索拉非尼降低放射对肝癌细胞的作用

Sorafenib Decreases the Anti-tumor Effect of Radiation in Hepatocelluar Carcinoma in vitro

目的:观察放射联合索拉非尼(Sorafenib)对人肝癌细胞的作用。方法:人肝癌细胞株SMMC-7721和BEL-7402,以单纯放射(IR)和放射前联合索拉非尼(IR+S)处理后,使用克隆形成实验检测照射后细胞克隆形成率并计算放射增敏比;细胞增殖抑制实验检测照射后细胞增殖抑制率;流式细胞仪检测放射后细胞凋亡率与细胞周期分布;免疫荧光染色观察照射后DNA损伤阳性细胞比例。结果:索拉非尼增加肝癌细胞放射后克隆形成,放射增敏在SMMC-7721和BEL-7402细胞中比分别为0.78和0.88。放射2天后,IR+S处理后肝癌细胞的细胞增殖抑制率与IR处理靠近。加入索拉非尼后出现:1)放射后24h肝癌细胞的细胞凋亡比增加(IR+S vs IR在SMMC-7721细胞中为18.3%±2.0% vs 6.1%±1.0%,在BEL-7402细胞中为17.0%±2.4% vs 8.2%±2.1%,P<0.05),但对放射后48h细胞凋亡比例无影响;2)延迟和延长放射后G_2/M期细胞比例升高;3)不影响放射后DNA受损阳性细胞比例,但是减少放射后6h DNA受损阳性细胞残留(DNA受损阳性细胞比例IR+S vs IR在SMMC-7721细胞中为23.8%±2.9% vs 59.9%±2.4%,在BEL-7402细胞中为25.0%±3.0% vs 46.4%±3.8%,P<0.001)。结论:放射前联合索拉非尼降低了放射对肝癌细胞的作用。进一步体内实验需要考虑更合适的联合方式。

Objective： To observe the effect of radiation combined with Somfenib on hepatocellular carcinoma cells. Methods： Human hepatocellular carcinoma cell （ HCC ） lines SMMC-7721 and BEL-7402 were studied. One set was treated with radiation alone （ 1R group ） and the other set was treated with Somfenib followed by radiation （IR＋S group）. A colony-forming assay was performed with the cell fractiom that survived radiation with and without Somfenib and the sensitivity enhancement ratio was calculated. The MTS assay was used to compare proliferation in the 2 groups after treatment. Flow cytometry was used to quantify apoptotic cells and cell cycle distribution. The tumor cells were irradiated and their DNA double stranded breaks （ DSbs ） were detected through γ-H2AX focus by immunofluorescence. Results： The IR＋S treated HCC cells formed more colonies. The sensitivity enhancement ratio （SERSF2） was 0.78 in the IR＋S treated SMMC-7721 cells and 0.88 in the IR＋S treated BEL-7402 cells. Curve analysis with data from the MTS assay showed that the inhibition in the IR＋S group was similar to that in the IR group at 2 days after radiation. Somfenib increased the apoptotie ceils at 24 hours after radiation. The proportion of apoptotic cells in the 1R＋S set was 18.3%±2.0%, while the proportion in the IR set was 6.1%±1.0% in SMMC-7721 cells. The proportion of apoptotic cells in the IR＋S set was 17.0%±2.4%, while the proportion in the IR set was 8.2%±2.1% in BEL-7402 cells （ P〈 0.05.）. Sorafenib had no effect on the proportion of apoptotic cells at 48 h ours after radiation. Sorafenib delayed and prolonged the arrest of cells in GgM. The percentage of cells positive for DNA damage was similar in the two groups at 30 hours after radiation but was higher in the IR group than in the IR＋S group at 6 hours after radiation. The proportion ofcells with DNA damage in the IR＋S group was 23.8%±2.9%, while the proportion in the IR group was 59.9%±2.4% in SMMC-7721 cells. The proportion of cells with DNA damage in the IR＋S set was 25.0±3.0%, while the proportion in the IR set was 46.4%±3.8% in the BEL-7402 cells （ P〈 0.001）. Conclusion： Pre-radiation Sorafenib decreases the effect of radiation on HCC cells. Further studiesin vivo are required to consider more suitable combinations to increase the radiation effect on HCC cells. Keywords Hepatocellular carcinoma; Radiation; Sorafenib