关键词: nutlin-3, 黑色素瘤，实验性, 细胞周期, 细胞凋亡, 细胞运动

Abstract: Ding Xiaojie, Wei Dapeng, Chen Juping*. *Department of Dermatology, Second Clinical Medical College of Yangzhou University, Yangzhou 225001, China Corresponding author: Chen Juping, Email: chenjuping@medmail.com.cn 【Abstract】 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.

Key words: Nutlin-3, Melanoma, experimental, Cell cycle, Apoptosis, Cell movement