三氧化二砷对人恶性黑色素瘤A375细胞增殖及新癌基因PPO表达的影响

摘要

目的 探讨三氧化二砷对人恶性黑素瘤A375细胞株细胞周期、增殖、凋亡及新癌基因PPO(proliferation and phosphorylation oncogene)表达的影响.方法 培养人恶性黑色素瘤A375细胞株,采用 MTT法检测三氧化二砷在不同浓度和不同时间下对A375细胞增殖的影响,流式细胞术分析三氧化二砷对细胞周期及凋亡的影响,倒置相差显微镜观察药物处理后对人A375细胞的细胞分化及形态的影响,免疫细胞化学(SP)法检测三氧化二砷对PPO蛋白表达的影响.结果 MTT法证实三氧化二砷在1～10 μmol/L浓度范围内对A375细胞有明显的增殖抑制作用,呈现明显的剂量和时间依赖效应关系.流式细胞术检测发现,随着药物浓度的增加,G0/G1的细胞逐渐减少(P＜0.01),S期的比例显著增加,出现S期阻滞.倒置显微镜观察发现细胞出现典型凋亡细胞形态学改变.免疫细胞化学法检测PPO蛋白主要表达于A375细胞胞浆内,染色呈棕黄色.三氧化二砷作用48 h后,随着药物浓度的增加,PPO的染色程度逐渐减弱,差异具有显著性(P＜0.01).结论 三氧化二砷能够显著抑制A375细胞株增殖,并诱导凋亡发生,且与三氧化二砷的浓度和作用时间成正相关,其作用机制可能是通过下调新癌基因PPO的表达发挥作用.%Objective To study the effect of arsenic trioxide on proliferation, apoptosis, cell cycles and phosphorylation oncogene (PPO) expression in the human melanoma cell line A375. Methods The human melanoma cell line A375 was cultured. MTT assay was used to detect the effect of arsenic trioxide on proliferation, apoptosis and cell cycles of A375 cells at different concentrations. The effects of arsenic trioxide on cell cycle and apoptotic rates were assessed by flow cytometry. The morphological changes of cells were determined by the reverse microscopy after the treatment with arsenic trioxide. The influence of arsenic trioxide on the expressions of PPO in the A375 cells were detected with immunohistochemical method. Results It was confirmed by MTT assay that within the concentration interval of 1 ～ 10 μmol/L, as the concentration and exposure time increased, the inhibition ratio of arsenic trioxide on the A375 cells gradually increased. The inhibitory effects of arsenic trioxide on the proliferation of the A375 cells showed a time-and concentration-dependent manner. The growth of the A375 cells were arrested at S stage after treatment with arsenic trioxide with flow cytometry. It could be seen by the reverse microscopy that the typical morphological change of apoptosis occurred in the A375 cells. PPO protein was expressed in the intracytoplasm of the A375 cells. The expression of PPO protein diminished in arsenic trioxide-treated cells compared with the controls. There was difference between the control and the experiment groups (P ＜ 0. 05). Conclusions Arsenic trioxide significantly inhibits the proliferation of the A.375 cells and there is a positive correlation between the concentration and effectiveness of the drug. The mechanism of action might be related to the apoptosis resulting from inhibition of PPO expression in the A375 cells.