Modulation Of Heparanase's Expression And Activity By Selective Inhibition Of EIF-4E In Human Colon Adenocarcinoma Cell Line: An Investigation Of The Molecular Mechanism

For a malignant tumor cell to metastasize, it must break away from its neighbors, force its way through the surrounding stroma, and penetrate basement membranes to enter the stroma, and penetrate basement membranes to enter the circulation. A critical event in the process of cancer invasion and metastasis is therefore degradation of various constituents of the extracellular matrix (ECM) including collagen, laminin,fibronectin, and heparan sulfate proteoglycans(HSPGs). The malignant cell is able to accomplish this task through the concerted sequential action of enzymes such as metalloproteinases, serine proteases, and endoglycosidases. Among these enzymes is an endo- P -glucuronidase (heparanase) that selectively degrades the heparan sulfate chains of HSPGs, essential and ubiquitous macromolecules associated with the cell surface and ECM of a wide range of cells and tissues. Heparanase degrades the heparan sulfate proteoglycans (HSPGs) and is a critical mediator of tumor metastasis and angiogenesis. Recently, it has been cloned as a single gene family and found to be a potential target for antimetastasis drugs. So, it's very helpful to investigate the molecular basis for the regulation of heparanase expression.Eukaryotic initiation factor 4E (eIF-4E) is the 25kDa mRNA cap-binding phosphoprotein that is rate-limiting for the initiation of cap-dependent mRNA translation by the eIF-4F translation initiation complex. A widespreadincreased expression of eIF-4E has been found in human carcinoma tissue and tumor cell lines. The increased eIF-4E activity provides a central regulatory step facilitating the collective overexpression of key malignancy-related gene products such as ODC, c-myc, cyclin D1,VEGF or MMP-9,etc. The cooperative overexpression of these potent molecules leads to the occurrence of tumorigenic phenotype that conspire to drive metastatic progression. However, the mechanisms underlying these regulatory events remain unclear. Since eIF-4E binds to the mRNA cap, it has the potential to alter gene expression at levels including translational initiation, mRNA splicing, mRNA 3'-end processing, mRNA nucleocytoplasmic transport, and protection against 5'-exonucleolytic degradation. The aim of this study were:?to observe whether the selective inhibition of eIF-4E leads to the alteration of heparanase expression at enzymatic activity and protein levels, and determine whether the eukaryotic initiation factor-4E(eIF-4E) is involved in the cap-dependent translational regulation of heparanase in human colon adenocarcinoma cell line :LS-174T cells. (2) To determine whether the over-expressed eukaryotic initiation factor-4E (eIF-4E) is involved in the nucleocytoplasmic transport of heparanase mRNA and explore the roles of eIF-4E in regulation of heparanase expression at post-transcriptional levels in LS-174T.(3) To determine whether the eukaryotic initiation factor-4E (eIF-4E) inhibition facilitate the degradation of heparanase mRNA and explore the alteration of heparanase protein expression in human colon adenocarcinoma cell line :LS-174T. ~~ > Effect of eIF-4E inhibition on the expression and activity of heparanase in LS-174T In order to repress the expression of eIF-4E, a 20-mer antisense s-oligodeoxynucleotide (asODN) complementary to the translation start site of eIF-4E mRNA were introduced into human colorectal cancer cell line LS-174T cells via lipofactin. The results revealed that this sequence specific asODN against eIF-4E specifically and significantly inhibited eIF-4E expression at the transcriptional and translational levels, which were detected by Western blot analysis and RT-PCR respectively. Following eIF-4E inhibition, both 50kDa and 65kDa heparanase proteinexpression were effectively retarded. (According to a published data, A 50 kDa protein correspond to active mature heparanase, while a 65kDa protein correspond to the precursor form of heparanase.) Meanwhile, the enzymatic activity of heparanase was markedly reduced as well. In these conditions, the invasiveness estimated by using mod...