Luteolin通过抑制Akt的活化和促进XIAP的降解敏化Fas-诱导的HepG2细胞凋亡

发布时间：2018-01-27 11:32

本文关键词： 木犀草素 Fas/FasL 蛋白激酶B X-连锁凋亡抑制蛋白　出处：《南京师范大学》2011年硕士论文　论文类型：学位论文

【摘要】：Fas属于肿瘤坏死因子(TNF)受体家族,是一种工型跨膜糖蛋白,其与天然配体FasL或拮抗性抗Fas抗体结合可以活化caspases级联反应,诱导细胞凋亡。但是现在存在的问题是：①并不是所有有Fas表达的细胞都对Fas/FasL诱导的细胞凋亡敏感,细胞内存在多种抗凋亡因子。②在体内的实验发现给小鼠静脉注射FasL对肝脏有很大的毒性,会引起严重的肝损伤,临床前试验中只能用于局部的治疗。因此寻找克服Fas-诱导细胞凋亡的抗性的机制并且降低FasL的用量是迫不及待的事情。木犀草素是广泛存在于蔬菜水果和药用植物中的一种黄酮类物质。木犀草素有两个苯环和一个含有两个氧原子的环,含有不饱和的键,有两个碳双键,在第四位有个羧基。研究表明Luteolin有抗癌、抗氧化、抗炎及保护心脏等作用。Luteolin的抗癌作用是通过干扰肿瘤细胞代谢、抑制细胞生长、抑制激酶活化及诱导细胞凋亡等多种方式实现的。现在越来越多的研究开始关注Luteolin在肿瘤细胞凋亡中的敏化作用。 Akt是一种丝氨酸/苏氨酸激酶。Akt的活化需要双重调控：①Akt需要转移到质膜上去；②Akt的Thr308和Ser473需要磷酸化。Akt通过磷酸化调节其下游靶蛋白参与调控如抑制细胞凋亡、促进细胞周期和细胞增殖等。Akt磷酸化凋亡抑制蛋白XIAP的Ser87,抑制它的泛素化降解从而抑制细胞凋亡。在这篇文章中我们发现在HepG2细胞中Luteolin增强FasL引起的细胞凋亡。这种敏化作用是通过抑制Akt的活化和促进凋亡抑制蛋白(XIAP)的降解实现的。XIAP作为Akt的下游靶点可以保护细胞免于细胞的凋亡。我们的实验发现高剂量的Luteolin可以直接下调Akt的蛋白水平,更有趣的是低剂量的Luteolin位点特异性的抑制Akt的Ser473位点的磷酸化而对Akt的整体水平没有影响。联用Luteolin和FasL处理细胞后发现Luteolin可以抑制FasL引起的Akt的磷酸化,我们过表达野生型Akt后Luteolin和FasL诱导的细胞凋亡明显减少。我们还发现Luteolin和FasL处理细胞后,Akt的下游的XIAP发生了明显的下调。过表达野生型Akt质粒后明显逆转了Luteolin和FasL诱导p-Akt和XAIP的下调。我们还用Akt的抑制剂LY294002作为一个阳性对照发现Luteolin与其有相同的抑制Akt磷酸化的作用。这些结果表明抑制Akt的活化和促进XIAP的降解在Luteolin敏化Fas-诱导的凋亡中发挥了重要的作用。
[Abstract]:Fas belongs to the TNF receptor family and is a transmembrane glycoprotein. It can activate the cascade reaction of caspases by binding with natural ligand FasL or antagonistic anti Fas antibody. But the problem now is that not all Fas-expressing cells are sensitive to Fas/FasL-induced apoptosis. A variety of anti-apoptotic factor-2 was found in vivo. It was found that intravenously injected FasL in mice was toxic to the liver and could cause severe liver injury. Preclinical trials can only be used for local treatment. Therefore, it is urgent to find mechanisms to overcome Fas-induced apoptosis resistance and reduce the dosage of FasL. Luteolin is a flavonoid found widely in vegetables, fruits and medicinal plants. Luteolin has two benzene rings and one ring containing two oxygen atoms, containing unsaturated bonds and two carbon double bonds. There is a carboxyl group in position 4th. Studies have shown that Luteolin has anti-cancer, anti-oxidative, anti-inflammatory and cardioprotective effects. Luteolin acts as an anticancer agent by interfering with the metabolism of tumor cells. Nowadays, more and more researches have focused on the sensitization of Luteolin in tumor cell apoptosis by inhibiting cell growth, inhibiting kinase activation and inducing apoptosis. The activation of Akt is a serine / threonine kinase. Akt needs to be transferred to the plasma membrane. 2 the Thr308 and Ser473 of Akt need phosphorylation. Akt regulates its downstream target proteins by phosphorylation, such as inhibition of apoptosis. Promote cell cycle and cell proliferation. Akt phosphorylated apoptosis inhibitor XIAP Ser87, inhibit its Ubiquitin degradation and inhibit cell apoptosis. In this paper, we found that Luteolin enhances apoptosis induced by FasL in HepG2 cells by inhibiting the activation of Akt and promoting apoptosis inhibitor proteins (. XIAP). As a downstream target of Akt, we found that high doses of Luteolin could directly down-regulate the protein level of Akt. What is more interesting is that low doses of Luteolin sites specifically inhibit the phosphorylation of Ser473 sites of Akt without affecting the overall level of Akt. L-treated cells showed that Luteolin could inhibit the phosphorylation of Akt induced by FasL. After overexpression of wild-type Akt, apoptosis induced by Luteolin and FasL was significantly reduced. We also found that Luteolin and FasL treated the cells. The down-regulation of XIAP downstream of Akt was observed. Overexpression of wild type Akt plasmid reversed the down-regulation of p-Akt and XAIP induced by Luteolin and FasL. LY294002, an inhibitor of Akt, as a positive control, found that Luteolin had the same inhibitory effect on Akt phosphorylation as Luteolin. These results suggest that Luteolin inhibits the activation of Akt and promotes XI. AP degradation plays an important role in Luteolin-sensitized Fas- induced apoptosis.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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