Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCysuPA(19-31)) directed against cysteine proteases and the urokinasetype plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the Nterminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCysuPA(19-31) or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OVMZ-6\#8). Additionally, OVMZ 6\#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitorproducing cells was significantly reduced compared to vectortransfected control cells. Thus, these novel, compact, and smallsize inhibitors directed against up to three different tumorassociated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis

Cell surface antigens in renal tumour cells: detection by immunoluminescence and enzymatic analysis

Two renal cell carcinoma cell lines (49RC 43STR and 75RC 2STR) were characterized by detection of the cell surface proteins: CD44(var), intercellular adhesion molecule-1 (ICAM-1), urokinase-type plasminogen activator (uPA) and its receptor and aminopeptidase N (APN). To detect their localization the immunoluminescent technique was used. In addition, the enzyme activity of uPA and APN was investigated in cell suspensions as well as in monolayers. The latter procedure was more advantageous since the additional use of HPLC permits a single registration of the fluorescent hydrolysis-product AMC (7-amino-4-methylcoumarin) without interference by cellular autofluorescence or non-reacted fluorescent substrate. Unlike 75RC 2STR, the cell line 49RC 43STR expressed high levels of uPA and APN. Contrary to that the cell line 75RC 2STR expressed high levels of ICAM-1 and CD44(v6), whereas 49RC 43STR showed a low level of ICAM-1 and no distinct light signal with anti-CD44(v6). The uPA activity was measured directly as well as indirectly (via plasmin) with the substrate Z-Gly-Gly-Arg-AMC. Both activator and plasmin activity were inhibited by D-Val-Phe-Lys-CMK and phenylmethylsulfonyl fluoride. The anti-catalytic antibody to uPA and that to uPA receptor were found to be inhibiting the uPA activity in a concentration-dependent manner. APN activity was assayed using alanine-p-nitroanilide. Peptidase activity was effectively inhibited by 1,10-phenanthroline and partly inhibited by ethylenediamine-tetraacetic acid. © 2001 Cancer Research Campaignhttp://www.bjcancer.co

Expression pattern of the urokinase-plasminogen activator system in rat DS-sarcoma: Role of oxygenation status and tumour size

The urokinase plasminogen activator system plays a central role in malignant tumour progression. Both tumour hypoxia and enhancement of urokinase plasminogen activator, urokinase plasminogen activator-receptor and plasminogen activator inhibitor type 1 have been identified as adverse prognostic factors. Upregulation of urokinase plasminogen activator or plasminogen activator inhibitor type 1 could present means by which hypoxia influences malignant progression. Therefore, the impact of hypoxia on the expression pattern of the urokinase plasminogen activator system in rat DS-sarcoma in vivo and in vitro was examined. In the in vivo setting, tumour cells were implanted subcutaneously into rats, which were housed under either hypoxia, atmospheric air or hyperoxia. For in vitro studies, DS-sarcoma cells were incubated for 24 h under hypoxia. Urokinase plasminogen activator and urokinase plasminogen activator-receptor expression were analysed by flow cytometry. Urokinase plasminogen activator activity was measured using zymography. Plasminogen activator inhibitor type 1 protein levels in vitro and in vivo were examined with ELISA. PAI-1 mRNA levels were determined by RT–PCR. DS-sarcoma cells express urokinase plasminogen activator, urokinase plasminogen activator-receptor, and plasminogen activator inhibitor type 1 in vitro and in vivo. The urokinase plasminogen activator activity is enhanced in DS-sarcomas compared to normal tissues and rises with increasing tumour volume. The oxygenation level has no impact on the urokinase plasminogen activator activity in cultured DS-sarcoma cells or in solid tumours, although in vitro an increase in plasminogen activator inhibitor type 1 protein and mRNA expression after hypoxic challenge is detectable. The latter plasminogen activator inhibitor type 1 changes were not detectable in vivo. Hypoxia has been demonstrated to contribute to the upregulation of some components of the system in vitro, although this effect was not reproducible in vivo. This may indicate that the serum level of plasminogen activator inhibitor type 1 is not a reliable surrogate marker of tumour hypoxia

Induction of plasminogen activator inhibitor type-1 (PAI-1) by hypoxia and irradiation in human head and neck carcinoma cell lines

