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Symmetrical and dissymmetrical bolaforms were prepared with good to high yields from unsaturated L-rhamnosides and phenolic esters (ferulic, phloretic, coumaric, sinapic and caffeic) using two eco-compatible synthetic strategies involving glycosylation, enzymatic synthesis and cross-metathesis under microwaves activation. Furthermore, some of these new compounds present good eliciting properties depending on the carbon chain length and on the nature of the hydrophilic head. Their respective antioxidant activities have been also evaluated as well as their cytotoxic properties on dermal cells for cosmetic uses.

Angiogenesis is defined as the formation of new capillaries by sprouting from the pre-existing microvasculature. It occurs in physiological and pathological processes particularly in tumor growth and metastasis. α1, α2, α3, and α6 NC1 domains from type IV collagen were reported to inhibit tumor angiogenesis. We previously demonstrated that the α4 NC1 domain from type IV collagen, named Tetrastatin, inhibited tumor growth in a mouse melanoma model. The inhibitory activity was located in a 13 amino acid sequence named QS-13. In the present paper, we demonstrate that QS-13 decreases VEGF-induced-angiogenesis in vivo using the Matrigel plug model. Fluorescence molecular tomography allows the measurement of a 65% decrease in Matrigel plug angiogenesis following QS-13 administration. The results are confirmed by CD31 microvessel density analysis on Matrigel plug slices. QS-13 peptide decreases Human Umbilical Vein Endothelial Cells (HUVEC) migration and pseudotube formation in vitro. Relevant QS-13 conformations were obtained from molecular dynamics simulations and docking. A putative interaction of QS-13 with α5β1 integrin was investigated. The interaction was confirmed by affinity chromatography, solid phase assay, and surface plasmon resonance. QS-13 binding site on α5β1 integrin is located in close vicinity to the RGD binding site, as demonstrated by competition assays. Collectively, our results suggest that QS-13 exhibits a mighty anti-angiogenic activity that could be used in cancer treatment and other pathologies with excessive angiogenesis such as hemangioma, psoriasis or diabetes.

Extracellular vesicles (EVs) like exosomes and shed microvesicles are generated by many different cells. However, among all the cells, cancer cells are now recognized to secrete more EVs than healthy cells. Tumor-derived EVs can be isolated from biofluids such as blood, urine, ascitic fluid, and saliva. Their numerous components (nucleic acids, proteins, and lipids) possess many pleiotropic functions involved in cancer progression. The tumor-derived EVs generated under the influence of tumor microenvironment play distant roles and promote cellular communication by directly interacting with different cells. Moreover, they modulate extracellular matrix remodeling and tumor progression. Tumor-derived EVs are involved in pre-metastatic niche formation, dependent on the EV-associated protein receptors, and in cancer chemoresistance as they transfer drug-resistance-related genes to recipient cells. Recent advances in preclinical and clinical fields suggest their potential use as biomarkers for diagnosis and prognosis as well as for drug delivery in cancer. In this Review, we discuss EV characteristics and pro-tumor capacities, and highlight the future crucial impact of tumor-derived EVs in pancreatic cancer diagnosis and prognosis.

Although conventional radiotherapy promotes the migration/invasion of cancer stem cells (CSCs) under normoxia, carbon ion (C-ion) irradiation actually decreases these processes. Unraveling the mechanisms of this discrepancy, particularly under the hypoxic conditions that pertain in niches where CSCs are preferentially localized, would provide a better understanding of the origins of metastases. Invasion/migration, proteins involved in epithelial-to-mesenchymal transition (EMT), and expression of MMP-2 and HIF-1α were quantified in the CSC subpopulations of two head-and-neck squamous cell carcinoma (HNSCC) cell lines irradiated with X-rays or C-ions. X-rays triggered HNSCC-CSC migration/invasion under normoxia, however this effect was significantly attenuated under hypoxia. C-ions induced fewer of these processes in both oxygenation conditions. The differential response to C-ions was associated with a lack of HIF-1α stabilization, MMP-2 expression, or activation of kinases of the main EMT signaling pathways. Furthermore, we demonstrated a major role of reactive oxygen species (ROS) in the triggering of invasion/migration in response to X-rays. Monte-Carlo simulations demonstrated that HO・ radicals are quantitatively higher after C-ions than after X-rays, however they are very differently distributed within cells. We postulate that the uniform distribution of ROS after X-rays induces the mechanisms leading to invasion/migration, which ROS concentrated in C-ion tracks are unable to trigger.

International audience; BACKGROUND: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases.METHODS: EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA.RESULTS: We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding.CONCLUSIONS: Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSAbinding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.

Tumor marker assays are daily practiced, for screening and follow up of cancers. Interassay precision is an important parameter for the interpretation of the kinetics of the markers, in order to conclude to the efficiency or failure of treatment. The aim of this study was to compare two automated Immunoassay analyzers, Lumipulse® G1200 and AIA® 2000. Both analyzers used an immunoassay system but with different antibodies. Six tumor markers commonly used were studied: AFP, PSA, CA 19-9, CA 15-3, CA 125 and CEA. 253 samples have been collected over a period of one month and analyzed by both analyzers. Regression of Passing-Badblock and Bland-Altman diagram were used to analyze the results for AFP (n=36), PSA (n=39), CA-125 (n=40), CA 15-3 (n=40), CA 19-9 (n=46) and CEA (n=52) were performed. Analytical performances of Lumipulse® G1200 highlighted the good inter-run and intra-run precision of the analyzer. We obtained a good correlation coefficient between Lumipulse G1200® and AIA 2000®, >0.96 for most markers except CA 19-9 which provided a correlation coefficient significantly lower than that obtained with other markers. The concordance for all markers was >94% except for CA 19-9 (83.7%). This study showed a good correlation between the two analyzers and, therefore, a transfer from one analyzer to the other is possible for the different markers studied. However, we found here the classical difficulty to transfer this type of analysis, due to the absence of method standardization. This difficulty was particularly illustrated by CA19-9.

