MAP4K3-mTOR信号在甘草查而酮A诱导的鳞癌细胞自噬与凋亡中的作用研究

Effect of MAP4K3-mTOR Signaling in Licochalcone A-induced Autophagy and Apoptosis of Human Oral Squamous Cell Carcinoma Cells

目的:探索MAP4K3-mTOR信号级联在甘草查而酮A诱导的人口腔鳞癌细胞自噬与凋亡中的作用。方法:分别采用0、25、50μmol/L甘草查尔酮A刺激人口腔鳞癌SCC-25系细胞,并于刺激后0、6及24h收集细胞,采用Western blot检测MAP4K3、AKT、mTOR通路分子,细胞自噬标识分子LC3-II、beclin1,以及细胞凋亡标记分子caspase-3、caspase-9及bcl-2等的蛋白表达水平,采用Annexin V-PI染色检测细胞凋亡状况。结果:甘草查尔酮A可剂量及时间依赖性地降低SCC-25细胞MAP4K3、p-mTOR及p-p70S6蛋白的表达水平,促进其LC3-II、beclin1、caspase-3及caspase-9蛋白表达水平,并抑制其bcl-2蛋白表达水平(P<0.05)。采用慢病毒过表达MAP4K3后,p-mTOR、caspase-3及caspase-9蛋白表达水平显著增高,LC3-II、beclin1及bcl-2蛋白表达水平则显著降低(P<0.05)。经甘草查尔酮A刺激后,SCC-25细胞早期凋亡数与晚期凋亡数均较对照组显著增多,该效应可被MAP4K3过表达处理显著增强(P<0.05)。结论:甘草查尔酮A可时间剂量依赖地抑制MAP4K3-mTOR信号级联,并促进人口腔鳞癌细胞的自噬与凋亡,当过表达MAP4K3活化mTOR信号级联后,鳞癌细胞自噬活动受到抑制,但其凋亡活动则显著增强。

Objective： To investigate the effect of MAP4K3--mTOR signaling in licochalcone A--induced autoph- agy and apoptosis of human oral squamous cell carcinoma cells （OSCC）. Methods.. OSCC SCC-25 cells were a- dopted and treated once with licochalcone A for 0, 6, and 24 h at 0, 25 and 50 μM. The cells were harvested for further investigation. Western blot analysis was adopted to detect the protein expression of MAP4K3, AKT, mTOR, and autophagy marker molecules LC3- II and beclinl, and apoptosis marker molecules caspase-3, caspase--9, and bcl-2. Annexin V--PI staining was used to detect the cell apoptosis. Results.. Exposure of SCC- 25 ceils to licochalcone A dose-- and time-- dependently decreased the protein expression of MAP4K3, p-mTOR, and p--p70S6, and increased the protein expression of LC3--Ⅱ, beclinl, caspase--3, and caspase--9. When the MAP4K3 was overexpressed, the protein expressions of p--mTOR, caspase--3, and caspase--9 were increased, while the expressions of LC3--Ⅱ, beclinl, and bcl--2 were decreased （P〈0.05）. In addition, licochalcone A stim- ulation increased the number of early and late apoptotic SCC--25cells, which could be obviously reversed by MAP4K3 overexpression （P〈0. 05）. Conclusion.. Licochalcone A dose-- and time-- dependently decreased MAP4K3--mTOR signaling, but increased the autophagy and apoptosis of SCC--25 cells. When MAP4K3 was overexpressed, the licochalcone A--induced autophagy decreased, but licochalcone A--induced apoptosis increased.