Effect Of Rac1 Gene On The Invasion Of HT1080 Fibrosarcoma Cell Across Collagen Barrier And Its Related Mechanisms

Background and Aim : During metastasis, invasive cells must traverse tissue barriers comprised largely of extracellular matrix (ECM). This process depends on the ability of tumor cells to degrade the surrounding collagen matrix and then migrate through the matrix defects.The actin dynamics regulated by Rho family of small GTPases play a critical role in cell migration. The initiation of cell migration is characterized by actin polymerization at the leading edge and extension of a lamella in the direction of motion. Rac1, a member of the Rho family proteins that regulates the assembly of a meshwork of actin filaments at the cell periphery to produce lamellipodia, has been implicated in oncogenic transformation and metastasis induction. Recent studies have demonstrated the direct role of Rac1 activity in cell invasiveness in type I collagen matrix. Transfection of non-invasive mammalian epithelial cells T47D with active Rac1 induces cell invasion through type I collagen. Similarly, overexpression of adaptor proteins p130Crk-associated substrate (CAS)1/c-CrkII (Crk) induces Rac1-dependent COS-7 cell invasiveness in three-dimensional collagen (3D-col) culture. However, the proteolytic activities during Rac1-promoted cell invasion through type I collagen barriers remain undefined.The invasive growth and degradation of ECM as well as matrix remodeling around cancer cells are highly coordinated events. MMPs are expressed in various cancer tissues, and their expressive intensity is closely associated with the properties of invasive growth and metastasis. Increasing evidence has suggested that proteolytic activities at cell surface promote cell invasion. MMP2(IV collagenase or gelatinase) is a kind of cell surface-associated type I collagen-degrading MMP, its activation ia associate with MT1-MMP and TIMP.The purpose of our research is to examine the effect of Rac1 gene expression on the invasion of HT1080 fibrosarcoma cell across collagen barrier, and further to investigate the relationship between the increasing invasive ability on type I collagen induced by Rac1 expression and the proteolytic cascade initiated by MT1-MMP and MMP2 activation.Methods:We investigated effect of expression of exogenous Rac1 HT1080 fibrosarcoma cell invasion across collagen barrier. Testing plasmids (Rac1V12N17, Rac1V12 and vector) and a plasmid construct containing the neomycinresistant gene were co-transfected into HT1080 fibrosarcoma cells. Exogenous Rac1 gene expression was detected with western blot analysis. Structure of actin cytoskeleton was stained with Texas Red-conjugated phalloidin to show the morphological characters of the cells cultured in 3D medium containing collagen protein. Assay of cell invasion across collagen barrier was performed on a thin layer of medium gel containing vitrogen covered the membrane of transwell chamber, and two kind of protease inhibitors were used to observe their effects on above-mentioned invasive assay.Then, we observed the effect of Rac1 gene expression on the activity of MMPs in the lysate and conditional medium from 3 D collagen or fibrin cell culture system with gelatin and collagen zymography, and the influence of Rac1 expression on MMP2 expression was studied with Western blot as well. Further more, different protease inhibitors were used to test their effects on MMP2 activation mediated by Rac1.Northern blot and Western blot were used to examine the transcription and expression of MT1-MMP and its degraded fragments. Collagen fibril dissolution test was used to observe ability of 3 kinds of cell to digest a thin layer of collagen. Finally, we observed the effect of specific MMP2 inhibitors on the invasion of HV cells across collagen barrier mediated by Rac1 with transwell test.Results: Exogenous Rac1 gene expression was detected with western blot analysis in the cellular lysate from cells transfected dominant negative (Rac1V12N17-HN), constitutively active Rac1(Rac1V12-HV). Cultured in 3D collagen,HT1080 cells stably expressing Rac1 mutant exhibit distinct morphological and invasive properties, and the increased invasive ability could be eradicated after using MMPs inhibitor.In the 3 D collagen or fibrin cell culture system, the enhancement of MMP2 activation in the cellular lysate or conditional medium from cells stably expressing constitutively active Rac1 could be observed, and also the activated MMP2 increased.However adverse effects can be seen from cells expressing dominant negative Rac1. Meanwhile, in the 3D fibrin cell culture system, we could not find activated MMP2 in the lysate or conditional medium from cells expressing empty vector, but that band could be found from cells expressing constitutively active Rac1.The enhancement of MMP2 activation mediated by Rac1 could be eradicated by SC68180, a kind of broad spectrum MMPs inhibitor, while aprotinin, the other kind of protease inhibitor, did not show this effect. In 3D collagen gel, the level of MT1-MMP transcription and expression in HV cells was relative higher than that in HW or HN cells, and the expression of MT1-MMP degraded fragments was also elevated. The ability of HV cells to degrade collagen fibril was increased. The enhancement of MMP2 activation mediated by Rac1 could be inhibited by CTD, which specifically inhibit cell-associated MMP-2 activation, and this effect showed a dose dependant fashion. The experiment of cell invasion across collagen barrier showed TIMP2, FI and CTD could significantly decrease the enhancement of invasive property mediated by Rac1in HV cells.