Collagen IV is a major component of the dermo-epidermal junction (DEJ). To study expression of collagen IV upon aging in the DEJ and dermal fibroblasts isolated from the same patients. A model of senescent fibroblasts was developed in order to identify biological compounds that might restore the level of collagen IV. Skin fragments of women (30 to 70 years old) were collected. Localisation of collagen IV expression in the DEJ was studied by immunofluorescence. Fibroblast collagen IV expression was studied by real-time PCR, ELISA, and western blotting. Premature senescence was simulated by exposing fibroblasts to subcytotoxic H2O2 concentrations. Collagen IV decreased in the DEJ and fibroblasts relative to age. TGF-β1 treatment significantly increased collagen IV gene and protein expression in fibroblasts and restored expression in the model of senescence. Addition of TGF-β1-neutralizing antibody to fibroblast cultures decreased collagen IV expression. Taken together, the results suggest that the decrease in collagen IV in the DEJ, relative to age, could be due to a decrease in collagen IV expression by senescent dermal fibroblasts and may involve TGF-β1 signalling.

International audience; The epithelio-mesenchymal transition (EMT) is the mechanism that allows cells to escape from the tumor to form metastases. While conventional radiotherapy promotes the invasion/migration of cancer stem cells (CSCs), carbon ion irradiation decrease these processes. Moreover, CSCs are localized in hypoxic tumor niches where hypoxia amplifies the biological effects associated with radioresistance. Thus, understanding the differential mechanisms involved in the response to photon and carbon ion irradiation, particularly in hypoxic conditions, would provide a better understanding of the tumor escape process.Motility, invasion/migration processes, and EMT signaling pathways were studied in response to photon and carbon ion irradiations for two HNSCC cell lines and their subpopulation of CSCs in normoxic and hypoxic conditions.After confirming, in normoxic conditions, the invasion/migration increase in response to photons and the decrease after irradiation with carbon ions, we have shown that under hypoxia, the two types of irradiation lead to a decrease in the processes. In order to understand this differential response to radiations, the phosphorylation profiles of the Akt/mTor, STAT3 and ERK/p38/MSK pathways involved in the EMT were established. In response to photons, the activation of the three kinase cascades is important, whereas it is weaker in hypoxia, and not at all in response to carbon ion irradiation regardless of the oxygen tension.Our results show a phosphorylation profile of the three main EMT pathways, depending on the type of irradiation and the oxygen tension. ROS production is essential to activate the EMT pathways. We propose a new paradigm where the spatial ROS distribution contribute to the activation or not of the signaling pathways. The ROS production, uniformly distributed in the cell in response to photons allows the activation of the pathways unlike carbon ions where the ROS are only located in the carbon ion tracks and not sufficient to activate the EMT pathways.Supported by LabEx PRIMES (ANR11LABX0063), France Hadron (ANR11INBS0007), Cancéropôle Rhone Alpes Auvergne.

Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.

Type XIX collagen is a minor collagen associated with basement membranes in vascular, neuronal, mesenchymal, and epithelial tissues. We demonstrated that the NC1, C-terminal, domain of collagen XIX inhibits the migration capacities of tumor cells and exerts a strong inhibition of tumor growth. Other basement membrane collagens or derived fragments were measured in biological fluids such as blood and urine of patients and appeared to be useful for diagnosis, prognosis, or treatment monitoring. The aim of this study was to develop and validate methods to measure collagen XIX and its fragments in human cell cultures, tissue extracts, and human biological fluids. For that purpose, we developed real-time PCR, Western blot, and competitive enzyme-linked immunosorbent assays. We demonstrated that the methods developed in this paper are specific for collagen XIX. We showed that it is expressed in human cell cultures, tissue extracts, and various biological fluids. These methods may be used in various human tissue extracts and biological fluids such as serum, amniotic fluid, cord blood, and many other fluids. Collagen XIX or its fragments could constitute new biomarkers for human diseases as well as for diagnosis and/or prognosis.

Type XIX collagen is a minor collagen that is associated with the basement membrane zone that belongs to the FACIT family (Fibril-Associated Collagens with Interrupted Triple helices). The FACIT family is composed of type IX, XII, XIV, XVI, XX, XXI, XXII and XIX collagens, which share many highly conserved structural motifs: a short NC1 domain, a thrombospondin-like N-terminal domain (TSPN), and numerous cysteine residues. The main role of FACITs is to ensure the integrity and stability of the extracellular matrix and its fibrillar collagen network by regulating the formation and size of the collagen fibrils. Type XIX collagen was discovered in a human rhabdomyosarcoma cell line. The collagen α1(XIX) chain is composed of 5 triple-helical domains (COL) interrupted by 6 non-triple-helical (NC) domains with a short, C-terminal, 19 amino acid non-collagenous domain (NC1). This collagen is involved in the differentiation of muscle cells, central nervous system development, and formation of the esophagus. Type XIX collagen is associated with the basement membrane zone, like type XVIII and XV collagens. Its short NC1(XIX) C-terminal domain inhibits the migration and invasion of melanoma cells. It also exerts a strong anti-angiogenic effect by inhibiting MMP-14 and VEGF expression. NC1(XIX) binding to αvβ3 integrin decreases the phosphorylation of proteins involved in the FAK (Focal Adhesion Kinase)/PI3K (PhosphoInositide 3-Kinase)/Akt (protein kinase B)/mTOR (Mammalian Target Of Rapamycin) pathway. On the other hand, NC1(XIX) induces an increase in GSK3β activity by decreasing its level of phosphorylation. The inhibition of this pathway could explain the anti-tumor properties of the NC1(XIX) domain.

Type XIX collagen is a minor collagen that localizes to basement membrane zones, together with types IV, XV, and XVIII collagens. Because several NC1 COOH-terminal domains of other chains from basement membrane collagens were reported to exhibit antitumor activity, we decided to study the effects of the NC1(XIX) collagen domain on tumor progression using an experimental in vivo model of mouse melanoma. We observed a 70% reduction in tumor volume in NC1(XIX)-treated mice compared with the corresponding controls. Histologic examination of the tumors showed a strong decrease in tumor vascularization in treated mice. In vitro, NC1(XIX) inhibited the migrating capacity of tumor cells and their capacity to invade Matrigel. It also inhibited the capacity of human microvascular endothelial cells to form pseudotubes in Matrigel. This effect was accompanied by a strong inhibition of membrane type-1 matrix metalloproteinase (matrix metalloproteinase-14) and vascular endothelial growth factor expression. Collectively, our data indicate that the NC1 domain of type XIX collagen exerts antitumor activity. This effect is mediated by a strong inhibition of the invasive capacities of tumor cells and antiangiogenic effects. NC1(XIX) should now be considered as a new member of the basement membrane collagen-derived matrikine family with antitumor and antiangiogenic activity. [Mol Cancer Ther 2007;6(2):506–14]

The term "matrikines" was coined for designating peptides liberated by partial proteolysis of extracellular matrix macromolecules, which are able to regulate cell activities. Among these peptides, some of them may modulate proliferation, migration, protease production, or apoptosis. In this review, we summarize the activity of matrikines derived from elastin and interstitial or basement membrane collagens on the regulation of matrix metalloproteinases expression and/or activation, and on the plasminogen/plasmin system. Due to their activity, matrikines may play a significant role in physiological or pathological processes such as wound healing or tumor invasion.

