白桦脂酸抑制鼻咽癌细胞CNE-1生长及其机制探讨

Inhibitory Effect of Betulinic Acid on Nasopharyngeal Carcinoma Cell Line CNE-1 and Possible Mechanism

目的研究白桦脂酸(betulinic acid)对鼻咽癌细胞CNE-1生物学行为的影响并探讨其可能机制。方法不同浓度(10、20、40、80、120、160μg/mL)白桦脂酸处理CNE-1细胞,作用不同时间(24、48、72 h)后,MTT法检测CNE-1细胞增殖变化;不同浓度白桦脂酸(10、20、40μg/mL)作用CNE-1细胞72 h后,流式细胞仪PI单染法和FITC-AnnexinⅤ/PI双染法检测细胞周期和凋亡变化;RT-PCR检测凋亡相关基因Bcl-2和Bax mRNA表达水平的变化;Western blot检测Bcl-2和Bax蛋白表达水平的变化。结果 MTT实验显示不同浓度白桦脂酸对CNE-1细胞均有抑制作用(均P<0.05),抑制效果呈时间和剂量依赖性,24、48和72 h的IC50分别为(37.46±2.51),(24.28±1.87),(21.15±1.42)μg/mL;流式细胞术结果显示10、20、40μg/mL白桦脂酸组与空白组比较均出现G0/G1期阻滞[(59.21±2.47)%、(72.20±4.28)%、(77.13±3.57)%vs(42.94±2.63)%,均P<0.05]和凋亡比例的增加[(14.95±2.11)%、(21.71±2.84)%、(38.49±3.26)%vs(4.02±0.96)%,均P<0.05];RT-PCR结果显示对照组与白桦脂酸20、40μg/mL组的Bcl-2与内参β-actin吸光度积分值之比(Bcl-2/β-actin)随白桦脂酸浓度增加而减小,而Bax与内参β-actin吸光度积分值之比(Bax/β-actin)随白桦脂酸浓度增加而增加;Western blot结果显示白桦脂酸下调抑凋亡基因Bcl-2蛋白的表达,同时上调促凋亡基因Bax蛋白表达。结论白桦脂酸通过介导细胞G0/G1期阻滞和诱导细胞凋亡抑制鼻咽癌细胞CNE-1增殖。其诱导凋亡作用可能与下调Bcl-2/Bax表达比例相关。

Objective To investigate the antitumor effect of betulinic acid on human nasopharyngeal carcinoma cell line CNE-1 and the possible mechanism.Methods Concentration-dependent effect and time-dependent effect of betulinic acid on CNE-1 cells were observed by MTT assay.Cell cycle and apoptosis rate were evaluated by flow cytometry.The mRNA and protein expression levels of Bcl-2 and Bax were determined by RT-PCR and Western blot.Results MTT assay showed that betulinic acid significantly inhibited the growth of CNE-1 cells in a dose-and time-dependent manner（P〈0.05）.IC50 of betulinic acid at 24,48 and 72 h was（37.46±2.51）,（24.28±1.87）,（21.15±1.42） μg/mL respectively.Flow cytometry indicated that as compared with normal control group,the G0/G1 percentage in 10,20,40 μg/mL betulinic acid-treated groups was（59.21±2.47）%,（72.20±4.28）% and（77.13±3.57）% respectively vs（42.94±2.63）%（P〈0.05）,and the apoptosis rate was（14.95±2.11）%,（21.71±2.84）% and（38.49±3.26）% respectively vs（4.02±0.96）%（P〈0.05）.RT-PCR and Western bolt revealed that betulinic acid down-regulated the Bcl-2 mRNA and protein expression,and meanwhile,up-regulated the Bax mRNA and protein expression.Conclusion Betulinic acid could inhibit the proliferation of CNE-1 via G0/G1 arrest and induction of cell apoptosis in vitro.The down-regulation of Bcl-2/Bax ratio may induce the apoptosis of CNE-1 cells.