elastin and Fibrosarcoma

The elastin-derived peptides (EDPs) exert protumoural activities by potentiating the secretion of matrix metalloproteinases (MMP) and the plasminogen-plasmin activating system. In the present paper, we studied heat-shock protein 90 (Hsp90) involvement in this mechanism.. HT-1080 fibrosarcoma cell migration and invasion were studied in artificial wound assay and modified Boyden chamber assay, respectively. Heat-shock protein 90 was studied by western blot and immunofluorescence. Matrix metalloproteinase-2 and urokinase plasminogen activator (uPA) were studied by gelatin ± plasminogen zymography and immunofluorescence. Heat-shock protein 90 partners were studied by immunoprecipitation. Messenger RNA expression was studied using real-time PCR. Small interfering RNAs were used to confirm the essential role of Hsp90.. We showed that kappa-elastin and VGVAPG elastin hexapeptide stimulated Hsp90, pro-MMP-2 and uPA secretion within 6 h, whereas AGVPGLGVG and GRKRK peptides had no effect. No increase of mRNA level was observed. Heat-shock protein 90-specific inhibitors inhibit EDP-stimulated HT-1080 cell-invasive capacity and restrained EDP-stimulated pro-MMP-2 and uPA secretions. The inhibitory effect was reproduced by using Hsp90-blocking antibody or Hsp90 knockdown by siRNA. Heat-shock protein 90 interacted with and stabilised uPA and pro-MMP-2 in conditioned culture media of HT-1080 fibrosarcoma cells.. Taken together, our results demonstrate that EDPs exert protumoural activities through an Hsp90-dependent mechanism involving pro-MMP-2 and uPA.

Topics: Cell Line, Tumor; Cell Movement; Elastin; Fibrosarcoma; HSP90 Heat-Shock Proteins; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Peptide Fragments; Transfection; Urokinase-Type Plasminogen Activator

Numerous antagonists of tumor necrosis factor alpha (TNFalpha) have been developed to attenuate inflammation and accompanying pain in many disease processes. Soluble TNF receptor type II (sTNFRII) is one such antagonist that sequesters TNFalpha away from target receptors and attenuates its activity. Systemic delivery of soluble TNF receptors or other antagonists may have deleterious side effects associated with immune suppression, so that strategies for locally targeted drug delivery are of interest. Elastin-like polypeptides (ELPs) are biopolymers capable of in situ drug depot formation through thermally-driven supramolecular complexes at physiological temperatures. A recombinant fusion protein between ELP and sTNFRII was designed and evaluated for retention of bivalent functionality. Thermal sensitivity was observed by formation of supramolecular submicron-sized particles at 32 degrees C, with gradual resolubilization from the depot observed at physiological temperatures. In vitro refolding of the sTNFRII domain was required and the purified product exhibited an equilibrium dissociation constant for interacting with TNFalpha that was seven-fold higher than free sTNFRII. Furthermore, anti-TNF activity was observed in inhibiting TNFalpha-mediated cytotoxicity in the murine L929 fibrosarcoma assay. Potential advantages of this ELP-sTNFRII fusion protein as an anti-TNFa drug depot include facility of injection, in situ depot formation, low endotoxin content, and functionality against TNFalpha.

Topics: Animals; Elastin; Escherichia coli; Fibrosarcoma; Humans; Inhibitory Concentration 50; Kinetics; L Cells; Mice; Peptides; Protein Renaturation; Protein Structure, Tertiary; Receptors, Tumor Necrosis Factor, Type II; Recombinant Fusion Proteins; Solubility; Temperature; Tumor Necrosis Factor-alpha

HT-1080 fibrosarcoma cells express at their plasma membrane the elastin-binding protein (EBP). Occupancy of EBP by elastin fragments, tropoelastin or XGVAPG peptides was found to trigger procollagenase-1 (proMMP-1) overproduction by HT-1080 cells at the protein and enzyme levels. RT-PCR analysis indicated that elastin peptides did not modify the MMP-1 mRNA steady state levels, suggesting the involvement of a post-transcriptional mechanism. We previously reported that binding of elastin peptides to EBP induced other matrix metalloproteinases (MMP-2 and MT1-MMP) expression. Since those peptides were here found to also accelerate the secretion of urokinase from HT-1080 cells, culture medium was supplemented with plasminogen together with elastin peptides at aims to induce or potentiate MMPs activation cascades. In such conditions, plasmin activity was generated and exacerbate proMMP-1 and proMMP-2 activation. As a consequence, elastin peptides and plasminogen-treated HT-1080 cells displayed a significant type I collagen matrix invasive capacity.

Topics: Collagen Type I; Collagenases; DNA Primers; Elastin; Enzyme Activation; Enzyme Precursors; Enzyme-Linked Immunosorbent Assay; Fibrinolysin; Fibrosarcoma; Gelatin; Gelatinases; Humans; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Metalloendopeptidases; Neoplasm Invasiveness; Peptide Fragments; Plasminogen; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

To delineate cis-acting regulatory elements of the human elastin gene, several elastin promoter region/chloramphenicol acetyltransferase reporter gene constructs were developed. The spectrum of inserts, spanning from -2260 to +2, was shown to contain several SP-1 and AP2 binding sites, as well as putative glucocorticoid, cAMP, and 12-O-tetradecanoylphorbol-13-acetate responsive elements. Assay of promoter activity in transient transfections of rat aortic smooth muscle cells, human skin fibroblasts, HT-1080 human fibrosarcoma cells, HeLa cells, or mouse NIH-3T3 cells allowed delineation of several functional subregions within 2.26 kilobases of the 5'-flanking DNA. The results suggest that the basic promoter element resides within the region -128 to -1, and the 5'-flanking DNA contains several functional regulatory subregions. Also, the regulatory function of three putative SP-1 binding sites was demonstrated by transfections with a plasmid devoid of such sequences. These findings attest to the complexity of transcriptional regulation of the elastin gene.

