cathepsin-g and batimastat

We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.

Topics: Adult; Amides; Calcimycin; Cathepsin G; Cathepsins; Cells, Cultured; Culture Media, Conditioned; Endothelium, Vascular; Enzyme Activation; Enzyme Precursors; Humans; Inflammation; Ionomycin; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Neoplasm Metastasis; Neutrophils; Oligopeptides; Pancreatic Elastase; Phenylalanine; Protease Inhibitors; Proteins; RNA, Messenger; Serine Endopeptidases; Substrate Specificity; Tetradecanoylphorbol Acetate; Thiophenes; Tissue Inhibitor of Metalloproteinase-2; Umbilical Veins

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