed for cells treated with bortezomib and radiation. Enhancement factor (EF) was 1.46 in Hep-2 xenografts receiving radiation and bortezomib. NF-kappaB activation was induced by radiation and inhibited by pretreatment with bortezomib, and was in a dose-dependent manner (r = 0.989, P < 0.05). Hep-2 cells treated with 100 nmol/L Bortezomib were arrested at G2-M phase (t = 22.31, P = 0.000) and resulted in all increased apoptosis with and without irradiation (P < 0.01). Morphological evidence of apoptotic cells could be distinguished under the fluorescence microscope after staining with Hochest 33342. Many nuclear fragments were observed in Hep-2 cells with bortezomib. CONCLUSION: Bortezomib could enhanced the radiosensitivity of Hep-2 laryngeal cancer cells by regulation of the distribution of cell cycle.