Nutlin-3对人A375黑素瘤细胞生物学行为的影响及可能机制

Effect of nutlin-3 on the biological behavior of A375 human melanoma cells and its mechanism

目的 观察顺式咪唑啉衍生物nutlin-3对人A375黑素瘤细胞生物学行为的影响,研究其机制.方法 将A375细胞分为实验组和对照组,实验组细胞接受2.5、5、10 μmol/L nutlin-3处理,对照组细胞采用二甲基亚砜处理.分别于作用24、48、72 h后,噻唑蓝（MTT）法检测细胞增殖情况;Western印迹检测p53蛋白表达的改变;流式细胞仪检测细胞周期分布以及细胞凋亡;Transwell法检测迁移性变化.采用重复测量的方差分析进行统计学检验.结果 2.5、5、10μmol/L nutlin-3处理A375细胞24、48、72 h后,MTT法显示不同时间点间的增殖抑制率差异有统计学意义（F=67.43,P＜ 0.01）,不同浓度间的抑制率差异有统计学意义（F=135.58,P＜ 0.01）,浓度越高抑制率越高;时间和浓度之间有交互作用（F=26.95,P＜0.01）.Western印迹、流式细胞仪、Transwell法检测显示,不同时间点之间A375细胞的p53表达、G2期细胞百分率、凋亡率、迁移抑制率差异均有统计学意义（F值分别为1255.00、831.38、809.45、1100.00,均P＜0.01）,除p53外,时间越长各项指标值越高;不同浓度间各项指标值差异均有统计学意义（F值分别为9196.00、267.99、723.83、1667.00,均P＜0.01）,浓度越高各项指标值越高;时间与浓度之间交互作用有统计学意义（F值分别为826.79、21.602、44.48、313.09,均P＜ 0.01）.结论 Nutlin-3可能通过p53蛋白积累途径抑制人A375细胞的增殖和迁移,促进细胞周期停滞和细胞凋亡.

Objective To estimate the effect of a cis-imidazoline derivative,nutlin-3,on the biological behavior of A375 human melanoma cells,and to investigate its mechanism.Methods Cultured A375 cells were divided into several test groups treated with nutlin-3 at different concentrations （2.5,5,10 μmol/L） for 24,48 and 72 hours,and a control group treated with dimethyl sulfoxide （DMSO） only.Then,methyl thiazolyl tetrazolium （MTT） assay was performed to evaluate cellular proliferative activity,Western blot to measure the expression of p53 protein,flow cytometry to estimate cell cycle phase distribution and apoptosis rate,and Transwell assay to evaluate migratory activity,of A375 cells.Statistical analysis was carried out by repeated-measures analysis of variance （ANOVA）.Results After treatment with nutlin-3 of 2.5,5 and 10 μmol/L for 24,48 and 72 hours,significant differences were observed among different time points at each concentration and among different concentrations at the same time point in proliferation inhibition rate （F =67.43,135.58,respectively,both P 〈 0.01）,p53 protein expression level （F =1255.00,9196.00,respectively,both P 〈 0.01）,percentage of cells at G2 phase （F =831.38,267.99,respectively,both P 〈 0.01）,apoptosis rate （F =809.45,723.83,respectively,both P 〈 0.01）,migration inhibition rate （F =1100.00,1667.00,respectively,both P 〈 0.01）.The influence of nutlin-3 on cellular proliferative activity increased with the increase in its concentration,and that on percentage of cells at G2 phase,apoptosis rate and migratory activity increased with the increase in its concentration and treatment duration.There was a significant interaction between the treatment duration and concentration of nutlin-3 for p53 protein expression level in （F =826.79,P 〈 0.01）,percentage of cells at G2 phase in （F =21.602,P 〈 0.01）,apoptosis rate in （F =44.48,P 〈 0.01）,migratory activity of （F =313.09,P 〈 0.01）,and cellular proliferative activity of （F =26.95,P 〈 0.01）,A375 cells.Conclusion Nutlin-3 may inhibit the proliferation and migration of,but promote cell cycle arrest and apoptosis in,A375 cells,through accumulation of p53 protein.