| Early metastasis is the primary cause of death in melanoma patients. A hallmark of melanoma progression is the ability of neoplastic cells to invade several extracellular matrix (ECM)barriers, including basement membrane and dermal connective tissue. ECM plays important roles in maintaining tissue structure and in regulating cell morphology, differentiation, migration, proliferation and functions. Information provided by ECM can control processes of embryonic growth and differentiation, tissue remodeling and repair. ECM is no longer an inert scaffold but plays an active role in the control of tumor cells and tumor growth. Earlier work also demonstrated that ECM molecules signal interacted with their respective transmembrane receptors and induced clustering of integrin receptors to form focal adhesion complexes, resulting in synergistic activation of intracellular signaling cascades. Matrix Metalloproteinases (MMPs) are a family of enzymes that specifically degrade ECM components , and MMPs play a key role in the turnover of ECM during various physiological and pathological processes, including tissue repair, wound healing, fibrosis and tumor invasion. To illustrate the importance of ECM in directing MMP activity and to delineate how those partners can be interrelated in cancer, we will focus on one member of the MMP family , i.e. MMP-2 or gelatinase A. It has been now widely documented that proMMP-2 activation first necessitates the formation of a ternary complex between proMMP-2, tissue inhibitor of metalloproteinase-2 (TIMP-2), and membrane type 1-matrix metalloproteinase (MT1-MMP) at the cell plasma membrane; enzyme activation would then require the participation of another free MT1-MMP molecule.The integrins are a family of cell surface glycoproteins which mediates several cellular adhesive functions. While in some cases integrins can mediate direct cell-cell interactions, they are primarily responsible for the adhesion of cells to the extracellul- ar matrix. Although integrins were originally characterized as a family of cell surface receptors that are responsible for anchoring cells to the ECM, they have recently been shown to impact on such dynamic processes, in normal and tumor cells, as intracellular signaling and gene expression that leads to cell migration, proliferation, differentiation and survival. Integrins are heterodimeric molecules comprised of one each of the 16 known α subunits and the 8 known β subunits, and integrins recognize the tripeptide Arg-Gly-Asp (RGD). Bidirectional signal transduction by integrins is a model of integrin function based on numerous observations that integrins can transmit information both out of cells (``inside-out'') and into cells (``outside-in'').The two melanoma cell lines used in this study (M21or and M21-L) were chosen because of their difference in expression ofαVβ3 integrin, that is ,M21-L is anαVβ3- integrin-negative(β3)cell line.Then,they are the useful rool for us to study the role ofαVβ3 integrin in regulating the expression and activation of MMP-2. M21 cells and M21-L cells were cultured on several types of substrats including, three dimensional (3-D) type I collagen gel,FN and ECM gel containing the basement membrane components(LN and type Ⅳ collagen ). To prepare serum-free conditioned medium (SFCM), cells were harvested by trypsinization and counted using a hemacytometer and 2×105 cells/well were seeded in 24-well tissue culture plates coated with these ECM components,respectively. After 12h, cells were washed three times with phosphate-buffered saline, followed by incubation in serum-free medium. SFCM was collected 48 h later and stored at -80°C until use.Then,we examined the expression of MMP-2 by zymography, and the expression of MT1-MMP mRNA were examined by reverse transcription polymerase chain reaction (RT-PCR).Results:1,M21 cells cultivated on culture dishes expressed proenzyme of MMP-2 stably.2,Culture of M21 cells on 3D-type I collagen gel,FN and ECM gel up-regulated the expression of pro-MMP-2 pro...
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