复合活性多肽通过PI3K/Akt/mTOR信号通路对皮肤鳞癌细胞增殖与凋亡的影响

Effects of compound active peptides on proliferation and apoptosis of cutaneous squamous cell carcinoma cells through PI3K/Akt/mTOR signaling pathway

目的探讨复合活性多肽(compound active peptides,CAP)对皮肤鳞癌细胞A431增殖和凋亡的影响及其作用机制。方法选用人皮肤鳞癌A431细胞为研究对象,采用细胞增殖检测法(CCK8)检测不同浓度的CAP(0、50、100、150、200和250μg/mL)分别作用12、24、36和48 h后对A431细胞增殖的影响;选择50、100和150μg/mL CAP作用24 h进行后续实验。采用集落形成实验检测不同浓度的CAP对A431细胞的增殖能力,细胞划痕实验和流式细胞术检测对A431细胞迁移和凋亡的影响,Western blot法检测对细胞凋亡及PI3K/Akt/mTOR通路相关蛋白的表达。结果与对照组相比,CAP显著抑制细胞增殖(P<0.05),且呈时间和浓度依赖性;CAP处理后A431细胞的克隆形成率显著降低,呈现剂量依赖性(P<0.05),且A431细胞的迁移能力随浓度增高而显著降低(P<0.05)。与对照组相比,经过CAP处理A431细胞24 h后,细胞凋亡率随浓度升高而增加,凋亡相关蛋白Bax和Cleaved-PARP的表达量显著增加(P<0.05),Bcl-2和cyclin-D1蛋白表达量随浓度升高显著降低(P<0.05)。此外,与对照组相比,CAP显著下调PI3K、磷酸化p70-S6K、S6、mTOR和Akt蛋白的表达(P<0.05)。结论CAP可以抑制皮肤鳞癌A431细胞的增殖,并通过抑制PI3K/Akt/mTOR信号通路诱导A431细胞的凋亡。

Objective To investigate the effect and mechanism of the compound active peptides(CAP)on the proliferation and apoptosis of cutaneous squamous cell carcinoma cells A431.Methods Human cutaneous squamous carcinoma A431 cells were selected and the cell proliferation assay(CCK8)was used to determine the effects of different CAP concentration(0,50,100,150,200 and 250μg/mL)on A431 cell proliferation after treatment for 12,24,36 and 48 h;CAP were selected for 24 h of development for subsequent experiments.Colony formation assay was used to determine the proliferation ability of A431 cells with different concentrations of CAP;cell scratch assay and flow cytometry were used to detect the influence on A431 cell migration and apoptosis;Western blot was used to detect the apoptosis and the expression of PI3K/Akt/mTOR pathway related proteins.Results CAP significantly(P<0.05)inhibited cell proliferation in a time-dependent and concentration-dependent manner compared with the control group.The clonogenic rate of A431 cells was significantly reduced after treatment with the CAP in a dose-dependent manner(P<0.05),and the migration ability of A431 cells was significantly reduced with increasing concentrations(P<0.05).Compared with the control group,after 24 h treatment of A431 cells with CAP the apoptosis rate with increasing concentrantion and the protein expressions of Bax and cleaved-PARP significantly increased(P<0.05),and the Bcl-2 and cyclin-D1 protein expressions with increasing concentrantion decreased(P<0.05).In addition,the expression of PI3K,phosphorylated p70-S6K,S6,mTOR,and Akt proteins were significantly downregulated by the CAP compared with the control(P<0.05).Conclusions CAP could inhibit the proliferation of cutaneous squamous cell carcinoma A431 cells and induce the apoptosis of A431 cells by inhibiting the PI3K/Akt/mTOR signaling pathway.