[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Skin aging is characterised by a progressive deterioration of its functional properties, linked to alterations of dermal connective tissue. Whereas many studies have been devoted to collagen alterations during aging, the situation is less clear concerning glycosaminoglycans and proteoglycans. Particularly, the alterations of the expression of small leucine-rich proteoglycans (SLRPs), a family of proteoglycans strongly implicated in cell regulation, have never been studied. In the present study we measured glycosaminoglycans and small leucine-rich proteoglycans synthesis by skin fibroblasts from donors of 1 month to 83 years old. [3H]-glucosamine and [35S]-sulfate incorporation did not show significant differences of sulfated GAG synthesis during aging. On the other hand, a significant positive correlation was found between hyaluronan secretion and donor's age. Northern blot analysis of SLRPs mRNAs showed a significant negative correlation of lumican mRNA with donor's age, whereas decorin and biglycan mRNAs were not significantly altered. Immunohistochemical study and quantitative image analysis confirmed a decreased lumican accumulation in aged human skin. Taken together, our results suggest that impairment of glycosaminoglycans and SLRPs synthesis might be involved in the functional alterations of aged skin.
[Show abstract][Hide abstract] ABSTRACT: Chondrocyte glycosaminoglycan (GAG) synthesis is regulated by the availability of UDP-glucuronate, the substrate of glucuronosyl transferases which form the GAG chains in proteoglycans and hyaluronan. UDP-glucose dehydrogenase (UDPGD) is therefore a key enzyme in the synthesis of UDP-glucuronate from glucose. However, the mechanisms regulating its expression in chondrocytes are not fully understood. We investigated the effect of c-Krox, a zinc-finger transcription factor previously shown to modulate several matrix genes, on the synthesis of GAG and transcriptional activity of several UDPGD gene promoter constructs, using transient transfection and decoy experiments in rabbit articular chondrocytes (RACs). We show that overexpression of c-Krox inhibits radiosulfate incorporation into neosynthesized GAG and that the effect was mediated by a cis-sequence located between +18 and +39bp of the UDPGD gene. Since that sequence can also bind Sp1/Sp3 factors, it is likely that c-Krox acts in concert with these proteins to modulate the UDPGD gene expression in articular chondrocytes.
Biochemical and Biophysical Research Communications 09/2005; 333(4):1123-31. DOI:10.1016/j.bbrc.2005.06.020 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Cancer Detection and Prevention 02/2005; 29(3):260-6. DOI:10.1016/j.cdp.2004.09.003 · 2.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The concentration of neuron-specific enolase (NSE) in serum and cerebrospinal fluid (CSF) has been used as a biomarker in some cancers and, more recently, in neurodegenerative diseases. Pre-analytical conditions are very important for the quality of returned results. In this study, we evaluated the effects of storage conditions (temperature and duration of storage) and hemolysis on the concentration of NSE in serum and CSF. Our results demonstrate that samples for NSE measurement may be stored at -80 degrees C for no more than 6 months in the case of CSF and 9 months in the case of serum samples. Even invisible hemolysis may increase NSE levels in samples. Consequently, an index of hemolysis should be determined before deciding whether or not to perform NSE measurement.
Clinical Chemistry and Laboratory Medicine 02/2005; 43(11):1215-7. DOI:10.1515/CCLM.2005.210 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Experimental Cell Research 01/2005; 301(2):251-65. DOI:10.1016/j.yexcr.2004.07.036 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lumican is a member of the small leucine-rich proteoglycan (SLRP) family. It contributes to the organisation of the collagen network and plays an important role in cell migration and tissue repair. The present study aimed to determine the influence of lumican expression on adhesion, anchorage-dependent and -independent growth, migration, in vitro invasion and in vivo melanoma growth. For that purpose, B16F1 mouse melanoma cells were stably transfected with an expression plasmid containing the complete lumican cDNA. Lumican expression by tumor cells did not change the proliferative activity of mouse melanoma cells in monolayer culture and did not influence either cell adhesion to extracellular matrix gel or type I collagen or cell spreading on these substrates. In contrast, lumican-transfected cells were characterized by a strong reduction of their anchorage-independent proliferation in agarose gel and capacity to invade extracellular matrix gel. After subcutaneous injections of transfected B16F1 cells in syngenic mice, lumican expression significantly decreased subcutaneous tumor formation in vivo, with a concomitant decrease of cyclin D1 expression. Lumican induced and/or increased the apoptosis of B16F1 cells. The results suggest that lumican is involved in the control of melanoma growth and invasion and may be considered, like decorin, as an anti-tumor factor from the extracellular matrix.
