阿托伐他汀对人舌鳞癌CAL-27细胞增殖、凋亡和迁移的影响及其机制

Effects of atorvastatin on proliferation,apoptosis,and migration of human tongue squamous cell carcinoma CAL-27 cells and their mechanisms

目的:探讨阿托伐他汀(ATO)对人舌鳞癌CAL-27细胞体外增殖、凋亡和迁移的影响,阐明其作用机制。方法:CAL-27细胞分为对照组和1、5、10、20及40μmol·L^(-1)ATO组。ATO作用后,CCK-8法检测各组CAL-27细胞存活率,克隆形成实验检测各组CAL-27细胞克隆形成率,Hoechst33342荧光染色和流式细胞术检测各组CAL-27细胞凋亡率,细胞划痕实验检测各组CAL-27细胞迁移率,Western blotting法检测各组CAL-27细胞中P53、P21、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-3、Caspase-9和周期蛋白依赖性激酶6(CDK6)蛋白表达水平。结果:培养24和48 h后,与对照组比较,不同浓度ATO组细胞存活率明显降低(P<0.05)。培养48 h后,与对照组比较,不同浓度ATO组细胞克隆形成率(P<0.01)和细胞迁移率(P<0.05)明显降低,40μmol·L^(-1)ATO组细胞基本丧失克隆和迁移能力;不同浓度ATO组细胞凋亡率明显升高(P<0.05),其中40μmol·L^(-1)ATO组细胞几乎全部凋亡。培养48 h后,与对照组比较,不同浓度ATO组细胞均出现不同程度细胞核浓染或碎片化,20和40μmol·L^(-1)ATO组细胞全部呈现碎片化。与对照组比较,培养48 h后,不同浓度ATO组细胞中P53、P21、Bax、Caspase-3和Caspase-9蛋白表达水平升高(P<0.01),CDK6和Bcl-2蛋白表达水平降低(P<0.05或P<0.01)。结论:ATO通过P53/P21/CDK6通路抑制CAL-27细胞增殖及迁移并促进凋亡。

Objective:To investigate the effects of atorvastatin(ATO)on the proliferation,apoptosis,and migration of the human tongue squamous cell carcinoma CAL-27 cells in vitro,and to clarify their mechanisms.Methods:The CAL-27 cells were divided into control group and 1,5,10,20,and 40μmol∙L^(-1) ATO groups.After treated with ATO,CCK-8 assay was used to detect the survival rates of the CAL-27 cells in various groups,clone formation assay was used to detect the clone formation rates of the CAL-27 cells in various groups;Hoechst33342 fluorescence staining and flow cytometry were used to detect the apoptotic rates of the CAL-27 cells in various groups;cell scratch test was used to detect the migration rates the CAL-27 cells in various groups;Western blotting method was used to detect the expression levels of P53,P21,B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),cysteinyl aspartate specific proteinase(Caspase)-3,Caspase-9,and cyclin-dependent kinase 6(CDK6)proteins in the CAL-27 cells in various groups.Results:Compared with control group,the survival rates of the cells in different concentrations of ATO groups were decreased after cutured for 24 and 48 h(P<0.05).After cultured for 48 h,compared with control group,the clone formation rates(P<0.01)and migration rates(P<0.05)of the cells in different concentrations of ATO groups were decreased,and the cells in 40μmol∙L^(-1) ATO group basically lost the abilities of cloning and migration;the apoptotic rates in different concentrations of ATO groups were increased(P<0.05),and almost all the cells in 40μmol∙L^(-1)ATO group were apoptotic.After cultured for 48 h,compared with control group,the cells in different concentrations of ATO groups showed nuclear staining or fragmentation,and all the cells in 20 and 40μmol∙L^(-1) ATO groups were fragmented.Compared with control group,after cultured for 48 h,the expression levels of P53,P21,Bax,Caspase-3,and Caspase-9 proteins in the cells in different concentrations of ATO groups were increased(P<0.01),and the expression levels of CDK6 and Bcl-2 proteins were decreased(P<0.05 or P<0.01).Conclusion:ATO inhibits the proliferation and migration of the CAL-27 cells and promotes the apoptosis of the CAL-27 cells through P53/P21/CDK6 pathway.