索拉非尼降低放射对肝癌细胞的作用

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24 h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48 h细胞凋亡比例无影响；2）延迟和延长放射后G2/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6 h 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。

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

Sorafenib Decreases the Anti-tumor Effect of Radiation in Hepatocelluar Carcinoma in vitroQiaoqiao LI , Mengzhong LIU, LiWANG, Jinqing LICorrespondence to: Mengzhong LIU, E-mail: liumz@sysucc.org.cnDepartment of Radiation Oncology, Cancer Center of Sun Yat-sen University, Guangzhou 510060, ChinaAbstract Objective: To observe the effect of radiation combined with Sorafenib on hepatocellular carcinoma cells. Methods: Humanhepatocellular carcinoma cell ( HCC ) lines SMMC-7721 and BEL-7402 were studied. One set was treated with radiation alone ( IR group )and the other set was treated with Sorafenib followed by radiation (IR+S group). Acolony-forming assay was performed with the cell fractionsthat survived radiation with and without Sorafenib and the sensitivity enhancement ratio was calculated. The MTS assay was used to compareproliferation in the 2 groups after treatment. Flow cytometry was used to quantify apoptotic cells and cell cycle distribution. The tumor cellswere irradiated and their DNA double stranded breaks ( DSbs ) were detected through γ-H2AX focus by immunofluorescence. Results: TheIR+S treated HCC cells formed more colonies. The sensitivity enhancement ratio (SERSF2) was 0.78 in the IR+S treated SMMC-7721 cellsand 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 groupwas similar to that in the IR group at 2 days after radiation. Sorafenib increased the apoptotic cells at 24 hours after radiation. The proportionof apoptotic cells in the IR+S set was 18.3%±2.0%, while the proportion in the IR set was 6.1%±1.0% in SMMC-7721 cells. The proportionof 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 cellsin G2/M. The percentage of cells positive for DNA damage was similar in the two groups at 30 hours after radiation but was higher in theIR group than in the IR+S group at 6 hours after radiation. The proportion of cells 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 DNAdamage in the IR+S set was25.0±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 Sorafenibdecreases the effect of radiation on HCC cells. Further studies in vivo are required to consider more suitable combinations to increase theradiation effect on HCC cells.Keywords Hepatocellular carcinoma; Radiation; Sorafenib