2,3-吲哚醌诱导人神经母细胞瘤SH-SY5Y细胞凋亡及周期阻滞

背景与目的:2,3-吲哚醌(isatin,ISA)是存在于哺乳动物体液及组织中的一种天然物质,已发现其对肿瘤细胞的生长有抑制作用,但具体作用机制尚不明。本研究以人神经母细胞瘤细胞SH-SY5Y为靶细胞,观察ISA对SH-SY5Y细胞的作用及其机制。方法:采用荧光染色、流式细胞术(flow cytometry,FCM)及Western blot等方法检测不同浓度ISA(0、100、200、400μmol/L)诱导SH-SY5Y细胞凋亡及细胞周期阻滞的作用机制。结果:400μmol/L ISA作用48h后,在荧光显微镜下观察到SH-SY5Y细胞出现核固缩、DNA断裂等典型的凋亡形态学改变。Western blot结果显示,随ISA浓度的增加,Bcl-2蛋白表达下降、Bax蛋白表达无明显改变,Bcl-2/Bax下降。100、200、400μmol/L ISA处理SH-SY5Y细胞48h,活化Caspase-3的表达率分别为19.28%、25.88%、33.43%,明显高于对照组(P<0.05)。Western blot进一步检测到其下游底物Caspase激活的脱氧核糖核酸酶抑制剂(inhibitor of caspase-activated DNase,ICAD)被降解。细胞周期分析表明,经100、200、400μmol/L ISA处理48h后,G1期SH-SY5Y细胞数明显增加,呈明显的G1期阻滞;磷酸化的ERK蛋白及Cyclin D1(CDK1)蛋白表达显著减少(P<0.05)。结论:ISA能明显诱导SH-SY5Y细胞凋亡和细胞周期G1期阻滞,该作用可能与Bcl-2/Bax降低、Caspase-3激活,以及下调磷酸化ERK和细胞周期因子CDK1表达有关。

Effects of isatin on cell cycle arrest and apoptosis of neuroblastoma cell line SH-SY5Y

BACKGROUND & OBJECTIVE: Isatin (ISA) is a natural material that exists in mammalian body fluids and tissues. ISA could inhibit the growth of tumors, but the mechanism remains unclear. This study aimed to explore the effects of ISA on the cell cycle and apoptosis of neuroblastoma cell line SH-SY5Y. METHODS: Fluorescent staining, flow cytometry, and Western blot were performed to analyze the cell cycle arrest and apoptosis of SH-SY5Y cells after treatment of ISA at different concentrations (0, 100, 200, 400 micromol/L). RESULTS: When treated with 400 micromol/L ISA for 48 h, SH-SY5Y cells showed typical apoptotic morphologic changes including chromatin condensation and DNA fragment. Along with the increase of ISA concentration, Bcl-2 expression was decreased, the ratio of Bcl-2 to Bax was significantly decreased (P<0.05). When treated with ISA (100, 200, 400 micromol/L) for 48 h, the positive rates of activated Caspase-3 in SH-SY5Y cells were significantly higher than that in control SH-SY5Y cells (19.28%, 25.88%, and 33.43% vs. 10.58%, P<0.05). Moreover, inhibitor of caspase-activated DNase (ICAD), the substrate of Caspase-3, was degraded. In addition, the proportion of SH-SY5Y cells at G1 phase was significantly increased with an apparent G1 phase arrest when treated with ISA (100, 200, 400 micromol/L) for 48 h. In the progress of cell cycle arrest induced by ISA, phosphorylated ERK and CDK1 expression were down-regulated (P<0.05). CONCLUSION: ISA can induce apoptosis and G1 phase arrest in SH-SY5Y cells, possibly by decreasing Bcl-2/Bax, activating Caspase-3 and down-regulating the expression of phosphorylated ERK and CDK1.