法尼基转移酶抑制剂Manumycin诱导HepG2细胞凋亡的研究

Apoptosis Induced by Farnesyltransferase Inhibitor Manumycin in Human Hepatoma Cancer Cell Line HepG2

目的:研究法尼基转移酶抑制剂Manumycin对人肝癌HepG2细胞的抗肿瘤作用,并探讨其诱导凋亡的分子机制。方法:采用MTT(Methythiazolyltetrazolium)法观察法尼基转移酶抑制剂Manumycin对肝癌细胞株HepG2细胞增殖的抑制作用,荧光显微镜、DNA凝胶电泳、流式细胞术等技术检测细胞凋亡,应用Westernblot方法检测bcl-2、p53、bax的蛋白水平变化。结果:法尼基转移酶抑制剂Manumycin能明显抑制HepG2细胞的生长且呈浓度依赖性,其IC50为(17.65±0.58)μmol/L。荧光显微镜检查显示Manumycin处理的HepG2细胞DAPI染色后,细胞核内可见浓染致密的颗粒荧光,典型细胞可见新月型改变,固缩或片段化的核。DNA凝胶电泳可见典型的DNA梯形带。流式细胞DNA直方图上出现典型的亚二倍体“凋亡峰”,细胞凋亡与Manumycin作用的时间和浓度相关。Manumycin能时间依赖性地诱导HepG2细胞发生G2/M期阻滞。Manumycin处理HepG2细胞后,Westernblot检测结果显示p53蛋白表达明显增加,而bcl-2蛋白和bax蛋白表达无明显变化。结论:法尼基转移酶抑制剂Manumycin对人肝癌细胞株HepG2有强烈的细胞毒作用,其分子机制可能是诱导HepG2细胞凋亡。Manumycin诱导HepG2细胞凋亡与bcl-2蛋白和bax蛋白表达水平无关。

Objective:To investigate the antitumor effect of farnesyltransferase inhibitor Manumycin in human hepatoma cell line HepG2, and to clarify its probable mechanisms. Methods: The growth inhibitory effect of farnesyltransferase inhibitor Manumycin on human hepatoma cell line HepG2 was observed by using MTT assay, Cell apoptosis was analyzed using flow cytometry, agrose gel electrophoresis of DNA and fluorescent microscopy. The protein expression of bcl 2, p53, and bax affected by Manumycin were determined by Western blot. Results: Farnesyltransferase inhibitor Manumycin significantly inhibited cell growth of human hepatoma cell line HepG2 with IC50 value of 17.65±0.58 μmol/L. DNA Ladder appeared on the agarose gel. And a typical subdiploid peak before Phase G0/G1 was detected by flow cytometry. The rates of apoptosis were correlated with the concentration of Manumycin and time of action. HepG2 cells were blocked at Phase G2/M which was correlated with time of action. Expression level of p53 in HepG2 was elevated after treatment with Manumycin, while there was no change for bcl 2 protein and bax protein. Conclusion: Manumycin can induce apoptosis in HepG2 cells which was associated with increasing of p53, but not bcl 2 and bax.