cathepsin-g and Fibrosarcoma

As part of a continuing search for potential anticancer drug candidates from antimicrobial peptides of marine organisms, tilapia (Oreochromis mossambicus) hepcidin TH2-3 was evaluated in several tumor cell lines. The results indicated that TH2-3, a synthetic 20-mer antimicrobial peptide, specifically inhibited human fibrosarcoma cell (HT1080 cell line) proliferation and migration. The way in which TH2-3 inhibited HT1080 cell growth was then studied. TH2-3 inhibited HT1080 cell growth in a concentration-dependent manner according to an MTT analysis, which was confirmed by a soft-agar assay and AO/EtBr staining. Scanning electron microscopy revealed that TH2-3 caused lethal membrane disruption in HT1080 cancer cells, and a wound healing assay supported that TH2-3 decreased the migration of HT1080 cells. In addition, c-Jun mRNA expression was downregulated after treatment with TH2-3 for 48-96 h compared to the untreated group. These findings suggest a mechanism of cytotoxic action of TH2-3 and indicate that TH2-3 may be a promising chemotherapeutic agent against human fibrosarcoma cells.

Topics: Animals; Antimicrobial Cationic Peptides; Antineoplastic Agents; Calpain; Caspase 3; Cathepsin G; Cathepsins; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Cell Survival; Chlorocebus aethiops; COS Cells; Down-Regulation; Fibrosarcoma; Fish Proteins; Gene Expression; HeLa Cells; Hepcidins; Humans; Proto-Oncogene Proteins c-jun; Serine Endopeptidases; Tilapia; Wound Healing

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

Activity of lysosomal (cathepsins A,B,C,D and E) and nonlysosomal proteases (cathepsin G, elastase, collagenase, prolidase, prolinase) was evaluated in fibrosarcoma induced in rats by methylcholanthrene. No differences were found in the activity of the examined proteases in tumours of different size in the external, intermediate and central spheres of these tumours. Activity of cathepsins A,B,C,D,E and G, prolidase and prolinase was higher in the fibrosarcoma and activity of collagenase and elastase was lower than in the rat skin.

Topics: Animals; Carboxypeptidases; Carcinogens; Cathepsin A; Cathepsin B; Cathepsin C; Cathepsin D; Cathepsin E; Cathepsin G; Cathepsins; Collagenases; Dipeptidases; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Fibrosarcoma; Lysosomes; Male; Methylcholanthrene; Rats; Rats, Wistar; Serine Endopeptidases; Skin; Skin Neoplasms

Activity of lysosomal and nonlysosomal proteases and contents of protein and its degradation products in the blood serum of rats with methylcholantrene fibrosarcoma were evaluated. Activity of lysosomal proteases and prolidase and prolinase as well in the blood serum of rats with methylcholanthrene tumour did not differ from the activity of these enzymes in the blood serum of control rats. Only the activity of elastase and collagenase in the blood serum of rats with methylcholanthrene tumour especially with tumour of intermediate and big mass was increased. Content of total protein was decreased in the blood serum of rats with tumour of intermediate and big mass and contents of glycoproteins and alfa-amin nitrogen were increased in comparison to the blood serum of control rats.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Blood Proteins; Carboxypeptidases; Carcinogens; Cathepsin A; Cathepsin B; Cathepsin C; Cathepsin D; Cathepsin E; Cathepsin G; Cathepsins; Collagenases; Dipeptidases; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Fibrosarcoma; gamma-Glutamyltransferase; Glycoproteins; Lysosomes; Male; Methylcholanthrene; Nitrogen; Pancreatic Elastase; Rats; Rats, Wistar; Serine Endopeptidases; Skin Neoplasms

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).

Topics: Amino Acid Sequence; Blotting, Western; Cathepsin G; Cathepsins; Chymotrypsin; Collagenases; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Precursors; Fibrosarcoma; Glycoproteins; Humans; Kinetics; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Molecular Sequence Data; Phenylmercuric Acetate; Serine Endopeptidases; Substrate Specificity; Tissue Inhibitor of Metalloproteinases; Trypsin; Tumor Cells, Cultured