Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been shown that the type IV collagenase with its two subtypes, 72 kDa/ MMP-2 and 92 kDa/MMP-9, plays an important role in tumor invasion and metastasis formation that occur through a mechanism of proteolytic degradation of collagen IV in the basement membrane. One possible method to specifically inhibit the function of the targeted protein of a cell is to express intracellular antibody combining site that can block the function or prevent the expression of the targeted molecule. Accordingly, intracellular antibodies against type IV collagenase may have a therapeutic use against tumor invasion and metastasis. As described in our previous reports, an anti-type IV collagenase monoclonal antibody (3D6) was obtained using the hybridoma approach, and its functional single-chain Fv fragment (scFv) named M97 was constructed based on recombinant phage display techniques. In this study, the endoplasmic reticulum (ER)-retained scFv antibody fragment was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acid (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector containing the CMV early-intermediate promoter/enhancer. The resulting plasmid was sequenced and then introduced by the lipofectamine method into PG cells, a highly metastatic human lung cancer cell line and G418-resistant cells were obtained by G418 selection. After transfection, the M97 mRNA expression was observed and the type IV collagenase expression was downregulated significantly as measured by ELISA. The biological behavior of PG cells, such as the ability of in vitro invasion of colony formation on soft agar through Matrigel, were also inhibited by scFv M97 transfection. The results indicate that intracellular antibody technology represents a novel and efficient way to selectively abrogate the activity of type IV collagenase, at least in vitro. We are presently exploring the efficacy of this approach in a xenograft model of human lung cancer.
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PMID:Antineoplastic effect of intracellular expression of a single-chain antibody directed against type IV collagenase. 1090 9

The expression and activation of matrix metalloproteinases (MMPs) by tumor cells is correlated with progression to invasive and metastatic status. The purpose of this study was to examine the role of increased MMP-2 (gelatinase A) expression in prostate cancer progression utilizing human prostate PC-3 cancer cells that overexpress MMP-2 using gene transfection. PC-3 cells were transfected with pCR-3 vector only and pCR-3 MMP-2 plasmids employing the LipofectAMINE method, and stable transfectants were selected with G418. The expression of MMP-2, tissue inhibitor of metalloproteinase-2 (TIMP-2), and membrane-type MMP 1 (MT1-MMP) in PC-3 parental and transfected cells under serum-free conditions was determined by zymography, immunoblotting, immunofluorescent microscopy, Northern blotting, and/or reverse transcriptase-polymerase chain reaction (RT-PCR). MMP-2 transfected cells produced primarily the proenzyme form of MMP-2; the parental and vector control transfected PC-3 cells did not express any MMP-2 that was detectable by the methods we employed. Treatment of PC-3 MMP-2 transfected cells with Concanavalin A (Con A), in contrast to HT-1080 cells, processed only a small amount of the secreted 72-kd proenzyme to a 62-kd intermediate and a cell-associated 59-kd active form. The low level of secreted pro-MMP-2 processing induced by Con A was inhibited by serine protease inhibitors and was unaffected by cyclic adenosine monophosphate (cAMP). Immunoblotting showed that these cells produced abundant TIMP-2 and lower amounts of MT1-MMP in comparison with Con A-responding HT-1080 cells. HT-1080 cells respond to Con A by translocating MT1-MMP from intracellular localization sites to the plasma membrane, an effect not observed in PC-3 cells. The molecular basis for the low level of processing of pro-MMP-2 by PC-3 cells may be due to an overabundance of TIMP-2 and/or a low level of cell surface active MT1-MMP.
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PMID:Limited processing of pro-matrix metalloprotease-2 (gelatinase A) overexpressed by transfection in PC-3 human prostate tumor cells: association with restricted cell surface localization of membrane-type matrix metalloproteinase-1. 1476 14

Angiotensin II is well implicated in neointimal proliferation and the resulting restenosis, however, the mechanisms involved remain unclear. The type 2 angiotensin II (AT2) receptor, largely unexpressed in the adult vasculature, however, appears at significant levels after vascular injury. To investigate the specific contribution of AT2 receptor and the interplay of the angiotensin system to neointima, we engineered rat vascular smooth muscle cells (VSMCs) to express the AT2 receptor in a tetracycline-regulated system. Several VSMC clones resistant to both hygromycin and G418 were selected, many of which showed high, but regulatable levels of AT2R expression within 48 h of doxycycline (Dox) exposure. In untransfected VSMCs and stable transfectants with no AT2R induction, Ang II significantly increased the expression of matrix metalloproteinase 2 (MMP-2), which is linked to neointimal growth. However, induction of AT2R by Dox addition markedly decreased MMP-2 levels (P<0.01) and this downregulation was further promoted by CV-11974, a specific antagonist of AT1 receptor. In contrast, the PD123319 compound, which selectively curtails the AT2 receptor, reversed the inhibition caused by CV-11974. We conclude that Ang II enhances the MMP-2 expression via AT1R, and that enforces AT2R inhibited the same. These data confirm that AT2R functions to downregulate the effects elicited by Ang II + AT1R signaling and point to the role of MMP and extracellular matrix in vascular injury. The findings provide fresh experimental approaches to prevent or control restenosis through transduction of VSMCs expressing optimal levels of AT2R.
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PMID:Conditional expression of type 2 angiotensin II receptor in rat vascular smooth muscle cells reveals the interplay of the angiotensin system in matrix metalloproteinase 2 expression and vascular remodeling. 1951 42