Skin fibrosis is one of the most common late adverse effects observed after radiation therapy for cancer. As a dose-limiting factor and hence a major hindrance to increase the amount of radiation delivered to the tumor, this problem can be addressed according to the very early steps of the fibrotic process: the oxygen free radical production. Reactive oxygen species (ROS) generated during radiotherapy result from both inflammatory response and water radiolysis. Many studies have demonstrated that the extracellular matrix molecules are potential targets for ROS, and that collagen metabolism and properties are deeply and permanently modified after irradiation, both in vitro and in vivo. It is therefore possible to design different therapeutic approaches such as the clinical use of liposomal superoxide dismutase able to reverse the imbalance between collagen matrix synthesis and degradation. Finally, the so-called oxidative stress induced by radiation represents a significant parameter leading to fibrosis and will undoubtedly serve to design further experimental and clinical studies.

Malignant melanoma is the leading cause of death from diseases of the skin. This review summarizes the data from the literature and our laboratory addressing the effects of type IV collagen on melanoma progression. Many different sequences from type IV collagen promote melanoma cell adhesion, migration and invasion. The triple helical conformation of the collagenous domain plays a critical role in some of these interactions. However, recent studies from our group demonstrated that a sequence from the alpha3(IV) NC1 domain inhibits melanoma cell proliferation, migration and invasion by decreasing MMP production and activation. Peptide sequences from the alpha1(IV), alpha2(IV) and alpha3(IV) chains named arresten, canstatin and tumstatin, respectively were shown to inhibit angiogenesis. Further investigations regarding the inhibitory effects of the alpha(IV) NC1 domains will have a paramount relevance for the design of efficient strategies to limit melanoma development.

Our previous studies demonstrated that a synthetic peptide encompassing residues 185-203 of the noncollagenous (NC1) domain of the alpha3 chain of type IV collagen, named tumstatin, inhibits in vitro melanoma cell proliferation and migration. In the present study, B16F1 melanoma cells were stably transfected to overexpress the complete tumstatin domain (Tum 1-232) or its C-terminal part, encompassing residues 185-203 (Tum 183-232). Tumstatin domain overexpression inhibited B16F1 in vitro cell proliferation, anchorage-independent growth, and invasive properties. For studying the in vivo effect of overexpression, representative clones were subcutaneously injected into the left side of C57BL6 mice. In vivo tumor growth was decreased by -60% and -56%, respectively, with B16F1 cells overexpressing Tum 1-232 or Tum 183-232 compared to control cells. This inhibitory effect was associated with a decrease of in vivo cyclin D1 expression. We also demonstrated that the overexpression of Tum 1-232 or Tum 183-232 induced an in vivo down-regulation of proteolytic cascades involving matrix metalloproteinases (MMPs), especially the production or activation of MMP-2, MMP-9, MMP-13, as well as MMP-14. The plasminogen activation system was also altered in tumors with a decrease of urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) and a strong increase of plasminogen activator inhibitor-1 (PAI-1). Collectively, our results demonstrate that tumstatin or its C-terminal antitumor fragment, Tum 183-232, inhibits in vivo melanoma progression by triggering an intracellular transduction pathway, which involves a cyclic AMP (cAMP)-dependent mechanism.

The term of "matrikines" was coined for designating peptides liberated by partial proteolysis of extracellular matrix macromolecules, which are able to regulate cell activities. Among these peptides, some of them may modulate proliferation, migration, protease production, or apoptosis, which suggest that they can play a significant role in the control of tumor progression. In this introduction, we present the best characterized matrikines, derived from elastin, connective tissue glycoproteins, or collagens.

Tumor progression may be controlled by various fragments derived from noncollagenous 1 (NC1) C-terminal domains of type IV collagen. We demonstrated previously that a peptide sequence from the NC1 domain of the alpha3(IV) collagen chain inhibits the in vitro expression of matrix metalloproteinases in human melanoma cells through RGD-independent binding to alpha(v)beta(3) integrin. In the present paper, we demonstrate that in a mouse melanoma model, the NC1 alpha3(IV)-(185-203) peptide inhibits in vivo tumor growth in a conformation-dependent manner. The decrease of tumor growth is the result of an inhibition of cell proliferation and a decrease of cell invasive properties by down-regulation of proteolytic cascades, mainly matrix metalloproteinases and the plasminogen activation system. A shorter peptide comprising the seven N-terminal residues 185-191 (CNYYSNS) shares the same inhibitory profile. The three-dimensional structures of the CNYYSNS and NC1 alpha3(IV)-(185-203) peptides show a beta-turn at the YSNS (188-191) sequence level, which is crucial for biological activity. As well, the homologous MNYYSNS heptapeptide keeps the beta-turn and the inhibitory activity. In contrast, the DNYYSNS heptapeptide, which does not form the beta-turn at the YSNS level, is devoid of inhibitory activity. Structural studies indicate a strong structure-function relationship of the peptides and point to the YSNS turn as necessary for biological activity. These peptides could act as potent and specific antitumor antagonists of alpha(v)beta(3) integrin in melanoma progression.

The regulation of cell metabolism by the surrounding environment is deeply altered by the posttranslational nonenzymatic modifications of extracellular proteins that occur throughout lifespan in vivo and modify their structural and functional properties. Among them are protein adducts formed by components generated from oxidative processes, such as malondialdehyde (MDA). We have investigated here the effects of MDA-binding to proteins on cultured fibroblast functions. Type I collagen and/or serum proteins were incubated with 0–100 mM MDA for 3 h before use in fibroblast cultures. In tridimensional lattice cultures, MDA-treated collagen inhibited the contracting activity of fibroblasts. A similar inhibition of lattice contraction was reproduced by the addition of MDA-treated serum to the culture medium. In monolayer cultures, the addition of MDA-modified serum proteins completely inhibited fibroblast multiplication without effect on initial adhesion steps. MDA-modified proteins decreased the proliferative capacities of cells, strongly altered cell cycle progression by blocking passage to G2/M phases, and induced apoptotic features in fibroblasts. Our results show, for the first time, that MDA-modified proteins are potentially as deleterious as free MDA, and could be involved in aging as well as in degenerative complications of diseases with increased oxidative stress such as diabetes mellitus or atherosclerosis. J. Cell. Physiol. 191: 227–236, 2002. © 2002 Wiley-Liss, Inc.

