【作者】 杨郁；

【导师】 顾洛；

【作者基本信息】 南京医科大学 ， 生理学， 2010， 硕士

【摘要】 以往的研究证实,EGF是人类乳腺癌细胞迁移过程中起着关键趋化作用的诱导因子,EGF能促进胞内ROS的产生。另一方面,ROS可以促进恶性肿瘤细胞的迁移,但目前ROS在恶性肿瘤细胞迁移中的分子调节机制尚不清楚。本研究在建立体外EGF诱导MDA-MB-231乳腺癌细胞迁移模型的基础上,研究了ROS和PI3K/Akt-PAK1信号通路在EGF诱导的MDA-MB-231细胞迁移中作用的可能机制,以期为深入阐明恶性肿瘤浸润和转移机制提供理论依据。本研究发现:1、EGF能明显促进MDA-MB-231细胞内ROS的产生,而ROS生成阻断剂NAC则能显著抑制EGF所诱导的ROS生成。2、分别使用特异性化学阻断剂预处理细胞,或瞬时转染负显性突变Rac1、Akt、PAK1质粒,或siRNA干扰Akt表达后,通过细胞划痕实验,证实ROS、Rac1、PI3K/Akt和PAK1参与EGF诱导的MDA-MB-231细胞迁移。3、因为EGF能诱导Rac1、PI3K/Akt和PAK1的活化,瞬时转染负显性突变体Rac1 T17N能有效抑制EGF引起的PI3K/Akt及PAK1活化,PI3K特异性抑制剂LY294002和siRNA干扰Akt均可以显著地降低PAK1磷酸化水平,结果证明EGF诱导的MDA-MB-231细胞迁移中存在着Rac1→PI3K/Akt→PAK1信号调控途径。因为ROS生成阻断剂NAC能明显抑制EGF诱导的PI3K/Akt及PAK1磷酸化,结果提示ROS中介PI3K/Akt和PAK1激活过程。综上所述,我们第一次发现Rac1-PI3K/Akt-PAK1信号通路中介EGF诱导的MDA-MB-231细胞迁移;在此信号通路中,ROS中介EGF诱导的PI3K/Akt和PAK1活化。更多还原

【Abstract】 Epidermal growth factor (EGF) plays an important role in human breast cancer cell migration. EGF can increase intracellular ROS production, but the ROS sensitivity of migration and of the signaling pathways leading to migration are largely unknown. Previous studies have shown that activated Rac1, PI3K/Akt and PAK1 contribute to progression, invasion, and maintenance of the malignant phenotype in human cancers, so we supposed they all might participate in ROS-dependent regulation of EGF-induced breast cancer cell migration. In MDA-MB-231 breast cancer cells with high metastatic ability, we reported that EGF promoted migration compared with nonstimulated cells, with a maximum increase at 10ng/mL. Pretreatment with the ROS inhibitor NAC siginificantly attenuated migration. Moreover, preincubated with the PI3K inhibitor LY294002 or siRNA Akt or dominant negative forms of Rac1 T17N, SRα-Akt (T308A/S473A) and PAK1 K299R all obviously decreased cell migration in response to EGF. Phosphorylation of PI3K/Akt and PAK1 (Thr423) were attenuated by ROS inhibitor, but Rac1 regulated EGF-stimulated ROS production. PI3K/Akt activated PAK1 at Thr423, and EGF stimulated PI3K/Akt and PAK1 kinase activity through a Rac1-dependent mechanism. Taken together, these finding suggest that EGF-induced MDA-MB-231 cell migration is ROS dependent and identify the PI3K/Akt/PAK1 signaling pathway as an essential ROS-sensitive mediator of migration.更多还原