新藤黄酸通过调控PI3K/Akt/mTOR信号通路诱导黑色素瘤B16细胞凋亡的研究

Study of gambogenic acid-induced apoptosis of melanoma B16 cells through PI3K /Akt /mTOR signaling pathways

目的:探讨新藤黄酸诱导黑色素瘤B16细胞凋亡的机制。方法:MTT试验检测新藤黄酸对B16细胞增殖抑制的作用;采用Hochest 33258荧光染色观察新藤黄酸对B16细胞的影响;透射电镜观察新藤黄酸对B16细胞的超微结构的改变;Western blot法检测PI3K,p-PI3K,Akt,p-Akt,p-mTOR,PTEN蛋白的变化以探讨新藤黄酸诱导B16细胞凋亡的分子机制。结果:新藤黄酸对黑色素瘤细胞B16生长和增殖有明显地抑制作用,并随着新藤黄酸浓度的增加和作用时间的延长细胞活力明显下降;Hochest 33258染色结果显示,新藤黄酸处理的细胞中呈现明显的凋亡特征;透射电镜观察发现新藤黄酸作用B16细胞后,细胞发生明显凋亡的形态学变化;Western blot检测表明p-PI3K蛋白表达呈时间依赖性下降;p-Akt蛋白表达呈时间依赖性下降,而PI3K,Akt蛋白表达量基本不变,p-mTOR蛋白的表达随着时间的延长有所下降,PTEN蛋白的表达呈时间依赖性增加。结论:新藤黄酸在一定的时间和浓度范围内能够抑制黑色素瘤B16细胞的增殖,诱导细胞凋亡,其诱导细胞凋亡的机制可能与调控PI3K/Akt/mTOR信号通路有关。

Objective： To discuss the mechanism of gambogenic acid （GNA） in inducing the apoptosis of melanoma B16 cells. Method： The inhibitory effect of GNA on the proliferation of B16 cells was measured by the methyl thiazolyl tetrazolium （MTF） assay. The effect of GNA on B16 cells was detected by the Hoechst 33258 staining. The transmission electron microscopy was used to observe the ultra-structure changes of B16 cells. The changes in PI3K, p-PI3K, Akt, p-Akt, p-mTOR, PTEN proteins were detected by the Western blotting to discuss the molecular mechanism of GNA in inducing the apoptosis of B16 cells. Result： GNA showed a significant inhibitory effect in the growth and proliferation of melanoma B16 cells. The cell viability remarkably decreased with the increase of GNA concentration and the extension of the action time. The results of the Hoechst 33258 staining showed that ceils processed with GNA demonstrated apparent apoptotic characteristics. Under the transmission electron microscope, B16 cells, after being treated with GNA, showed obvious morphological changes of apoptosis. The Western blot showed a time-dependent reduction in the p-PI3K and p-Akt protein expressions, with no change in p-PI3K and p-Akt protein expression quantities. The p-mTOR protein expression de- creased with the extension of time, where as the PTEN protein expression showed a time-dependent increase. Conclusion： GNA could inhibit the proliferation of melanoma B16 cells and induce their apoptosis within certain time and concentration ranges. Its mechanism in inducing the cell apoptosis may be related to PI3K/Akt/mTOR signaling pathways.