乌骨藤提取物通过调控PI3K/AKT/mTOR信号通路抑制黑色素瘤细胞的活力并诱导细胞凋亡

Marsdenia tenacissima extract inhibits viability and induces apoptosis of melanoma cells via regulating PI3K/AKT/mTOR signaling pathway

目的:探究乌骨藤提取物(Marsdenia tenacissima extract,MTE)对黑色素瘤细胞活力及凋亡的作用及机制。方法:用不同浓度(0、50、100和200 mg/L) MTE处理小鼠皮肤黑色素瘤B16-F10细胞24 h或不同浓度MTE处理不同时间(0、24、48、72和96 h),MTT法检测细胞活力。流式细胞术检测细胞凋亡情况,Western blot法检测细胞增殖、凋亡和PI3K/AKT/mTOR通路相关蛋白的表达,同时检测通路激动剂胰岛素样生长因子1(IGF-1)处理细胞后p-PI3K及细胞增殖和凋亡相关蛋白的表达。结果:根据预实验结果,选择MTE作用时间为72 h。MTE浓度为100 mg/L和200 mg/L时能明显抑制B16-F10细胞活力及增殖相关蛋白Ki67和PCNA的表达,并且还可诱导B16-F10细胞凋亡,提高凋亡相关蛋白cleaved caspase-3和cleaved caspase-9的蛋白水平;同时,MTE还可显著降低p-PI3K、p-AKT和mTOR的蛋白水平;此外,激动剂IGF-1可明显减弱MTE抑制细胞活力及诱导细胞凋亡的作用。结论:MTE可通过下调PI3K/AKT/mTOR通路活性抑制黑色素瘤细胞活力,诱导细胞凋亡。

AIM:To evaluate the effects of Marsdenia tenacissima extract(MTE)on the viability and apoptosis of mouse skin melanoma cell line B16-F10.METHODS:B16-F10cells were treated with MTE at different doses for24h or at different doses for different time,and the cell viability was measured by MTT assay.The apoptosis was analyzed by flow cytometry.The protein levels were determined by Western blot.Meanwhile,the cells were treated with insulin-like growth factor-1(IGF-1)and the protein levels were measured again.RESULTS:The cells were treated with MTE for72h for further study according to the results of pre-experiments.MTE at100and200mg/L inhibited the viability of B16-F10cells and decreased the protein expression of Ki67and PCNA significantly.MTE induced the apoptosis of B16-F10cells as demonstrated by increasing cleaved caspase-3and cleaved caspase-9.Meanwhile,MTE down-regulated the protein levels of p-PI3K,p-AKT and mTOR.In addition,IGF-1,the activator of PI3K/AKT/mTOR pathway,alleviated the effects of MTE on the viability and apoptosis markedly.CONCLUSION:MTE inhibits the viability and induces the apoptosis of melanoma cells by down-regulating PI3K/AKT/mTOR signaling pathway.