Basement membrane collagens or derived fragments are measured in biological fluids such as blood and urine of patients and appear to be useful for diagnosis, prognostication, or treatment monitoring as proposed for endostatin, a fragment of collagen XVIII, or tumstatin, a fragment of collagen IV. Tetrastatin, the NC1 alpha 4 collagen IV domain, was previously reported to inhibit tumor growth and angiogenesis. The aim of this study was to develop and validate a method to measure tetrastatin concentrations in human fluids. We developed a competitive enzyme-linked immunosorbent assay (ELISA). It allowed measuring tetrastatin levels in human serum, bronchial aspiration and bronchoalveolar lavage fluids, and lung tissue extracts. The tetrastatin level was significantly higher in tumor tissues than in healthy lung tissues. Tetrastatin competitive ELISA could be useful to quantify tetrastatin in tissues and biological fluids for the diagnosis or prognostication of diseases in which basement membrane metabolism may be altered, especially tumor progression.

International audience; The basement membrane (BM) is the first barrier encountered by tumor cells when they become invasive. Moreover, some invasive tumor clusters are surrounded by a remnant or neosynthetized BM material. We have previously reported the presence of a particular alpha chain of type IV collagen, the alpha3(IV) chain, in bronchopulmonary carcinomas. This chain was not detected in the normal bronchial epithelium, but was found around some invasive tumor cluster BM. In the present study, we examined the effects of the alpha3(IV) chain on the invasive properties of bronchial tumor cell lines, with special emphasis on their expression of matrix metalloproteinase-2 (MMP-2) and its activator, membrane type 1-matrix metalloproteinase (MT1-MMP), which is largely involved in tumor progression. Two epithelial bronchial cell lines (16HBE14o- and BZR), showing different invasive abilities, were evaluated. Using the Boyden chamber invasion assay, we demonstrated that the alpha3(IV) chain inhibits the invasive properties of BZR cells and modifies their morphology by inducing an epithelial cell shape. In the presence of the recombinant NC1 domain of the alpha3(IV) chain, the expression of MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) was not modified in either cell line. The NC1 alpha3(IV) domain did not modulate the MT1-MMP expression of noninvasive 16HBE14o- cells, whereas a 50% decrease of MT1-MMP mRNA was observed in invasive BZR cells. Accordingly, Western blot analyses showed a disappearance of the 45-kd MT1-MMP form when BZR cells were treated with the recombinant NC1 alpha3(IV) domain. These findings suggest that the alpha3 chain of type IV collagen may play a role in tumor invasion, at least by decreasing the expression and synthesis of MT1-MMP.

We have recently identified integrin alpha(v)beta(3) and the associated CD47/integrin-associated protein (IAP) together with three other proteins as the potential tumor cell receptors for the alpha(3) chain of basement membrane type IV collagen (Shahan, T.A., Ziaie, Z., Pasco, S., Fawzi, A., Bellon, G., Monboisse, J. C., and Kefalides, N. A. (1999) Cancer Res. 59, 4584-4590). Using different cell lines expressing alpha(v)beta(3), alpha(IIb)beta(3), and/or CD47 and a liquid phase receptor capture assay, we now provide direct evidence that the synthetic and biologically active alpha3(IV)185-206 peptide, derived from the alpha3(IV) chain, interacts with the beta(3) subunit of integrin alpha(v)beta(3), independently of CD47. Increased alpha3(IV) peptide binding was observed on transforming growth factor-beta(1)-stimulated HT-144 cells shown to up-regulate alpha(v)beta(3) independently of CD47. Also, incubation of HT-144 melanoma cells in suspension induced de novo exposure of ligand-induced binding site epitopes on the beta(3) subunit similar to those observed following Arg-Gly-Asp-Ser (RGDS) stimulation. However, RGDS did not prevent HT-144 cell attachment and spreading on the alpha3(IV) peptide, suggesting that the alpha3(IV) binding domain on the beta(3) subunit is distinct from the RGD recognition site. alpha3(IV) peptide binding to HT-144 cells in suspension stimulated time-dependent tyrosine phosphorylation, while the RGDS peptide did not. Two major phosphotyrosine proteins of 120-130 and 85 kDa were immunologically identified as focal adhesion kinase and phosphatidylinositol 3-kinase (PI3-kinase). A direct involvement of PI3-kinase in alpha3(IV)-dependent beta(3) integrin signaling could be documented, since pretreatment of HT-144 cells with wortmannin, a PI3-kinase inhibitor, reverted the known inhibitory effect of alpha3(IV) on HT-144 cell proliferation as well as membrane type 1-matrix metalloproteinase gene expression. These results provide evidence that the alpha3(IV)185-206 peptide, by directly interacting with the beta(3) subunit of alpha(v)beta(3), activates a signaling cascade involving focal adhesion kinase and PI3-kinase.

Glycation and glycoxidation processes, which are increased in diabetes mellitus, are generally considered causative mechanisms of long-term complications. With reference to our previous studies, type-I and -IV collagens could induce differentially the adhesion and stimulation of polymorphonuclear leucocytes (PMNs). As PMNs play a role in sustained diabetic oxidative stress, the present study was designed to determine whether in vitro glycoxidation of these macromolecules could alter PMN adhesion, activation and migration. The adhesion of PMNs to in vitro-glycoxidized collagens was significantly increased when compared with control collagens: +37% (P < 0.05) and +99% (P < 0.01) for collagens I and IV, respectively. Glycoxidized type-I collagen increased the chemotactic properties of PMNs without significant stimulatory effect on respiratory burst, whereas pre-incubation of PMNs with glycoxidized type-I collagen induced a priming on subsequent stimulation by N-formyl-methionyl-leucyl-phenylalanine. Glycoxidation of type-IV collagen suppressed its inhibitory effect on further PMN stimulation or migration. Collectively, these results indicate that glycoxidation of two major extracellular-matrix collagens considerably alters their ability to modulate PMN migration and production of reactive oxygen species. This imbalance in PMN metabolism may be a major event in the increased oxidative status that characterizes diabetes mellitus.

