二烯丙基二硫对人白血病HL-60细胞凋亡启动过程的影响

Proteomics analysis of apoptosis initiation induced by diallyl disulfide in human leukemia HL-60 cells

背景与目的:二烯丙基二硫(diallyl disulfide,DADS)作为一种抗肿瘤物质日益受到关注。本研究在建立DADS启动HL-60细胞凋亡模型的基础上,研究DADS启动人白血病HL-60细胞凋亡的相关蛋白。方法:提取未处理HL-60细胞和3.6mg/LDADS处理2d的HL-60细胞的总蛋白,双向电泳分离细胞总蛋白质,绘制图谱,PDQuest软件分析,切割差异蛋白质,进行质谱分析、生物信息学分析,Western blot以及RT-PCR验证部分差异蛋白。结果:对照组和处理组蛋白质表达量相差2倍以上的点有29个,其中22个上调,7个下调。质谱分析和数据库搜索鉴定了9个蛋白质点,其中7个蛋白质点有意义,这些蛋白功能分别与信号转导、基因转录及调控、细胞骨架、细胞新陈代谢等有关。Western blot结果显示DADS处理后Rac2(ras-related C3 botulinum toxin substrate2)蛋白表达明显上调,RT-PCR显示DADS处理后Rac2表达增强。结论:Rac2等蛋白参与了DADS诱导的HL-60细胞凋亡,但其具体机制还有待于进一步研究。

Background and Objective= Diallyl disulfide （DADS）, an antitumor reagent, has increasingly gained attention. This study was to explore the related proteins in DADS-induced apoptosis initiation in human leukemia HL-60 cells. Methods= Total protein of HL-60 cells with or without 2-day treatment of 3.6 mg/L DADS was extracted, separated by two- dimensional polyacrylamide gel etectrophoresis, and analyzed by PDQuest 2-DE software. Differentially expressed proteins were separated and identified by peptide mass fingerprinting analysis and bioinformatics, and verified by Western blot and reverse transcription-polymerase chain reaction （RT-PCR）. Results. As compared with those in untreated HL-60 cells, 29 proteins were differentially expressed in DADS-treated HL-60 cells： 22 were up- regulated and seven were down-regulated. Among nine proteins which were randomly selected for peptide mass fingerprinting analysis and bioinformatics, seven were meaningful. These proteins were associated with cellular responses, gene transcription and regulation, cytoskeleton, metabolism, and so on. Western blot showed that Ras-related C3 botulinum toxin substrate 2 （Rac2） was up-regulated in DADS-treated HL-60 cells; this result was accordant with RT-PCR results. Conclusion. Some proteins, including Rac2, may be involved in DADS-induced apoptosis in human leukemia HL-60 cells, but the exact mechanism needs to be explored.