| CD82 coded by a tumor-suppressor gene KAI1 is membrane glycoprotein belonged to transmembrane 4 superfamily or tetraspan superfamily(TM4SF) and taked part in a lot of biological events such as tumor invasion,adhesion and migration. CD82 existed in a variety of normal tissues and it appeared deficiency in tumor tissues.A plethora of research results showed that CD82 was broad spectrum of tumor metastasis suppressor, because CD82 inhibited metastasis of a variety source of tumor cells in the body. The expression level of CD82 contributed to evaluate the metastatic capacity and prognosis of tumor.The mechanism of CD82 suppressing tumor metastasis was becoming more and more attention, but the detailed molecular mechanism how it work was still unclear.The CD82 consisted of extracellular domains, transmembrane domains and intracellular domains.The transmembrane domains included four hydrophobic peptides. The extracellular domains were comprised of a small extracellular loop(SEL) and a large extracellular loop(LEL).The intracellular domains existed a N-terminal tail, a C-terminal tail and a intracellular loop. The molecular structure was the basis of function of proteins.The extracellular domains of CD82 was important physiological function structure,CD82 recognizing mutually,combining and interacting with extracellular matrix molecules and other cell directly depend on its extracellular domains. The study for the function of the extracellular domains of CD82 contributed to clear how CD82 inhibiting tumor metastasis.However, there were few studies on the function of the extracellular domain of CD82,especially the function of SEL of extracellular domains was not clear. In this paper,for study the extracellular domains of CD82 in the role of CD82 inhibiting tumor metastasis, CD82-LEL and CD82-SEL mimic peptides were acpuired by the genetic recombination technolo gy and chemical synthesis methods and the studies wes performed as the following.1.Effects of mimic peptides of CD82 extracellular domains in vitro on the migration of tumor cells. CD82 inhibited cell movement and migration was the main mechanism of CD82 inhibiting tumor cell metastasis, so the CD82-LEL and CD82- SEL mimic peptides in vitro inhibiting cell migration was observed through scratching and Boyden chamber culture.Results: The CD82-LEL mimic peptides and CD82-SEL mimic oligopeptides were obtained by gene recombination technique and chemical synthesis method.CD82-LEL mimic peptides could not inhibit SW620 tumor cell migration and invasion in vitro obviously. CD82-SEL mimic oligopeptides significantly inhibited SW620 tumor cell migration and invasion in vitro, and concentration of CD82-SEL mimic oligopeptides higher, the inhibition level was better.Conclusions: CD82-LEL mimic peptides in vitro could not inhibit tumor cell movement and migration. CD82-SEL mimic oligopeptides inhibited tumor cells movement and migration in vitro.The inhibition strengthened with increasing of concentration of CD82-SEL mimic oligopeptides. Results indicated that CD82-SEL is important function structure of CD82 inhibiting tumor metastasis.2. Effect of mimic peptides of CD82 extracellular domains on tumor cells recognition and adhesion with extracellular matrix molecules. CD82 recognition and direct bind of cells and extracellular matrix relied on its extracellular domains. Therefore, extracellular domains was important functional structure of CD82 inhibiting tumor metastasision.so, in this part, the effect of mimic peptides of CD82 extracellular domains on SW620 tumor cells adhesion with Laminin(Laminin, LN), Fibronectin(Fibronectin, FN) and Matrigel were observed.Results: CD82-LEL mimic peptides could not inhibit SW620 tumor cell adhesion with LN, FN and Matrigel obviously. CD82-SEL mimic oligopeptides significantly inhibited SW620 tumor cell adhesion with LN, FN and Matrigel, and there was positive correlation between its inhibition and treatment concentration.Conclusions: CD82-LEL mimic peptides could not participate in tumor cells recognition and adhesion with extracellular matrix molecules alone that may be caused by losting the sugar chain structure.CD82-SEL mimic oligopeptides inhibited tumor cells recognition, adhesion with extracellular matrix molecules, which may related to CD82-SEL mimic oligopeptides inhibiting tumor cells migration.3. Effect of CD82-SEL mimic oligopeptides on the integrin interaction with extracellular matrix molecules. Iintegrin was only adhesion molecule on cell surface that ligand was extracellular matrix molecules, including FN, LN, Vitronecin(VN), Collagen(CA) and mediated cell-extracellular matrix adhesion.Literatures was reported, CD82 inhibited intigrins with the combination of LN and found CD82 with intigrins, growth factor receptors, and other TM4 SF members combined into complex family played a role in cell surface.To explore the mechanism of CD82-SEL mimic oligopeptides inhibition cell migration and adhesion cell with extracellular matrix,in this part, preliminary study on the role of integrins in the CD82-SEL mimic oligopeptides inhibition recognition and adhesion of tumor cells with extracellular matrix molecules was carried out.Results:(1) The expression profiles of subunits types of integrins in SW620 tumor cells were detected by Western Blot technique. The results showed that SW620 cells express α3, α4, α5, α6, β1 and β3 subunits of integrins.(2) The extracellular matrix molecule LN promoted expression of α3, α6 and β1 subunits of integrins in the SW620 cell, FN promoted expression of α4, α5 and β1 subunits of integrins in the SW620 cell. Matrigel promoted expressiion of α3 and β1 subunits of integrins in the SW620 cell.(3)The CD82-SEL mimic oligopeptides inhibited expression of α3, α4, α5, α6 and β1 subunits of integrins which induced expression by LN, FN and Matrigel in SW620 cell respectively.The effect of inhibition enhanced with the increase of the concentrations of CD82-SEL mimic oligopeptides.(4) The subunits α3, α6, β1 of integrins combined with LN.The subunits α4, α5 and β1 combined with FN. The subunits α3 and β1 combined with Matrigel.(5) The CD82-SEL mimic oligopeptides inhibited subunits of integrins combination with extracellular matrix molecules LN, FN and Matrigel.Conclusions:The CD82-SEL mimic oligopeptides inhibited expression of subunits of integrins in SW620 cell which induced expression by extracellular matrix molecules, as well as inhibited direct combination of subunits of integrins and extracellular matrix molecules LN, FN, and Matrigel.4. Effects of CD82-SEL mimic oligopeptides on the phosphorylation of FAK tyrosine residues in SW620 cells. Focal adhesion kinase(FAK) was key signalling molecules in signalling pathways mediated by integrin. FAK was activated by integrin dependent cell-extracellular matrix adhesion and then regulated cell growth, proliferation, division and migration movement.To investigate the mechanism of CD82-SEL mimic oligopeptides inhibition movement and migration of SW620 cell,we furture observed the effect of CD82-SEL mimic oligopeptides on the phosphorylation of FAK in SW620 cell.Results:(1) The phosphorylation of tyrosine residues of FAK in SW620 tumor cells were detected by Western Blot. The results showed that 397,407,576,577,861 and 925 tyrosine residues of FAK in SW620 cells were phosphorylated.(2)The extracellular matrix molecules LN, FN, Matrigel promoted the phosphorylation of 397, 407 and 861 tyrosine residues of FAK,but did not promoted the phosphorylation of 925 tyrosine residues of FAK.(3)The CD82-SEL mimic oligopeptides inhibited the phosphorylation of 397, 407 and 861 tyrosine residues of FAK which were promoted by extracellular matrix molecules LN, FN and Matrigel.Conclusions: The CD82-SEL mimic oligopeptides inhibited the phosphorylation of FAK by inhibiting integrins of SW620 cell combination with extracellular matrix that may be one of important mechanisms of the CD82-SEL oligopeptides inhibiting the migration of tumor cells. |
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