batimastat and Fibrosarcoma

Collagenase-3 (MMP-13) is characterized by an exceptionally wide substrate specificity and restricted expression. MMP-13 is 1 of the few MMPs primarily expressed by tumor cells in malignant tumors, e.g., squamous cell carcinomas and its expression correlates with their invasion capacity. In this work, we have constructed an expression vector and a recombinant adenovirus harboring human MMP-13 cDNA to investigate the role of MMP-13 in cancer cell invasion. Our results show that constitutive expression of MMP-13 by HT-1080 cells stably transfected with MMP-13 expression vector or transduced with MMP-13 adenovirus markedly increased their invasion both through type I collagen and reconstituted basement membrane (Matrigel) with no alterations in expression or activation of collagenase-1 (MMP-1), gelatinase-A (MMP-2), or gelatinase-B (MMP-9). The enhanced invasion capacity of MMP-13 expressing HT-1080 cells was dependent on MMP activity, as it was blocked by MMP inhibitor Batimastat (BB-94) and tissue inhibitor of metalloproteinases-3 (TIMP-3). Our data provide direct evidence for the role of MMP-13 as a potent invasion proteinase, which alone can enhance the ability of malignant cells to penetrate through both basement membrane and fibrillar collagen.

Topics: Adenoviridae; Basement Membrane; Carcinoma, Squamous Cell; Collagen; Collagen Type I; Collagenases; DNA, Complementary; Drug Combinations; Fibrosarcoma; Genetic Vectors; Humans; Laminin; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Models, Genetic; Neoplasm Invasiveness; Neoplasm Metastasis; Phenylalanine; Proteoglycans; Thiophenes; Tissue Inhibitor of Metalloproteinase-3; Transfection; Tumor Cells, Cultured

Matrix metalloproteinase-2 degrades a variety of basement membrane components and is essential for tumor invasion. We have previously reported that membrane type-1 matrix metalloproteinase (MT1-MMP) cooperates with neutrophil-derived serine proteinases (NDPs; elastase, cathepsin G, protease-3) to activate matrix metalloproteinase-2. We therefore hypothesized that NDPs enhance tumor-cell invasion.. Clones of human HT1080 fibrosarcoma cells transfected with MT1-MMP sense (HT-SE) or antisense CDNA (HT-AS) were used. These cells express either high (HT-SE) or extremely low levels (HT-AS) of MT1-MMP relative to nontransfected HT1080 cells (HT-WT). The cells were incubated in the presence or absence of purified NDP, with or without alpha 1-antitrypsin or the MMP inhibitor batimastat. Cell invasion was measured with the use of Boyden chambers with polycarbonate membranes coated with a reconstituted extracellular matrix.. Under control conditions HT-WT and HT-SE cells were 4-fold more invasive than HT-AS cells. The addition of NDP increased HT-WT and HT-SE cell invasion 60% to 100% but had no effect on HT-AS cells. alpha 1-antitrypsin or batimastat did not decrease the baseline invasiveness of HT-WT and HT-SE cells; however, they abrogated the stimulatory effect of NDP.. HT1080 cell invasion depends on MT1-MMP expression. MT1-MMP overexpression does not increase invasiveness by itself. NDPs increase invasion by MT1-MMP expressing cells by activating matrix metalloproteinase-2.

Topics: alpha 1-Antitrypsin; Cathepsin G; Cathepsins; Culture Media, Conditioned; Fibrosarcoma; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Myeloblastin; Neoplasm Invasiveness; Neutrophils; Pancreatic Elastase; Phenylalanine; Serine Endopeptidases; Serine Proteinase Inhibitors; Thiophenes; Tumor Cells, Cultured

Matrix metalloproteinase-2 (MMP-2), an enzyme involved in tumor invasion, is secreted as an inactive proenzyme and requires interaction with membrane-type 1 MMP (MT1-MMP) for activation. We have previously demonstrated that polymorphonuclear neutrophils (PMNs) release a soluble factor(s) that activates pro-MMP-2. Therefore, we tested the hypothesis that PMN-derived proteinases act in concert with MT1-MMP to activate pro-MMP-2.. Human HT-1080 cells transfected with MT1-MMP cDNA (HT-SE) or the corresponding antisense cDNA (HT-AS) or an empty vector (HT-V), which expressed differing levels of MT1-MMP, were incubated with serum-free, human PMN-conditioned medium with or without proteinase inhibitors. The culture supernatants were analyzed by gelatin zymography.. Ht-1080 cells expressing basal (HT-V) or low levels (HT-AS) of MT1-MMP secreted MMP-2 in proenzyme from (72 kd). Ht-1080 cells with high levels of MT1-MMP (HT-SE) secreted pro MMP-2 and a 68 kd intermediate activation product. Addition of PMN-conditioned medium to either HT-SE or HT-V clones resulted in dose-dependent generation of active, 62 kd MMP-2. In contrast, when PMN-conditioned medium was added to HT-AS clones, no MMP-2 activation occurred.. PMN-derived serine proteinases act in concert with MT1-MMP to activate proMMP-2. This finding indicates a potential role for inflammatory cells in promoting extracellular matrix breakdown during tumor invasion.

Topics: alpha 1-Antitrypsin; Aprotinin; Collagenases; Cysteine Proteinase Inhibitors; Endopeptidases; Enzyme Activation; Enzyme Precursors; Fibrosarcoma; Gelatinases; Humans; Leucine; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Neoplasm Invasiveness; Neutrophils; Pepstatins; Phenylalanine; Protease Inhibitors; Serine Proteinase Inhibitors; Thiophenes; Transfection; Tumor Cells, Cultured