EGCG诱导人胃癌MGC-803细胞凋亡相关基因的差异表达

Effects of epigallocatechin-3-gallate on the differential expression of apoptosis-associated genes in human gastric cancer MGC-803 cells

目的:研究表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)诱导人胃癌MGC-803细胞凋亡的生物学效应,并探讨其分子机制。方法:运用DNA含量分析、AnnexinⅤ/PI双标记法、DNA琼脂糖凝胶电泳以及透射电子显微镜形态学方法来观察细胞凋亡。通过SuperArray人细胞凋亡基因芯片检测100μmol/L EGCG作用于MGC-803细胞12 h后凋亡相关基因的表达谱,采用RT-PCR和Western blot技术验证上调基因Fas-L和下调基因Bag-1。结果:25、50、100、200μmol/L EGCG作用于MGC-803细胞24 h后,DNA含量分析出现了明显的亚二倍体峰;100μmol/L EGCG作用4、8、12、24 h后,AnnexinⅤ/PI双标记法表明早期凋亡细胞显著增加。100μmol/L EGCG作用24 h后,在琼脂糖凝胶上电泳可见DNA凋亡特征性的阶梯状条带。电子显微镜观察到细胞体积缩小,核浓缩和凋亡小体形成等典型的形态学改变。通过基因芯片检测发现,100μmol/L EGCG作用12 h后有8个凋亡相关基因表达差异显著,选择其中Fas-L和Bag-1运用RT-PCR和Western blot技术进行验证,其结果与基因芯片一致。结论:EGCG能够诱导人胃癌MGC-803细胞凋亡,这可能是多个基因和多条信号转导通路共同作用的结果。

Objective： This study investigates the biological effects and explores the molecular mechanisms of epigallocate-chin-3-gallate （EGCG） on the apoptosis of the human gastric cancer MGC-803 cells. Methods： After treatment with EGCG, cell apopto-sis was verified by flow cytometry with Annexin V and propidium iodide staining, DNA agarose gel electrophoresis, and transmission electron microscopy. The expression profiles of the apoptosis-related genes in the MGC-803 cells with or without treatment by EGCG for 12 h （100 μmol/L）, was identified using SuperArray Human Apoptosis Gene Array. The upregulated Fas-L gene and down-regulated Bag-1 gene were confirmed by reverse transcription polymerase chain reaction （RT-PCR） and Western blot. Results： When the MGC-803 cells were treated with EGCG at 25, 50, 100, and 200 μmol/L for 24 h, evident sub-diploid peaks were observed. Under treat-ment with 100 μmol/L for 4, 8, 12, and 24 h, the number of early apoptotic cells was greatly increased. When the cells were treated with 100 μmol/L for 24 h, the DNA extracted from the cells displayed a characteristic ladder pattern with agarose gel electrophoresis. Typi-cal morphological changes were observed by electron microscopy, including cell shrinkage, karyo-pyknosis, and the formation of apop-totic bodies. The differential expressions of eight apoptosis-associated genes were determined by gene array detection. The results of Fas-L and Bag-1 selected for RT-PCR and Western blot were consistent with those of gene array. Conclusion： EGCG induces apoptosis in MGC-803 cells, which might be mediated by a number of specific genes and various signal transduction pathways.