Contains fulltext : 53187.pdf ( ) (Open Access)BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) often contain highly radioresistant hypoxic regions, nonetheless, radiotherapy is a common treatment modality for these tumours. Reoxygenation during fractionated radiotherapy is desired to render these hypoxic tumour regions more radiosensitive. Hypoxia additionally leads to up-regulation of PAI-1, a protein involved in tumour progression and an established prognostic marker for poor outcome. However, the impact of reoxygenation and radiation on PAI-1 levels is not yet clear. Therefore, we investigated the kinetics of PAI-1 expression and secretion after hypoxia and reoxygenation, and determined the influence of ionizing radiation on PAI-1 levels in the two human SCCHN cell lines, BHY and FaDu. METHODS: HIF-1alpha immunoblot was used to visualize the degree of hypoxia in the two cell lines. Cellular PAI-1 expression was investigated by immunofluorescence microscopy. ELISA was used to quantify relative changes in PAI-1 expression (cell lysates) and secretion (cell culture supernatants) in response to various lengths (2-4 h) of hypoxic exposure (< 0.66% O2), reoxygenation (24 h, 20% O2), and radiation (0, 2, 5 and 10 Gy). RESULTS: HIF-1alpha expression was induced between 2 and 24 h of hypoxic exposure. Intracellular PAI-1 expression was significantly increased in BHY and FaDu cells as early as 4 h after hypoxic exposure. A significant induction in secreted PAI-1 was seen after 12 to 24 h (BHY) and 8 to 24 h (FaDu) hypoxia, as compared to the normoxic control. A 24 h reoxygenation period caused significantly less PAI-1 secretion than a 24 h hypoxia period in FaDu cells. Irradiation led to an up-regulation of PAI-1 expression and secretion in both, BHY and FaDu cells. CONCLUSION: Our data suggest that both, short-term (approximately 4-8 h) and long-term (approximately 20-24 h) hypoxic exposure could increase PAI-1 levels in SCCHN in vivo. Importantly, radiation itself could lead to PAI-1 up-regulation in head and neck tumours, whereas reoxygenation of hypoxic tumour cells during fractionated radiotherapy could counteract the increased PAI-1 levels

Fluoration catalytique d'aromatiques chlorés par simple échange chlore-fluor en présence d'acides de Lewis et d'acide fluorhydrique

La présence de fluor dans les biomolécules peut augmenter leur activité biologique (thérapeutique, phytosanitaire).Leur préparation nécessite la mise au point de nouveaux systèmes catalytiques à base d'acides de Lewis et d'acide fluorhydrique. La fluoration de deux molécules modèles (trichloroanisole et bis-1,3-trichlorométhylbenzène) est très rapide et dépend des conditions opératoires. Le trichloroanisole est totalement fluoré en présence d'une quantité stoechiométrique d'HF et d'un acide de Lewis (de degré d'oxydation +V) en très faible quantité. La monofluoration du trichloroanisole est observée en présence de solvants basiques. En revanche, la fluoration du bis-1,3-trichlorométhylbenzène est beaucoup plus complexe. Le schéma réactionnel a été établi quelles que soient les conditions opératoires et conforté par des calculs théoriques. Enfin, la formation du 1-trichlorométhyl-3-trifluorométhylbenzène est favorisée en présence d'un défaut d'acide fluorhydrique et d'un acide de Lewis.The presence of fluorine in biomolecules can increase their biological activity (therapeutical, phytosanitary). Their preparation requires the development of new Lewis acid and hydrofluoric acid-based catalytic systems. The fluorination of two model molecules (trichloromethoxybenzene and bis-1,3-trichloromethylbenzene) is very fast and depends on the operating conditions. Trichloromethoxybenzene is totally fluorinated in the presence of a stoichiometric amount of HF and of a Lewis acid (oxidation state +V) in very small amount. The monofluorination of trichloromethoxybenzene is observed in the presence of basic solvents. On the other hand, the fluorination of bis-1,3-trichloromethylbenzene is more complex. The reaction scheme was established whatever the operating conditions and was confirmed by theoretical calculations. Finally, the preparation of 1-trichloromethyl-3-trifluoromethylbenzene is favored in the presence of small amount of HF and of a Lewis acid.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

Biosens. Bioelectron.

The painting Nasturtium Landscape - enamel on plywood and recycled timber, 30cm x 30cm, exhibited in Flores et Fructus, 2017 at the Robin Gibson Gallery, was formed by amalgamating both pre-painted panels, and recycled timber which had an existing patina of brush strokes, into a ‘loose’ grid formation. The work did not utilise an aesthetic of overlapping, nor did it follow a strict grid for its configuration. The aesthetic outcome was however, indebted to a methodology of ‘fitting’ by which sections of the work had to be adjusted in shape to align with neighboring components. Given the vibrant orange colour of the top half of the work, and the long horizontal alignment of it to the bottom of the work, the notion, or illusion of a optical horizon can be seen to exist in the work, and thus the works title