Previous reports from our laboratories showed that type IV collagen from anterior lens capsule (ALC) inhibited stimulated neutrophil function. This property was shown to reside in the region comprising residues 185-203 of the non-collagenous domain (NC1) of the alpha 3(IV) chain. We also reported that ALC-type IV collagen or the synthetic alpha 3(IV) 185-203 peptide, induced a rise in intracellular cAMP which persisted for up to 60 minutes. In the present work we extend our previous studies on signal transduction by alpha 3(IV) 185-203 and we provide new data showing the involvement of cAMP-dependent PKA and protein phosphatases. The data also show that the alpha 3(IV) peptide triggered a rise in intracellular calcium that was dependent on phospholipase C activation. Inhibitors of the Ca(2+)/calmodulin system suppressed both the alpha 3(IV) 185-203 peptide-induced cAMP increase and the inhibitory activity of the peptide on f-Met-Leu-Phe triggered O(2)(-) generation. When alpha 3(IV) 185-203 peptide-induced calcium mobilization was blocked by U-73122, an inhibitor of phospholipase C activation, or by BAPTA/AM, a chelator of intracellular calcium, the inhibitory effect of the peptide on PMA-triggered O(2)(-) production was also abolished. These findings provide evidence that signal transduction by the alpha 3(IV) peptide occurs via pathways which involve calcium. Indeed, the cAMP increase was shown to be mediated by adenosine and adenosine A2 receptors and required calcium elevation, since adenosine deaminase, theophilline, dimethylpropargylxanthine, trifluoperazine or autocamtide-2 related inhibitory peptide, suppressed the activity of the alpha 3(IV) peptide. The inhibitory effect of the peptide on f-Met-Leu-Phe-induced O(2)(-) generation was slightly affected by 1 microM KT5720 or H89, two inhibitors of cAMP-dependent PKA, but was completely suppressed by 10 nM calyculin A or 10 microM okadaic acid, two inhibitors of ser/thr phosphatases. These results suggest that Ser/Thr protein phosphatases and/or cAMP-dependent PKA are involved in signal transduction by the alpha 3(IV) 185-203 peptide and is consistent with the concept that adenosine receptor occupancy modulates neutrophil function.

Nonenzymatic glycation of extracellular matrix (ECM) proteins is increased in diabetes mellitus and aging and triggers cellular events leading to an imbalance in ECM homeostasis. We studied the influence of collagen glycation on matrix metalloproteinase production by dermal fibroblasts using the model of lattice cultures. Contraction of glycated collagen lattices was strongly reduced when compared to controls. Meanwhile, fibroblasts synthesized lower amounts of interstitial collagenase (MMP-1). Gelatinase A (MMP-2) production was not modified, but its activation was strongly inhibited. These effects were independent from the intensity of lattice contraction and from any simultaneous modification of tissue inhibitors of metalloproteinase (TIMP-1 and 2) production. These results demonstrate that the impaired ability of fibroblasts to remodel and contract a glycated extracellular matrix coincides with a decrease in MMP production.

We have shown that basement membrane (BM) collagen (type IV), and specifically the peptide CNYYSNSYSFWLASLNPER (a.a. 185-203), from the non-collagenous domain of the alpha3 chain inhibits PMN. We examined the role of this peptide on PMN damage to BM in a vessel wall model. The presence of the endothelial monolayer as well as treatment of PMN with the alpha3(IV) 185-203 peptide reduced damage to BM by non-activated but not by activated PMN. The damage inhibition is unique to the alpha3(IV) peptide and not exhibited by comparable alpha1(IV) and alpha2(IV) chain peptides. A shorter peptide alpha3(IV) 185-191, containing the -SNS- triplet, reduced damage, whereas the one lacking the triplet, residues 194-203, was not effective. The CD47-alphavbeta3 integrin complex is the receptor for the alpha3(IV) peptide. Incubation of PMN with CD47 reactive mAb followed by the alpha3(IV) peptide abolished its protective effect on BM damage.

Tumour invasion is associated with strong remodelling of the extracellular matrix, including the basement membrane (BM). The major structural component of BMs is type IV collagen, which is composed of an association of three a chains. In this study, the distribution of the a1 and a3 chains in both normal and neoplastic lung tissues has been examined by immunohistochemistry, using specific monoclonal antibodies. In normal tissues, the a1(IV) chain was found in all BMs, whereas the a3(IV) chain was only found in alveolar BMs. In 36 lung tumours, the a1(IV) chain was detected in all cases, with irregular positivity around tumour clusters and in the stroma. It was noteworthy that this stromal distribution was particularly associated with the presence of cancer cells, whatever their invasive properties. In contrast, in 22 tumours out of 36, the a3(IV) chain was only found at the interface between invasive tumour clusters and stroma, with a linear and disrupted pattern. These data show a distinctive distribution of type IV collagen chains in lung tumours, with expression of a1(IV) chain and likely neosynthesis of the a3(IV) chain around some invasive tumour clusters. The results suggest the involvement of these BM components in the process of tumour invasion.

Objectives: Serum Lp(a) levels are generally considered unaffected by non-insulin-dependent diabetes mellitus (NIDDM). However, high Lp(a) concentrations as well as an increased rate of nonenzymatic glycation of proteins may be involved in degenerative diabetic complications. Design and Methods: We measured serum glycated Lp(a) levels in 17 NIDDM patients, as compared to 14 normoglycaemic controls. Glycated proteins were separated from nonglycated ones by boronate affinity chromatography, and specific proteins assayed by immunonephelometric methods in both fractions. Results: The percentage of glycated Lp(a) was 1.5 ± 0.4% (mean ± SD) in the control group, and was significantly higher in NIDDM patients: 4.3 ± 1.5% (p < 0.01). The basal level of Lp(a) glycation was lower than that of other proteins, particularly apo B (4.0 ± 0.7%). By contrast, the variations of glycated Lp(a) levels were of greater amplitude (+187%) than those of glycated apo B (+67%). Glycated Lp(a) values were significantly elevated in patients with micro and macrovascular complications in comparison with uncomplicated patients. Conclusions: These results suggest that glycated Lp(a) may be considered a potentially interesting parameter in the pathophysiology of diabetic vascular complications.