Topics: Animals; Aorta; Base Sequence; Binding Sites; Chromosome Deletion; Cloning, Molecular; Cyclic AMP; DNA-Binding Proteins; Elastin; Fibroblasts; Fibrosarcoma; Glucocorticoids; HeLa Cells; Humans; Molecular Sequence Data; Muscle, Smooth, Vascular; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Rats; Regulatory Sequences, Nucleic Acid; Sp1 Transcription Factor; Tetradecanoylphorbol Acetate; Transcription Factors; Transfection; Tumor Cells, Cultured

Artificial blood vessel walls constructed by the addition of bovine arterial endothelial cells to multilayers of rat smooth muscle cells were used as substrates for the human fibrosarcoma cell line HT1080. The extracellular matrix proteins elaborated by the smooth muscle cells were prelabeled with [3H]-proline; therefore, their subsequent digestion could be followed by the appearance of radioactivity in the culture medium. The fibrosarcoma cells rapidly hydrolyzed smooth muscle multilayers in the absence of endothelial cells, but an endothelial layer markedly retarded the destructive ability of the tumor cells. The protective effect of the endothelium was not due to a lack of penetration of this cell layer, since HT1080 cells were observed by light and electron microscopy to be in the subendothelial area 24 hr after plating. Subsequently, the tumor cells multiplied in the region between the endothelial and smooth muscle layers and, although their degradative ability was retarded, they were ultimately capable of destroying the structure. Endothelial cells also inhibited hydrolysis of the smooth muscle layers if added simultaneously or up to 1 week after HT1080 cells, but the degree of inhibition was not as great as that seen with a preestablished endothelial layer. Measurable inhibition of tumor cell degradative activity was observed at fibrosarcoma:endothelial cell ratios of 25:1, demonstrating the potency of endothelial cells in modulating this aspect of the invasive phenotype. Although the HT1080 cells only slowly degraded the preexisting matrix proteins in artificial vessel wall cultures, they interfered with the production of new connective tissue proteins which occurred in control cultures. These experiments therefore suggest that endothelial cells have profound effects on tumor cell proteolytic activity, and the significance of these observations to tumor cell extravasation in vivo is discussed.

Topics: Animals; Cell Communication; Cell Line; Collagen; Elastin; Endothelium; Fibrosarcoma; Humans; Mice; Models, Biological; Muscle, Smooth, Vascular; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplastic Cells, Circulating; Proline

Four human tumor cell lines were grown in direct contact with the extracellular matrix proteins which had previously been produced by cultured rat smooth muscle cells. The extracellular matrix contained glycoproteins, elastin, and collagen, and its digestion by the tumor cells was followed by the appearance of radioactive breakdown products in the supernatant medium. All four tumor lines tested digested glycoproteins present in the matrix, whereas human fibroblasts were inactive in glycoprotein digestion. The human fibrosarcoma cell line (HT1080) demonstrated elastolytic and collagenolytic activity in addition to a plasmin-induced hydrolysis of glycoproteins. Removal of glycoproteins from the matrix was necessary for the maximal digestion rate of elastin and collagen, and plasmin generation by the tumor cell plasminogen activator therefore played a pivotal role in the hydrolysis of all of the matrix components. The elastolytic and collagenolytic activities were localized to the plasma membrane since no matrix digestion occurred unless the tumor cells were grown in direct contact with the connective tissue proteins. These activities were not inhibited by a wide spectrum of protease inhibitors. The degradation of elastin and collagen required active protein synthesis suggesting a relatively short half-life for the degradative enzyme(s). These quantitative studies, in which tumor cells were grown in contact with a complex extracellular matrix possessing some of the characteristics of connective tissue, should have a bearing on tumor cell invasion.

Topics: Antimetabolites, Antineoplastic; Cell Line; Cell Membrane; Collagen; Connective Tissue; Elastin; Extracellular Space; Fibrosarcoma; Glycoproteins; Humans; Muscle Proteins; Peptide Hydrolases; Plasminogen Activators; Protease Inhibitors; Substrate Specificity

Extracellular matrices produced by cultured rat smooth muscle cells in the presence or absence of ascorbic acid wee used as substrates for the human fibrosarcoma cell line HT1080. The matrix elaborated by smooth muscle cells in the presence of ascorbic acid contained glycoproteins, elastin, and collagen, and all of these components were digested by the tumor cells. In contrast, the matrix elaborated in the absence of ascorbic acid which contained glycoproteins and underhydroxylated elastin but no collagen was more resistant to tumor-induced hydrolysis. The underhydroxylated elastin was particularly refractory to the tumor proteases, suggesting that the elastolytic activity produced by HT1080 cells showed a marked preference for the natural substrate containing hydroxyproline. The digestion by HT1080 cells of elastin from living cultures of smooth muscle cells was also retarded if the extracellular proteins were produced under ascorbic acid-deficient conditions. These experiments therefore do not support the notion that connective tissues made under scorbutic conditions are inherently more susceptible to tumor hydrolysis.

Topics: Ascorbic Acid; Cell Line; Collagen; Connective Tissue; Elastin; Extracellular Space; Fibrosarcoma; Glycoproteins; Humans; Hydrolysis; Kinetics; Muscle Proteins; Muscle, Smooth; Peptide Hydrolases