三七姜挥发油诱导(CNE-2)细胞周期G1期阻滞及相关基因表达的影响

目的:研究三七姜挥发油对人鼻咽癌 (CNE-2) 细胞周期的调控作用,并探讨其可能的机制。方法:体外培养人鼻咽癌细胞株(CNE-2),采用MTT法观察三七姜挥发油对CNE-2细胞增殖的抑制作用;流式细胞术分析三七姜挥发油处理CNE-2细胞后对细胞周期分布的影响;实时荧光定量PCR检测细胞周期调控相关基因的表达水平。结果:不同浓度的三七姜挥发油作用CNE-2细胞24,48,72,96 h后,均能明显抑制细胞增殖(P<0.05),且呈浓度与时间依赖性,其IC₅₀分别为71.12,60.034,53.167,34.75 μg·ml^-1;三七姜挥发油诱导CNE-2细胞发生G₁期阻滞,与阴性对照相组比,200 μg·ml^-1加药处理CNE-2细胞72 h后G₁期细胞增加了13.94%(P<0.01);伴随ATM,CCND1/E1,CDK6,CDC25A,E2F1等mRNA表达水平下调,而Tp53基因表达显著增强(P<0.01)。结论:三七姜挥发油诱导人鼻咽癌CNE-2细胞G₁期阻滞、抑制细胞增殖,其作用可能与抑制了介导DNA损伤修复的ATM-Chk2-Cdc25A通路,以及激活p53通路有关,从而抑制CDC25A,cyclinD1/E1、CDK6、E2F1等基因的表达而实现的。

Induction of G1 arrest of CNE-2 cells by volatile oils from Stahlianthus involucratus and effects on related gene expression

OBJECTIVE To investigate the regulating effects of volatile oils from Stahlianthus involucratus on cell cycle of human nasopharyngeal carcinoma cell line CNE-2 in vitro and the underlying potential mechanism.METHODS CNE-2 cells were cultured in vitro, and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to observe the inhibiting effects of volatile oil from Stahlianthus involucratus on proliferation of CNE-2 cell lines. The distribution of cell cycle of CNE-2 cells were determined by flow cytometry (FCM). Expression of cell cycle regulation-related genes were assessed by real-time polymerase chain reaction (RT-PCR) method.RESULTS Incubation with volatile oils from Stahlianthus involucratuson for 24, 48, 72 and 96 h resulted in a significant inhibition of CNE-2 cell proliferation in a dose and time dependent manner (P<0.05), and IC50 values for them were 71.12, 60.034, 53.167 μg·ml^-1 and 34.75 μg·ml^-1, respectively. Volatile oils from Stahlianthus involucratuson induced G₁ phase arrest, compared with the untreated control group, G₁ phase of the cells treated by 200 μg·ml^-1 drug increased by 13.94% after 72 h (70.03±1.15)% vs. (56.09±2.12)%(P<0.01). The mRNA levels of ATM, CCND1/E1, CDK6, CDC25A, and E2F1 decreased, while Tp53 elevated (P<0.01).CONCLUSION Volatile oils can potently disturb proliferation of CNE-2 cells in vitro and induce cell cycle arrest at G₁ phase, which may be realized by inhibiting ATM-Chk2-Cdc25A pathway which mediates DNA injury restoration, activating p53 pathway, and then inhibiting expression of genes of CDC25A, cyclinD1/E1, CDK6 and E2F1.