| Objective: Hepatocyte growth factor is one kind of polypeptide cytokines generated from mesenchymal cells(such as mechanocyte and macrophage etc.). HGF has many important biological functions, such as promoting proliferation and division of cells; suppressing the formation of cell gap junctions and cell adhere lead to cell scattering ; up-regulating the expression of urokinase plasminogen activator and its receptor (uPA/uPAR), promoting the degradation of extracellular matrix; increasing the phosphorylation of cytoskeletal protein and interfering the rearrangment of cytoskeleton, which make cells apt to migration. So, HGF is also named as mitogenic factor,scattering factor and morphogen. HGF plays an important role in regulation of embryo development,organic formation,cells growth,proliferation and migration . It is also concerned with generation,development,diffusion and metastasis of tumor.The receptor of HGF is coded by proto-oncogene c-met and is also called c-Met. c-Met is one member of receptor tyrosine protien kinase family and often expresses in epithelial cell(such as liver,kidney,enteron). The expression of c-Met in tumor cells is higher and has positive correlation with metastasis potential of tumor cell. HGF acts on target cell by paracrine after secretion from mesenchymal cells. Once conjugated with HGF, the receptors are dimerizated and activated, then ignite the signaling pathway in cells. It is known that, at least, there are three signaling pathways activated by HGF/c-Met, including MAPK signaling pathway, PLCγ1/DAG/PKC signaling pathway and PI3K/PKB signaling pathway. Among these, PI3K/PKB signaling pathway and PLCγ/PKC signaling pathway are the key pathways for regulating the invation and metastasis of tumor cell. Both of KAI-1/CD82 and MRP-1/CD9 belong to the tetraspanin or trans- membrane-4 superfamily protens. CD82 is coded by tumor metastasis suppressor gene KAI-1. At present, It is konwed as a broad-spectrum suppressor of metastasis. CD9 is also called cell motility-related protein-1. Both of them can down-regulate the cell motility, and inhibit the invation and metastasis of tumor cell. A lot of experiment date has shown that CD82/CD9 can interact with c-Met, and thereby inhibits HGF-induced Met tyrosine kinase activity, as well as integrin to Met cross-talk, down-regulate PI3K/PKB and PLCγ/PKC signaling pathway. This is the mechanism by witch CD82 and CD9 down-regulate the cell motility, and inhibit the invation and metastasis of tumor cell.Both of Hca-F cell strain and Hca-P cell strain are established from the same parental cell line. Hca-F is the high lymphatic metastasis potential cell strain and its lymphatic metastasis rate is 80%; Hca-P is low lymphatic metastasis potential cell strain and its lymphatic metastasis rate is 0-20%. However the molecular mechanism involved in the different lymphatic metastasis potential between these two cell strains remains unclear. To investigate the molecular mechanism of tumor lymphatic metastasis, The expression of CD8, CD9 and c-Met on Hca-F and Hca-P cells was examined and the activity of PI3K/PKB and PLCγ/PKC signaling pathway in two cell lines was analyzed comparatively.Method: Hca-F cell and Hca-P cell were cultured in mouse abdominal cavities. Then the cells were harvested and the cytosol proteins and plasma membrane proteins were prepared. The expression of CD82/CD9 and activity of PI3K/PKB signaling pathway and PLCγ/PKC signaling pathway were analysed by using Western Blotting and computer scanning technique.Result: 1)expression of CD82/CD9:The contents of CD9 have no significant difference between Hca-F cell and Hca-P cell, while the content of CD9 in Hca-P cell increased by 1.31 fold comparing with Hca-F cell, It suggests that CD9 maybe the key factor effecting the lymphatic metastasis potential of Hca-F and Hca-P cells.2) PI3K/ PKB signaling pathway: The distribution of PDK and the phosphorylation of PKB in Hca-F and Hca-P cell were analysed by using Western Blotting. The results shown that there were no significant differences on the expression of the phosphorylated PKB between the two cell lines; It is the same with the expression and distribution of PDK; The results suggest that PI3K/PKB signaling pathway isn't the main pathway effecting the tumor lymphatic metastasis potential. 3) PLCγ1/PKC signaling pathway:The acti- vety of PLCγ1and different hypotypes of PKCs among these two cell strains were analyzed by using Western Blotting. The results displayed that phosphorylated PLCγ1 in Hca-P cell significantly decressed by 45.23% comparing with Hca-F cell. The contents of PKCα,PKCβ1and PKCβ2 were obviously decressed by 71.75%,76.80% and 73.16% comparing with Hca-F cell; The results showed that the activity of PLCγ1/PKC signaling pathway in Hca-F cell is higher than that of Hca-P cell. Thus, PLCγ1/PKC signaling pathway maybe the major pathway effecting the the tumor lym- phatic metastasis potential.Conclusion:The expression of the tumor metastasis suppressor CD9 in Hca-P cell is higher comparing with Hca-F cell;Meantime, activity of PLCγ1/PKC signaling pathway in Hca-P cell has lower than that of Hca-F cell. These may be the important factors that effect the lymphatic metastasis potential of Hca-F and Hca-F cells.
|
PDF Full Text Request