羊栖菜多糖通过激活Caspase途径诱导Lovo细胞凋亡

Apoptosis in Lovo Cells Induced by SFPS was Associated with a Activation of Caspase-3 Mediated by Caspase-9

研究了羊栖菜多糖(SargassumFusiformePolysaccharides,SFPS)诱导人大肠癌lovo细胞凋亡及凋亡过程中caspase-3、caspase-8、caspase-9的活性变化。MTT法检测SFPS对lovo细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western印迹法测定caspase-3酶原和caspase-9的变化;RT-PCR检测caspase-3mRNA表达;caspase-3、caspase-8、caspase-9活性检测试剂盒观察caspase-3、caspase-8、caspase-9的活性改变。结果显示,SFPS对lovo细胞增殖有显著抑制作用,经形态变化、DNA条带和流式细胞分析,可见明显的细胞凋亡特征。SFPS处理lovo细胞后,发现caspase-3酶原蛋白表达降低,caspase-3mRNA高表达,并具有剂量和时间的依赖性。而在检测蛋白中,也发现caspase-9被激活进而形成具有活性的片段。另外,caspase的活性检测也进一步发现caspase-3、caspase-9的活性逐步增高。实验结果提示SFPS在体外诱导lovo细胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,并且是通过激活启动caspase-9,进而激活下游效应caspase-3的级联反应来实现的。

Human colorectal cancer （lovo） cells were chosen to study the anti-tumor effects of Sargassum fusiforme polysaccharides （SFPS） and explore the significance of caspase-3, caspase-8 and caspase-9 in the apoptosis of lovo cells induced by SFPS. Inhibition of the cell proliferation was measured by MTT assay. SFPS induced apoptosis of lovo cells was observed by electron microscopy, flow cytometry and DNA electrophoresis. And the expressions of caspase-3 mRNA and pro-caspase-3, caspase-9 were tested by RT-PCR and Western blot. Furthermore, caspase-3, caspase-8, caspase-9 protease activity was measured by colorimetric assay kit. The results suggested that SFPS exhibited obvious anti-proliferative activity. Morphological examination, DNA ladders and flow cytometry analysis obviously showed cell apoptosis with characteristic morphological changes and DNA fragmentation. Furthermore, the expression of pro-caspase-3 decreased and the level of caspase-3 mRNA increased with the time and dose-dependent manner. However, Western analysis also showed the cleavage of caspase-9, an initiator caspase in SFPS-treated cells. Otherwise, caspase activity assay suggested that SFPS could induce cell apoptosis, which was closely accompanied with increase caspase-3 and caspase-9 activation. In conclusion, our study suggested that SFPS could induce the apoptosis of lovo cells in vitro resulting in the inhibition of proliferation and SFPS-induced apoptosis was associated with activation of caspase-3 mediated by cleavage of caspase-9.