冬凌草甲素对人鼻咽癌HONE-1细胞增殖、迁移和凋亡的影响

Effect of oridonin on cell proliferation,migration,and apoptosis of human nasopharynx carcinoma HONE-1 cells

目的:探讨冬凌草甲素对人鼻咽癌HONE-1细胞增殖、迁移、上皮-间质转化(EMT)和凋亡的影响,阐明其相关抗肿瘤机制。方法:鼻咽癌HONE-1细胞经不同浓度(0、5、10、20、40、80和160 mg·L^(-1))冬凌草甲素处理48 h后,采用CCK-8法检测各组细胞增殖抑制率,确定后续实验的用药浓度。HONE-1细胞分为对照组、3 mg·L^(-1)冬凌草甲素组和6 mg·L^(-1)冬凌草甲素组,培养24和48 h后,采用CCK-8法检测各组细胞增殖活性,5-乙炔基-2'-脱氧尿嘧啶核苷(EdU)法检测各组细胞中EdU阳性细胞率,克隆形成实验检测各组细胞中克隆形成数,Transwell小室实验和细胞划痕实验检测各组细胞中迁移细胞数和划痕愈合率,实时荧光定量PCR(RT-qPCR)法检测各组细胞中细胞周期蛋白依赖性激酶1(CDK1)和细胞周期蛋白依赖性激酶4(CDK4)mRNA表达水平,Western blotting法检测各组细胞中E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)和多腺苷二磷酸核糖聚合酶1(PARP1)蛋白表达水平。结果:CCK-8法确定冬凌草甲素48 h半数抑制浓度(IC50)为12.18 mg·L^(-1),以1/4 IC50和1/2 IC50值为后续实验用药浓度。与对照组比较,24和48 h时3和6 mg·L^(-1)冬凌草甲素组细胞增殖活性降低(P<0.05或P<0.01),EdU阳性细胞率降低(P<0.05或P<0.01),细胞中克隆形成数和迁移细胞数减少(P<0.05或P<0.01),划痕愈合率降低(P<0.05或P<0.01),细胞中CDK1和CDK4 mRNA表达水平降低(P<0.05或P<0.01),E-cadherin、Caspase-3和PARP1蛋白表达水平升高(P<0.05或P<0.01),Vimentin蛋白表达水平降低(P<0.05)。结论:冬凌草甲素可通过抑制细胞周期相关蛋白的表达及EMT,进而抑制人鼻咽癌HONE-1细胞增殖、克隆形成和迁移能力,促进细胞凋亡,发挥抗肿瘤作用。

Objective:To discuss the effect of oridonin on the proliferation,migration,epithelialmesenchymal transition(EMT),and apoptosis of the human nasopharyngeal carcinoma HONE-1 cells,and to clarify its related antitumor mechanism.Methods:The HONE-1 cells were treated with different concentrations(0,5,10,20,40,80,and 160 mg·L^(-1))of oridonin for 48 h.CCK-8 method was used to detect the inhibitory rates of proliferation of the cells in various groups and the drug concentration for subsequent experiment was confirmed.The HONE-1 cells were divided into control group,3 mg·L^(-1) oridonin group,and 6 mg·L^(-1) oridonin group.After 24 and 48 h of culture,CCK-8 method was used to detect the proliferation activities of the cells in various groups;5-ethynyl-2'-deoxyuridine(EdU)method was used to detect the rates of EdU-positive cells in various groups;colony formation assay was used to detect the numbers of clone formation in the cells in various groups;Transwell chamber experiment and cell wound assay were used to detect the numbers of migration cells and the scratch healing rates of the cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of cyclin-dependent kinase 1(CDK1)and cyclin-dependent kinase 4(CDK4)mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of E-cadherin,Vimentin,Caspase-3,and poly ADP-ribose polymerase 1(PARP1)proteins in the cells in various groups.Results:The CCK-8 method results showed that the half-maximal inhibitory concentration(IC50)of oridonin at 48 h was 12.18 mg·L^(-1),and 1/4 IC50 and 1/2 IC50 values were used as the concentrations for subsequent experiments.Compared with control group,after treated for 24 and 48 h,the proliferation activities of the cells in 3 and 6 mg·L^(-1) oridonin groups were decreased(P<0.05 or P<0.01),the rate of EdU-positive cells were decreased(P<0.05 or P<0.01),the numbers of clone formation and migraton cells were decreased(P<0.05 or P<0.01),the scratch healing rates were decreased(P<0.05 or P<0.01),the expression levels of CDK1 and CDK4 mRNA in the cells were decreased(P<0.05 or P<0.01),the expression levels of E-cadherin,Caspase-3,and PARP1 proteins were increased(P<0.05 or P<0.01),and the expression levels of Vimentin protein were decreased(P<0.05).Conclusion:Oridonin can inhibit the proliferation,clone formation,and migration of the human nasopharyngeal carcinoma HONE-1 cells by downregulating the expression of cell cycle-related proteins and EMT,and promote the apoptosis to exert an antitumor effect.