新藤黄酸诱导人鼻咽癌细胞CNE-1凋亡以及对p-p38和p-ERK1/2蛋白的影响

目的探讨新藤黄酸对人鼻咽癌细胞CNE-1凋亡的作用以及对磷酸化p38、p-ERK1/2蛋白的影响。方法倒置显微镜观察新藤黄酸不同时间和不同浓度处理CNE-1细胞形态变化;采用四甲基偶氮唑蓝(MTT)比色法检测不同浓度新藤黄酸(0.25、0.5、1.0、2.0、4.0和8.0μmol.L-1)处理24 h、48 h和72 h对人鼻咽癌细胞CNE-1增殖的抑制作用;新藤黄酸作用CNE-1细胞24 h后的IC50为2.34μmol.L-1,再结合倒置显微镜观察的结果故选用2.0μmol.L-1作为药物作用浓度。Annexin V-FITC/PI双染流式细胞仪检测细胞凋亡率,并应用透射电子显微镜检测细胞凋亡形态改变及线粒体损伤;Western blot检测p-p38、p38、ERK1/2和p-ERK1/2蛋白和线粒体凋亡相关蛋白Cytochrome C和Caspase-3蛋白的表达。结果新藤黄酸对人鼻咽癌细胞CNE-1生长和增殖有抑制作用,并随着新藤黄酸浓度的增加或作用时间的延长细胞活力明显下降。通过电镜可见2.0μmol.L-1新藤黄酸作用CNE-1细胞后细胞体积皱缩变圆,细胞核发生典型核染色质固缩等细胞凋亡形态学的变化;与control组相比包括对线粒体的损伤。Western blot表明p-p38蛋白表达有所上调,特别是在短时间(120 min)内,p-ERK1/2蛋白表达呈现时间依赖性下降;而p38和ERK1/2表达则基本不变。Cytochrome C和Caspase-3蛋白表达也呈现时间依赖性增加。结论新藤黄酸能诱导人鼻咽癌细胞CNE-1凋亡,增强Cytochrome C和Caspase-3蛋白表达,同时对p-p38和p-ERK1/2蛋白也有影响。

Gambogenic acid induced apoptosis in human nasopharyngeal carcinoma CNE-1 cells and effect of p-p38 and p-ERK1/2 protein

Aim To study the effect of Gambogenic acid on human nasopharyngeal carcinoma CNE-1 cells and the proteins of phospho-p38,phospho-ERK1/2.Methods Inverted microscope method was used to observe morphological changes of Gambogenic acid treated with CNE-1 cells at different time points and concentrations.The inhibitory rate of human nasopharyngeal carcinoma CNE-1 cell proliferation was determined by MTT assay after Gambogenic acid(0.25,0.5,1.0,2.0,4.0 and 8.0 μmol·L-1)treatment at 24 h,48 h and 72 h;The 50% inhibitory concentration(IC50) value for CNE-1 cells was 2.34 μmol·L-1 at 24 h after Gambogenic acid treatment,and considering the results of inverted microscope we chose 2.0 μmol·L-1 as the drug concentration.Annexin V-FITC/PI double staining was employed to detect cell apoptosis rate by flow cytometry,and using transmission electron microscopy was used to observe morphological changes of cell apoptosis and mitochondrial damage;Western blot detected protein expression of p-p38,p38,ERK1/2,p-ERK1/2 protein and mitochondrial apoptosis-related protein expression of Cytochrome C and Caspase-3 protein.Results Gambogenic acid produced effect on human nasopharyngeal carcinoma cell CNE-1 by inhibiting their growth and proliferation,and with the increase of drug concentration treatment time,the cell viability was significantly decreased.Transmission electron microscopy showed effect on CNE-1 cells by 2.0 μmol·L-1 Gambogenic acid;cell volume shrinkage typical nuclear chromatin condensation and other morphological changes of apoptosis;compared with the control group,gambogen group had the mitochondrial damage.Western blot showed that the expression of p-p38 protein has raised,especially within the 120 min period;p-ERK1/2 protein expression showed time-dependent decrease;the p38 and ERK1/2 protein levels were basically unchanged.Cytochrome C and Caspase-3 protein expression was also on the increase in a time-dependent manner.Conclusion Gambogenic acid may induce apoptosis in human nasopharyngeal carcinoma CEN-1 cells and enhance the expression of Cytochrome C and Caspase-3 protein,while affecting p-p38 and p-ERK1/2 proteins.