Background Tumor microenvironment is a complex system composed of a largely altered extracellular matrix with different cell types that determine angiogenic responses and tumor progression. Upon the influence of hypoxia, tumor cells secrete cytokines that activate stromal cells to produce proteases and angiogenic factors. In addition to stromal ECM breakdown, proteases exert various pro- or anti-tumorigenic functions and participate in the release of various ECM fragments, named matrikines or matricryptins, capable to act as endogenous angiogenesis inhibitors and to limit tumor progression. Scope of review We will focus on the matrikines derived from the NC1 domains of the different constitutive chains of basement membrane-associated collagens and mainly collagen IV. Major conclusions The putative targets of the matrikine control are the proliferation and invasive properties of tumor or inflammatory cells, and the angiogenic and lymphangiogenic responses. Collagen-derived matrikines such as canstatin, tumstatin or tetrastatin for example, decrease tumor growth in various cancer models. Their anti-cancer activities comprise anti-proliferative effects on tumor or endothelial cells by induction of apoptosis or cell cycle blockade and the induction of a loss of their migratory phenotype. They were used in various preclinical therapeutic strategies: i) induction of their overexpression by cancer cells or by the host cells, ii) use of recombinant proteins or synthetic peptides or structural analogues designed from the structure of the active sequences, iii) used in combined therapies with conventional chemotherapy or radiotherapy. General significance Collagen-derived matrikines strongly inhibited tumor growth in many preclinical cancer models in mouse. They constitute a new family of anti-cancer agents able to limit cancer progression. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

The NC1 domains from the different α(IV) collagen chains were found to exert anti-tumorigenic and/or anti-angiogenic activities. A limitation to the therapeutic use of these matrikines is the large amount of purified recombinant proteins, in the milligram range in mice that should be administered daily throughout the experimental procedures. In the current study, we developed a new therapeutic approach based on tumstatin (NC1α3(IV)) overexpression in vivo in a mouse melanoma model. Gene electrotransfer of naked plasmid DNA (pDNA) is particularly attractive because of its simplicity, its lack of immune responsiveness and its safety. The pDNA electrotransfer in muscle mediates a substantial gene expression that lasts several months. A pVAX1© vector containing the tumstatin cDNA was injected into the legs of C57BL/6 mice and submitted to electrotranfer. Sera were collected at different times and tumstatin was quantified by ELISA. Tumstatin secretion reached a plateau at day 21 with an expression level of 12 μg/mL. For testing the effects of tumstatin expression on tumor growth in vivo, B16F1 melanoma cells were subcutaneously injected in mice 7 days after empty pVAX1© (Mock) or pVAX1©-tumstatin electrotransfer. Tumstatin expression triggered a large decrease in tumor growth and an increase in mouse survival. This new therapeutic approach seems promising to inhibit tumor progression in vivo.

Many biological processes such as cell differentiation, cell migration or gene expression are tightly controlled by cell-cell interactions or by various cytokines. During tumor progression, cancer cells are in contact with extracellular matrix (ECM) macromolecules involving specific receptors such as integrins. The different stages of tumor progression, and mainly the proteolytic cascades implicated in extracellular matrix degradation and cell migration, may be controlled by the extracellular matrix macromolecules or by domains released by directed and limited proteolysis of these molecules. In this review, we summarise the biological effects of various peptides, named matrikines, derived from basement membranes (BM) components, such as laminins (LN), proteoglycans or collagens. These peptides may control tumor progression by regulating the proteolytic cascades leading to cancer cell dissemination and metastasis.

Tumor microenvironment is a complex system composed of a largely altered Extracellular Matrix (ECM) with different cell types that determine tumor progression. Upon the influence of hypoxia, tumor cells secrete cytokines that activate stromal cells to produce proteases and angiogenic factors. The proteases degrade the stromal ECM and participate in the release of various ECM fragments, named matrikines or matricryptins, capable to control tumor invasion and metastasis dissemination. The putative targets of the matrikine action are the proliferation and invasive properties of tumor or inflammatory cells, and the angiogenic and lymphangiogenic responses. In the present review, we will describe pro-tumorigenic effects triggered by soluble elastin or Elastin-Derived Peptides (EDPs), as well as the anti-tumorigenic or anti-angiogenic activities the matrikines derived from basement membrane associated collagens and several proteoglycans such as perlecan or lumican. Matrikines constitute a new family of potent anticancer agents that could be used under various therapeutic strategies: i) induction of their overexpression by cancer cells or by the host, ii) use of recombinant proteins or synthetic peptides or structural analogs designed from the structure of the active sequences. Matrikines could be used in combination with conventional chemotherapy or radiotherapy to limit tumor progression.

Monoclonal antibodies to the αLβ2 integrin inhibit the binding of type I collagen to PMN (polymorphonuclear neutrophil leukocytes) as well as the subsequent stimulation of superoxide production and enzyme secretion elicited by this collagen. Pepsinized collagen still binds PMN but no longer stimulates them. The I domain of the α chain of the integrin is involved in the binding. Two sequences of the α1(I) polypeptide chain of collagen participate in the process. Experiments of competitive inhibition by synthetic peptides showed that the sequence RGD (915-917) is used for binding to the cells and DGGRYY (1034-1039) serves to stimulate PMN. Experiments of radioactive labeling of the cells and affinity chromatography on Sepharose-collagen confirmed the presence in PMN extracts of two proteins, 95 and 185 kDa, respectively, corresponding to the molecular weights of the β2 and αL chains of the integrin and recognized by their specific monoclonal antibodies. The transduction pathways depending on the αLβ2 integrin do not involve a G protein (ruled out by the use of cholera and pertussis toxins), whereas the cytoskeleton was found to participate in the process, as evidenced by inhibition by cytochalasin B. After collagen stimulation, cytoplasmic inositol trisphosphate and calcium ion increased sharply for less than 2 min. The use of the inhibitors staurosporine and calphostin C demonstrated that protein kinase C was involved. Evaluation of the activity of this enzyme showed that, upon stimulation of PMN with collagen I, it was translocated to plasma membrane. Acrylamide gel electrophoresis of the protein bands corresponding to the integrin αLβ2, followed by immunoblotting using monoclonal antibodies to phosphotyrosine, permitted us to demonstrate that, prior to stimulation by type I collagen, there was no phosphorylation, whereas after stimulation, both αLand β2 chains were stained by anti-phosphotyrosine antibodies. The adhesion of PMN to pepsinized type I collagen triggered tyrosine phosphorylation of the β2 chain of the integrin, without stimulating O⨪2 production by these cells, whereas their stimulation by complete type I collagen induced the tyrosine phosphorylation of both αL and β2 subunits. The tyrosine phosphorylation of both integrin subunits during transduction of stimuli is a heretofore undescribed phenomenon that may correspond to a new system of transmembrane communication.

