毛花苷C对肝癌细胞Na+/K+ -ATP酶A1亚基膜定位的调控及其相关抑癌机制

Subcellular localization of protein is the basis for the implementation of protein function. Prior studies on chemotherapy drugs targets focused on the changes in protein expression or protein variants, and subcellular localization of proteins are not involved. Found our preliminary study, lanatoside C significantly promotes liver cancer cells apoptosis and reduces Na+/K+ -ATPase subunit alpha 1 (ATP1A1) expression in the cell membrane, suggesting that lanatoside C acts as cancer suppressor maybe closely related to the regulation of ATP1A1 subcellular localization. In this project we are trying to observe LAMP3 expression level and its role in the pathogenesis of liver cancer. That include: 1.The measure of LAMP3 expression level in liver cancer tissues by real-time PCR and IHC. 2 The investigation of LAMP3 functional effect on liver cancer metastasis in vitro and in vivo by gene expression regulation. 3. The role of Rho, Rac1, Cdc42 in this association mechanism. The finish of this project will suggest a potential targeting application of LAMP3 in prognosis and cancer treatment and shield a beacon light on our understanding of liver cancer metastasis.In this project we are trying to observe ATP1A1 expression level and subcellular localization and its role in the pathogenesis of lanatoside c inducing liver cancer apoptosis. That include: 1.The measure of ATP1A1 expression level and subcellular localization in liver cancer tissues by real-time PCR or IHC. 2 The investigation of ATP1A1 functional effect on lanatoside c inducing liver cancer apoptosis in vitro and in vivo by gene expression regulation and mutation. 3. The role of PI3K-Akt-CDC42 pathway in this association mechanism.The finish of this project will suggest a potential targeting application of ATP1A1 in prognosis and cancer treatment and shield a beacon light on our understanding of lanatoside c inducing liver cancer apoptosis.

细胞蛋白的亚细胞定位是其执行相关生命功能的基础，既往对化疗药物靶向蛋白的研究多侧重于蛋白表达改变或蛋白变异，而对蛋白的亚细胞定位鲜有涉及。我们的前期研究中发现，毛花苷C明显促进肝癌细胞凋亡并抑制Na+/K+ -ATP酶A1亚基在细胞膜上的表达，提示毛花苷C的抑癌作用可能与其调控A1亚基的亚细胞定位密切相关。为阐明其抗肝癌机制，本课题拟开展如下研究：1、在肝癌组织样本中研究A1亚基的表达及亚细胞定位，并分析其相关临床意义；2、通过基因过表达及RNA干扰技术，验证A1亚基的表达差异在毛花苷C诱导肝癌细胞凋亡过程中的作用；构建A1亚基突变体，研究A1亚基细胞定位的改变在毛花苷C诱导肝癌细胞凋亡过程中的作用；3、重点研究毛花苷C对肝癌细胞内吞循环的影响，并通过反证实验阐明磷脂酰肌醇3激酶-蛋白激酶B在其中的作用。本课题研究的完成，将为强心苷类药物抗肝癌临床应用，提供新的理论与实验依据。

强心苷可能成为一类新型的肿瘤治疗药物,强心苷类物质抗癌机制众多，包括抑制Na+/K+-ATP酶活性、抑制缺氧诱导因子1活性、抑制FGF-2和NFκB、阻断雌激素受体、抑制拓扑异构酶的活性、诱导细胞凋亡等。毛花苷C选择性抗肝癌作用近年来已有初步研究，但其作用机制尚不明确，本课题研究发现毛花苷C可以抑制survivin（凋亡抑制基因）的表达，诱导Huh-7细胞凋亡执行蛋白Caspase-7的活化,促进肝癌SMMC-7721细胞凋亡。此外，毛花苷C可以明显促进Huh-7的I 型微管相关蛋白1轻链3（LC3-I）向II 型微管相关蛋白1轻链3（LC3-II）的转化,下调p62蛋白的表达， 上调AMPKβ1蛋白与Raptor蛋白的磷酸化水平，损伤在Huh-7细胞线粒体, 明显升高细胞胞浆内细胞色素C含量，增强Huh-7细胞的自噬；裸鼠皮下荷瘤动物实验证明：腹腔注射，给药9次，毛花苷C低剂量组抑瘤率为44.17%，毛花苷C高剂量组抑瘤率为67.22%，与对照组进行卡方比较，均有有显著性差异（p<0.05）。研究提示毛花苷C抗肝癌作用与其诱导肝癌细胞凋亡，通过损伤细胞线粒体，活化AMPK-Raptor通路，进而阻滞mTOR与Raptor的相互结合，抑制mTOR-Raptor复合体的活性，促进肝癌细胞自噬的作用有关。