摘要
背景与目的:2,3-吲哚醌(2,3-dioxoindoline,isatin,ISA,以下简称吲哚醌)是存在于哺乳动物体液及组织中的一种天然物质,化学结构明确,是我国独创Ⅰ类天然抗癌新药靛玉红的单体结构。前期工作已显示ISA对肿瘤细胞有抑制增殖的作用,但具体作用机制尚不明确。本研究以人神经母细胞瘤细胞SH-SY5Y为靶细胞,采用体外培养细胞(体外实验)及建立裸鼠模型(体内实验)方法从分子水平、细胞水平及动物整体水平探讨ISA的抗肿瘤活性及其作用机制,为最终将该药开发成一种新型的具有我国自主知识产权的天然抗癌药物奠定基础。
方法:1.体外实验:体外培养人神经母细胞瘤细胞SH-SY5Y,采用MTT比色法、荧光染色、琼脂糖凝胶电泳、流式细胞术(flow csytometry,FCM)、RT-PCR、Westernblot等方法,观察不同浓度ISA对SH-SY5Y细胞增殖和凋亡的影响。2.体内实验:培养SH-SY5Y细胞接种裸鼠建立荷瘤裸鼠模型,观察ISA对荷瘤裸鼠移植瘤生长的抑制情况,检测ISA处理后瘤组织内Bcl-2/Bax的表达及活化caspase-3的表达,探讨ISA体内外的抗肿瘤活性及其作用机制。
结果:1.体外实验:ISA(0、50、100、200μmol/L)作用SH-SY5Y细胞48 h后,在荧光显微镜下观察到200μmol/L ISA处理组细胞出现典型的凋亡形态学改变:细胞核固缩、有DNA碎片;琼脂糖凝胶电泳显示100、200μmol/L ISA处理组细胞出现梯形条带;RT-PCR及Western blot结果显示,随ISA浓度的增加,Bcl-2 mRNA和蛋白表达均下降、Bax mRNA和蛋白表达无明显改变,Bcl-2/Bax比值下降;流式细胞术及Western blot检测到随着ISA浓度的增加,线粒体膜电位下降、细胞色素c由线粒体向细胞质释放增加,caspase-3的活化率逐渐增加,明显高于对照组(P＜0.05);Westernblot进一步检测到caspase-3的下游底物ICAD(inhibitor of caspase-activated dnase,Caspase激活的脱氧核糖核酸酶抑制剂)被降解。MTT比色结果显示ISA能抑制SH-SY5Y细胞增殖;细胞周期分析表明,经50、1 00、200μmol/L ISA处理48 h后,G_1期SH-SY5Y细胞数明显增加,呈明显的G_1期阻滞;Western blot结果显示随ISA浓度增加,磷酸化的ERK蛋白及细胞周期因子CyclinD_1(CD1l)蛋白表达逐渐减少(P＜0.05)。2.体内实验:结果显示ISA能抑制荷瘤裸鼠移植瘤的增长;降低Bcl-2/Bax比值;增加caspase-3的活化率。
结论:ISA能明显诱导SH-SY5Y细胞凋亡和细胞周期G_1期阻滞,该作用可能与Bcl-2/Bax降低、线粒体膜电位下降、Caspase-3激活以及下调磷酸化ERK和细胞周期因子CDK1表达有关。
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 antitumor effects and the related mechanism of ISA in vitro and in vivo with neuroblastoma cell line SH-SY5Y as target cell.
METHODS:in vitro experiment:MTT,Fluorescent staining,argerose electrophoresis, flow cytometry,RT-PCR and Western blot were performed to analyze the cell cycle arrest and apoptosis of SH-SY5Y cells after treatment of ISA at different concentrations(0,50, 100,200μmol/L).In vivo experiment:Establish the murine exnograft model,the growth of tumor was messured in vivo.RT-PCR and Western blot were performed to analyze Bcl-2 mRNA and protein
RESULTS:in vitro experiment:When treated with 200μmol/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).Dym Depolarization in SH-SY5YCells was decreased when treated with ISA(P＜0.05). Cytosyl Cyt-c Proteins increased in SH-SY5Y cells after 48-hour treatment of ISA detected by Western blot.When treated with ISA(50、100,200μmol/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 G_1 phase was significantly increased with an apparent G_1 phase arrest when treated with ISA(50、100,200μmol/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).In vivo experiment:The growth of tumor was visibly suppressed in vivo.RT-PCR and Western blot results indicated expression reduced in excised tumors.In contrast,there were no obvious changes in negative control.
CONCLUSION:ISA can induce apoptosis and G_1 phase arrest in SH-SY5Y cells, possibly by decreasing Bcl-2/Bax,activating Caspase-3,down-regulating the expression of phosphorylated ERK and CDK1.
引文
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