We have previously shown that glutamine stimulates the synthesis of collagen in human dermal confluent fibroblast cultures (Bellon, G. et al. [1987] Biochim. Biophys. Acta, 930, 39-47). In this paper, we examine the effects of glutamine on collagen gene expression. A dose-dependent effect of glutamine on collagen synthesis was demonstrated from 0 to 0.25 mM followed by a plateau up to 10 mM glutamine. Depending on the cell population, collagen synthesis was increased by 1.3-to 2.3-fold. The mean increase in collagen and non-collagen protein synthesis was 63% and 18% respectively. Steady-state levels of alpha 1(I) and alpha 1(III) mRNAs, were measured by hybridizing total RNA to specific cDNA probes at high stringency. Glutamine increased the steady-state level of collagen alpha 1(I) and alpha 1(III) mRNAs in a dose-dependent manner. At 0.15 mM glutamine, collagen mRNAs were increased by 1.7-and 2.3-fold respectively. Nuclear run-off experiments at this concentration of glutamine indicated that the transcriptional activity was increased by 3.4-fold for the pro alpha 1(I) collagen gene. The effect of glutamine on gene transcription was also supported by the measurement of pro alpha 1(I) collagen mRNA half-life since glutamine did not affect its stability. Protein synthesis seemed to be required for the glutamine-dependent induction of collagen gene expression since cycloheximide suppressed the activation. The effect of glutamine appeared specific because analogues and/or derivatives of glutamine, such as acivicin, 6-diazo-5-oxo-L-norleucine, homoglutamine, ammonium chloride and glutamate did not replace glutamine. The influence of amino acid transport systems through plasma membrane was assessed by the use of 2(methylamino)-isobutyric acid and beta 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. The glutamine-dependent induction of collagen gene expression was found to be independent of transport system A but dependent on transport system L whose inhibition induced a decrease in pro alpha 1(I) collagen gene transcription by an unknown mechanism. Thus, glutamine, at physiological concentrations, indirectly regulates collagen gene expression.

Tumor microenvironment is a complex system composed of a largely altered extracellular matrix (ECM) with different cell types that determine the angiogenic response. Upon the influence of hypoxia, tumor cells secrete cytokines that activate stromal cells to produce proteases and angiogenic factors. The proteases degrade the stromal ECM and participate in the release of various ECM fragments, named matrikines or matricryptins, capable to control tumor invasion and metastasis dissemination. We will focus on the matrikines derived from the NC1 domains of the different constitutive chains of basement membrane-associated collagens and mainly collagen IV. The putative targets of the matrikine action are the proliferation and invasive properties of tumor or inflammatory cells, and the angiogenic and lymphangiogenic responses. For example, canstatin, tumstatin and tetrastatin, respectively derived from the NC1 domains of α2, α3 and α4 chains of collagen IV, inhibit in vivo tumor growth in various experimental cancer models. Their anti-cancer activity comprises an anti-proliferative effect on tumor cells and on endothelial cells by induction of cell apoptosis or cell cycle blockade and the induction of a loss of their migratory phenotype. Matrikines constitute a new family of potent anticancer agents that could be used under various therapeutic strategies: i) induction of their overexpression by cancer cells or by the host cells, ii) use of recombinant proteins or synthetic peptides or structural analogues designed from the structure of the active sequences. These matrikines could be used in combination with conventional chemotherapy or radiotherapy to limit tumor progression.

Background NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far. Methodology/Principal Findings We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (–38%) and invasive (–52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (–80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (KD = 148±9.54 nM). Conclusion/Significance Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine.

Tumor microenvironment is a complex system composed of a largely altered extracellular matrix (ECM) with different cell types that determine the angiogenic response. Upon the influence of hypoxia, tumor cells secrete cytokines that activate stromal cells to produce proteases and angiogenic factors. The proteases degrade the stromal ECM and participate in the release of various ECM fragments, named matrikines or matricryptins, capable to control tumor invasion and metastasis dissemination. We will focus on the matrikines derived from the NC1 domains of the different constitutive chains of basement membrane-associated collagens and mainly collagen IV. The putative targets of the matrikine action are the proliferation and invasive properties of tumor or inflammatory cells, and the angiogenic and lymphangiogenic responses. For example, canstatin, tumstatin and tetrastatin, respectively derived from the NC1 domains of α2, α3 and α4 chains of collagen IV, inhibit in vivo tumor growth in various experimental cancer models. Their anti-cancer activity comprises an anti-proliferative effect on tumor cells and on endothelial cells by induction of cell apoptosis or cell cycle blockade and the induction of a loss of their migratory phenotype. Matrikines constitute a new family of potent anticancer agents that could be used under various therapeutic strategies: i) induction of their overexpression by cancer cells or by the host cells, ii) use of recombinant proteins or synthetic peptides or structural analogues designed from the structure of the active sequences. These matrikines could be used in combination with conventional chemotherapy or radiotherapy to limit tumor progression.

Our initial observation that type I collagen activates polymorphonuclear leukocytes (PMN) prompted the testing of the activating potential of type IV collagen. It was noted, however, that type IV collagen isolated from bovine lens capsule did not activate PMN but rather prevented their stimulation by N-formylmethionyl-leucyl-phenylalanine, phorbol myristate acetate, or type I collagen. This observation led to the present study, which demonstrates that the inhibitory effect of lens capsule type IV collagen resides in the noncollagenous (NC1) domain of the alpha 3 chain and specifically in the region comprising residues 185-203 of the NC1 domain of both the human and bovine molecules. Synthetic peptides from the same region of the NC1 domains of the alpha 1, alpha 2, alpha 4, and alpha 5 chains did not possess the inhibitory effect seen with the alpha 3 chain. The sequence S-N-S (residues 189-191) is unique to the peptide of the alpha 3 chain, and substitution of either serine with alanine abolishes the inhibition. Type IV collagen isolated from the mouse Engelbreth-Holm-Swarm (EHS) tumor, a molecule that lacks the alpha 3 chain, did not prevent PMN activation but instead stimulated the secretion of elastase and type IV collagenase. Incubation of PMN with intact lens capsule type IV collagen or a peptide comprising residues 185-203 of the alpha 3 (IV) chain resulted in a 3-fold increase of intracellular cAMP, whereas, Ca2+ levels remained unchanged. Incubating PMN with forskolin or with dibutyryl-cAMP resulted in the inhibition of O2- production and degranulation by PMN, thus mimicking the effects of type IV collagen and the alpha 3 (IV) 185-203 peptide. The data suggest that type IV collagen, through its alpha 3 chain, down-regulates PMN activation and thus decreases the potential for damage as these cells traverse the capillary wall. Our in vitro experiments suggest that the higher the content of the alpha 3 (IV) chain is in a basement membrane, the wider would be its capacity for self-protection.

The tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ (GHK-Cu) was first described as a growth factor for differentiated cells. Recent in vitro data showed that it possesses several properties of a potential activator of wound repair. We investigated the effects of GHK-Cu in vivo, using the wound chamber model described previously (Schilling, J.A., W. Joel, and M.T. Shurley, 1959. Surgery [St. Louis]. 46:702-710). Stainless steel wire mesh cylinders were implanted subcutaneously on the back of rats. The animals were divided into groups that received sequential injections into the wound chamber of either saline (control group) or various concentrations of GHK-Cu. At the end of the experiments, rats were killed, wound chambers were collected, and their content was analyzed for dry weight, total proteins, collagen, DNA, elastin, glycosaminoglycans, and specific mRNAs for collagens and TGF beta. In the GHK-Cu-injected wound chambers, a concentration-dependent increase of dry weight, DNA, total protein, collagen, and glycosaminoglycan contents was found. The stimulation of collagen synthesis was twice that of noncollagen proteins. Type I and type III collagen mRNAs were increased but not TGF beta mRNAs. An increase of the relative amount of dermatan sulfate was also found. A control tripeptide, L-glutamyl-L-histidyl-L-proline, had no significant effect. These results demonstrate that GHK-Cu is able to increase extracellular matrix accumulation in wounds in vivo.

Type XIX collagen is a minor collagen that localizes to basement membrane zones. We previously demonstrated that the C-terminal NC1 domain of type XIX collagen inhibits tumor growth in vivo. In the present study, we analyzed the effects of the NC1(XIX) collagen domain on migratory behaviour of melanoma B16F10 cells. We found that NC1(XIX) do not inhibit melanoma cell proliferation. On the contrary, NC1(XIX) strongly inhibited the migratory capacities of melanoma cells in the scratch wound model and in Ibidi® devices: cell migration speed was 7.69 ± 1.49 μm/h for the controls vs 6.64 ± 0.82 μm/h for cells incubated with 30 μmol/L NC1(XIX) and 5.72 ± 0.67 μmol/h with 60 μmol/L NC1(XIX). Similar results were obtained with UACC 903 human melanoma cells. Further work will be necessary to elucidate the molecular mechanisms of this migration inhibition. It may, however, explain, at least partially, the inhibition of tumor growth that we observed in vivo.

International audience; We previously demonstrated that the CNYYSNS peptide derived from tumstatin inhibited in vivo tumor progression. The YSNS motif formed a β-turn crucial for biological activity. More recently, a YSNSG cyclopeptide with a constrained β-turn on the YSNS residues was designed. Intraperitoneal administration of the YSNSG cyclopeptide inhibited in vivo melanoma progression more efficiently than the native linear peptide. In the present article, we showed that the YSNSG cyclopeptide also triggered an inhibition of in vivo tumor neovascularization and we further analyzed its in vitroantiangiogenic effect. The YSNSG cyclopeptide did not alter endothelial cell proliferation but inhibited cell migration by 83% in an in vitro wound healing model. The inhibition was mediated by a decrease in active MT1-MMP at the migration front as well as a decrease in u-PA and u-PAR expression. The cyclopeptide also altered β1-integrin distribution in endothelial cell lamellipodia, induced a strong decrease in the phosphorylated focal adhesion kinase (p125FAK), disorganized F-actin stress fibers and decreased the number of lamellipodia, resulting in a non migratory phenotype. Our results confirm the YSNSG cyclopeptide as a potent antitumor agent, through both the inhibition of invasive properties of tumor cells and the antiangiogenic activity.

We previously demonstrated that the α1(l) polypeptide chain of collagen can bind and activate polymorphonuclear neutrophils (PMN). In the present experiments, performed in culture grade 96-well plastic plates coated with collagen, fibronectin, or other proteins, adhesion was assessed by staining the adhering cells after 30 min with crystal violet and measuring absorbance at 560 nm, and activation of PMNs was assessed by measuring the amount of O2- formed. Adhesion occurred at 17 and 37°C but activation at 37°C only. Monoclonal antibody anti-CD 18 inhibited adhesion, showing that the receptor of collagen I on PMNs is a β2 integrin. On the other hand, adhesion of PMNs to fibronectin was inhibited by monoclonal antibodies to CD18 and to CD11b.

Interstitial 2q36 deletion is a rare event. We report on a patient with a de novo del(2)(q36.2q36.3) interstitial deletion of the long arm of chromosome 2 diagnosed by classical banding. The phenotype comprised facial dysmorphism, enlarged kidneys with multiple renal cysts, abnormal minora labia, asymmetric lower limbs with dysplastic patella, and severe mental retardation. By physical mapping, using array-comparative genomic hybridisation (CGH) confirmed by Fluorescent In Situ Hybridisation (FISH), the breakpoints of the deletion were mapped and the size of the deletions was measured: 5.61+/-0.19Mb. A skin biopsy was analysed using electronic microscopy showing an alteration of the structure and organisation of the dermal and peri-neuronal basement membrane. The relation between the phenotype and the deletion of both COL4A4 and COL4A3 genes, located in 2q36.3 loci, as well as the disruption of TRIP12 were discussed.

We previously demonstrated that the NC1[alpha3(IV)185-191] CNYYSNS peptide inhibited in vivo tumor progression. The YSNS motif formed a beta turn crucial for biological activity. The aim of the present study was to design a YSNSG cyclopeptide with a constrained beta turn on the YSNS residues more stable than CNYYSNS. By nuclear magnetic resonance and molecular modeling, we demonstrated that the YSNSG cyclopeptide actually adopted the expected beta-turn conformation. It promoted melanoma cell adhesion and prevented their adhesion to the native peptide. It inhibited in vitro cell proliferation and migration through Matrigel by downregulating proteolytic cascades. Moreover, intraperitoneal administration of the YSNSG cyclopeptide inhibited melanoma progression far more efficiently than the native peptide. The increased solubility and stability at low pH of the YSNSG cyclopeptide suggest this peptide as a potent antitumor therapeutic agent.