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The Comparative Analysis Of The Phosphorylation Of Tyrosine Residues Of C-Met In Murine Ascitic Hepatic Cancer Cell Lines With Different Lymph Node Metastatic Potentia

Posted on:2011-07-25Degree:MasterType:Thesis
Country:ChinaCandidate:Y LiFull Text:PDF
GTID:2144360305975954Subject:Biochemistry and Molecular Biology
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Objective:CD82 (KAI1), as well as homologous proteins CD9 belong to the four transmembrane protein family, can inhibit both tumor cells in vitro movement and migration. Clinical studies have found that the metastatic rate of a variety of tumor (in particular, lymph node metastasis) and the expression of these two proteins have a negative correlation, therefore, CD82 and CD9 are considered broad-spectrum inhibitor of tumor metastasis. Studies of its mechanism of action have shown that the CD82 and CD9 mainly inhibit the activation of a number of growth factor receptor, such as hepatocyte growth factor receptor (HGFR, c-Met), epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) and so on, down-regulate the activity of some signaling parthway which are involved in regulation of the tumor metastasis. As we known, two signaling parthway, PI3K/AKT and PLCγ/DAG/PKC signaling parthway in cells play a crucial role in tumor metastasis, which regulate the expression of a number of tumor metastasis-related metastasis protein factors, such as cell adhesion molecules, extracellular matrix molecules, metalloproteinases, and cytoskeletal proteins, thus affecting tumor invasion and metastasis. Recent studies have also found that gangliosides GM2/GM3 on the plasma membrane have synergistic effect with CD82 and CD9. GM2 and GM3 could form dimmer with CD82 and CD9, respectively and enhance the inhibiting action of tumor metastasis. Therefore, it is important to illuminate molecular mechanism of GM2/GM3→CD82/CD9→GFR→PI3K/AKT and PLCγ/DAG/PKC regulating axes.In our previous work, we have compared the expression of gangliosides and CD82/CD9 between two cell lines with different metastatic potential in lymph node and found that the expression of gangliosides and CD82/CD9 in two cell lines are significantly different, Hca-F cell line with high metastatic potentia express mainly ganglioside GM2, but Hca-P cell line with poor metastatic potential express mainly ganglioside GM3; Hca-P cells express CD9 higher than that of Hca-P cells; There is no significant difference in expression of CD82 between two cells. We compared further the activity of the PI3K/AKT and PLCγ/DAG/PKC signaling pathway in two cells. The results showed that the content of phosphorylated PLCyl is higher in Hca-F cells that in Hca-P cells. It is surgested that the activity of PLCγ/DAG/PKC signaling pathways in Hca-F cells is higher than that of low metastatic Hca-P cells. We also analyzed comparatively the expression and phosphorylation of hepatocyte growth factor receptor between two cell lines and found that there was no significant difference in the phosphorylation of c-Met between two cell lines. It is well known that, after binding with ligand, the c-Met receptors are dimerized and phosphorylated at some tyrosine residues. The phosphorylated tyrosine residues can be recognized and bind by some signal molecules with SH2 doman in cells. Different phosphorylated tyrosine residues can be recognized by different signal molecules and activate different downstream signaling pathway, thus, play a different regulatory role, have different biological effects. Therefore, it is the phosphorylate tyrosine residues that could determine which signaling pathway can be activated and what kind of regulating action can be generated. But, it is not known that the tyrosine residues of receptor are phosphorylated by all or none or select phosphorylation manner. If the tyrosine residues of receptor are phosphorylated by the selective phosphorylation manner, what are the factors that affect the selectivity and what is the mechanism are not known. Although the phosphorylation of c-Met between two cell lines is at the same level, we suppose that the tyrosine residues which can be phosphorylated should be different, which lead to the higher avtivity of PLCγ/DAG/PKC signaling pathway in Hca-F cells than in Hca-P cells. In this work, to Validate the presumption, we analyzed comparatively the phosphorylated tyrosine residues of c-Met and activity of PLCγ/DAG/PKC and PI3K/AKT signaling pathway between the two cell lines under different treatment conditions.Method:The murine ascitic hepatic cancer cell lines HCa-F cells(high metastatic potential) and HCa-P cells(low metastatic potential) were cultured in 1640 medium containing 10%, after 10 hours of hunger in serum-free 1640 medium, cells were divided into control group and treatment group. After treatment of HGF, laminin, fibronectin, the phosphorylated tyrosine residues of c-Met and activity of PLCγ/DAG/ PKC and PI3K/AKT signaling pathway between the two cell lines were analyzed by SDS-PAGE, Western Blotting technology and computer quantitative.Result:(1) After treatment of HGF, the level of phosphorylation of 1365 tyrosine residue and 1313 tyrosine residue is higher in high metastatic Hca-F cells than in low metastatic Hca-P cells; The level of phosphorylation of 1349 tyrosine residue is higher in low metastatic Hca-P cells than in high metastatic Hca-F cells; The content of p-plcyl (Tyr783) in high metastatic Hca-F cells is higher than that in low metastatic Hca-P cells; The content of p-Akt (Thr308) and p-Akt (ser473) in low metastatic Hca-P cells is higher than that in high metastatic Hca-F cells.(2) After treatment of FN, the level of phosphorylation of 1313 tyrosine residue is higher in high metastatic Hca-F cells than in low metastatic Hca-P cells; The level of phosphorylation of 1365 tyrosine residue and 1349 tyrosine residue has no significant difference between two cell lines; The content of p-plcyl (Tyr783) in high metastatic Hca-F cells is higher than that in low metastatic Hca-P cells; The content of p-Akt (Thr308) and p-Akt (ser473) between two cell lines has no significant difference. (3) After treatment of LN, the level of phosphorylation of 1365 tyrosine residue is higher in high metastatic Hca-F cells than in low metastatic Hca-P cells; The level of phosphorylation of 1313 tyrosine residue and 1349 tyrosine residue has no significant difference between two cell lines; The content of p-plcyl (Tyr783) in high metastatic Hca-F cells is higher than that in low metastatic Hca-P cells; The content of p-Akt (Thr308) and p-Akt (ser473) between two cell lines has no significant difference.Conclusion:(1) After cMet is activated, the tyrosine residues in cMet is phosphorylated by the manner of selective phosphorylation, but not by the all or none manner. (2) Different ligands could cause phosphorylation at different tyrosine residues of cMet. (3) The phosphorylation of 1313 and 1365 tyrosine residues of cMet may be involved in avtivation of PLCγDAG/PKC signaling pathway; Thr phosphorylation of 1349 tyrosine residues of cMet may be closely related to PI3K/AKT signaling pathway activation. (4) The metastatic potential of HCa-F cells in lymph node may be associated with PLCγ/DAG/PKC signal pathway activation.
Keywords/Search Tags:HGF, LN, FN, c-Met, signaltransduction
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