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

摘要

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。

Abstract

Objective To discuss the effect of oridonin on the proliferation， migration， epithelial-mesenchymal 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.

关键词

冬凌草甲素 / 鼻咽肿瘤 / 细胞周期 / 上皮-间质转化 / 细胞凋亡

Key words

Oridonin / Nasopharyngeal neoplasm / Cell cycle / Epithelial-Mesenchymal transit / Apoptosis

Author summay

梁 超（1998-），女，山东省临沂市人，在读硕士研究生，主要从事肿瘤发病机制方面的研究。