雷氏大疣蛛蛛毒对人肝癌BEL-7402细胞增殖的抑制作用及其机制的研究

Inhibitory Effect of the Venom of Spider Macrothele raveni on Proliferation of Human Hepatocellular Carcinoma Cell Line BEL-7402 and Its Mechanism

背景与目的蜘蛛毒素抗肿瘤的研究迄今还是未知领域。本研究探讨雷氏大疣蛛(Macrotheleraveni)蛛毒对BEL-7402细胞增殖的抑制作用,进一步探讨其作用机制。方法采用MTT法测定了雷氏大疣蛛蛛毒(10、20、40、80滋g/ml)对BEL-7402细胞增殖作用的影响;采用[3H]-TdR掺入法检测蛛毒加入前后BEL-7402细胞DNA的变化;利用流式细胞光度术(FCM)探讨雷氏大疣蛛蛛毒对BEL-7402细胞凋亡率和细胞周期的影响;利用Westernblot方法进一步检测雷氏大疣蛛蛛毒对细胞周期相关基因c-myc的变化。结果雷氏大疣蛛蛛毒对BEL-7402细胞增殖有较强的抑制作用(P<0.05),IC50为20滋g/ml,时效和量效关系良好。雷氏大疣蛛蛛毒可以抑制BEL-7402细胞DNA的合成。流式细胞仪检测发现经过雷氏大疣蛛蛛毒作用下的BEL-7402细胞凋亡率增加,细胞周期阻滞在G0/G1期。Westernblot方法进一步检测表明雷氏大疣蛛蛛毒作用于BEL-7402细胞72h后,c-myc基因表达减弱。结论雷氏大疣蛛蛛毒就可以抑制人肝癌BEL-7402细胞的增殖和DNA的合成。其药理作用机制可能是除了诱导凋亡外,主要是使细胞周期相关基因c-myc表达减弱,导致细胞周期的变化。

BACKGROUND & OBJECTIVE: Antitumor effect of spider venom has not been reported yet. This study was to explore inhibitory effect of the venom of spider Macrothele raveni on proliferation of human hepatocellular carcinoma cell line BEL-7402 and its molecular mechanism. METHODS: When treated with the venom of spider Macrothele raveni, proliferation of BEL-7402 cells was examined by MTT assay; DNA synthesis of BEL-7402 cells was tested by 3H-TdR incorporation; cell apoptosis and cell cycle were detected by flow cytometry; expression of C-myc was detected by Western blot. RESULTS: The spider venom inhibited proliferation of BEL-7402 cells in time-dependent and concentration-dependent manners with 50% inhibitory concentration (IC50) of 20 ?滋g/ml (48 h), and inhibited DNA synthesis of BEL-7402 cells. After treatment of the spider venom, apoptosis of BEL-7402 cells was enhanced, and cells were arrested in G0/G1 phase; expression of C-myc was obviously down-regulated. CONCLUSION: The venom of spider Macrothele raveni could inhibit proliferation and DNA synthesis of BEL-7402 cells, which might through inducing cell apoptosis, down-regulating C-myc, and arresting cell cycle progression of BEL-7402 cells.