Experimental Cell Research 07/2004; 296(2):294-306. DOI:10.1016/j.yexcr.2004.02.005 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Glycosaminoglycans (GAGs) and proteoglycans (PGs) belong to a class of extracellular macromolecules necessary for the growth of any multicellular structures, including tumours. Transformed cells induce stromal reaction either per se or by activation of the mesenchymal cells. Tumour stroma contains several chondroitin sulphate and heparan sulphate proteoglycans. These proteoglycans and their glycosaminoglycan chains modify cell behaviour by interacting with different molecules such as growth factors, cytokines, chemokines, proteinases and their inhibitors. This review describes the main proteoglycans of tumour stoma and discusses their implication in the regulation of the activity of extracellular proteins and peptides.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The degradation of basement membranes by tumor cells involves secretion and activation of proteinases, such as matrix metalloproteinases (MMPs) and the plasminogen activation system (uPA, tPA, PAI-1), and results from an imbalance between their inhibitors and activators, controlled by various growth factors or cytokines. Among them, the TGF-beta family is one of the most intriguing because it has been reported either to decrease or promote cancer progression. In the present paper, we studied the effect of TGF-beta1 in a mouse melanoma model. In vivo, TGF-beta1 inhibited tumor growth after subcutaneous injection of B16F1 cells in syngenic mice. In vitro, TGF-beta1 did not alter B16F1 cell proliferation, but strongly decreased their migration through Matrigel-coated membranes. The protease production was analyzed by zymography, Western blot, or RT-PCR. MMP-2 and TIMP-2 expression were not altered by TGF-beta1. In contrast, TGF-beta1 triggered a large decrease of uPA and tPA, as well as a decrease of uPA and uPAR mRNAs. By Western blot and RT-PCR analyses, TGF-beta1 was shown to induce a strong increase of PAI-1 synthesis. Collectively, these results suggest that TGF-beta1 may inhibit melanoma tumor growth by specifically decreasing plasmin activity of tumor cells and play a protective role during the earliest stages of tumor progression.
Experimental Cell Research 12/2003; 291(1):1-10. DOI:10.1016/S0014-4827(03)00336-7 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: UDP-glucose dehydrogenase (UGDH) is a key enzyme of the unique pathway for the synthesis of UDP-glucuronate, the substrate for the numerous glucuronosyl transferases, which act on the synthesis of glycosaminoglycans and glucuronidation reaction of xeno- and endobiotics. Using the bacterial artificial chromosome approach, we have cloned and characterized the human UGDH promoter. The core promoter of -644 nucleotides conferred reporter gene activity in transient transfection assay of a variety of cell types, including MRC5 fibroblasts and the HepG2 hepatoma cell line. The minimal promoter of -100 nucleotides contains a functional inverted TATA box. No consensus CAAT sequence was found up to -2133 nucleotides. The expression of UGDH was up- and down-regulated by transforming growth factor (TGF)-beta and hypoxia, respectively. TGF-beta enhanced the activity of all the deletion constructs, except the minimal promoter. Hypoxia slightly increased the activity of the short promoter-containing constructs but decreased that of the -374 nucleotides and core promoter constructs. The core promoter contained numerous GC-rich sequences for the binding of Sp1 transcription factor. Bisanthracycline, an anti-Sp1 compound, decreased UGDH mRNA expression and inhibited the core promoter constructs activity. Gel mobility shift and supershift assays after TGF-beta stimulation demonstrated an increased DNA binding of the nuclear extract proteins to the two Sp1 sequences located in the -374-bp promoter. By contrast, nuclear extract proteins from hypoxia-treated cells demonstrated a decreased binding of the consensus Sp1 sequence. These results indicate that numerous Sp1 cis-acting sequences of the UGDH core promoter are responsible for up- and down-regulation of the gene after TGF-beta stimulation and in hypoxic conditions, respectively.
[Show abstract][Hide abstract] ABSTRACT: Various biological events, such as cell differentiation, cell migration or gene expression, are controlled by cell-cell interactions or by cytokines, as well as by interactions between cells and extracellular matrix. The regulation of these events involves a directed and limited proteolysis of matrix macromolecules, that induces the release of proteic domains and peptides exhibiting biological activities. In this review, we summarise several data from our laboratory showing that peptides from type I and type IV collagens play an important role in the control of inflammation and tumor progression. Type I collagen peptides stimulate respiratory burst, granule exocytosis and cytokine secretion by human leukocytes (polymorphonuclear neutrophils or monocytes) for the detersion of inflammatory sites and then for the chemoattraction of various cell types needed for wound healing. A peptide of the NC1 domain of the alpha 3(IV) collagen chain prevents leukocyte activation. In addition, this peptide is also capable of limiting tumor progression by downregulating in vitro and in vivo invasive properties of melanoma cells.
Journal de la Société de Biologie 02/2003; 197(1):31-9.
[Show abstract][Hide abstract] ABSTRACT: The degradation of extracellular matrix (ECM) during physio-pathological processes involves, essentially, two proteolytic systems: the plasmin (ogen) system and the matrix metalloproteinase (MMP) family. Enzyme activity necessitates the formation of proteolytic cascades acting in the pericellular environment. Several proteins (proteases, integrins, matrix, inhibitors, activators...) participate to enzyme catalysis forming assemblies within specialized plasma membrane structures (invadopodia, caveolae). MMP-mediated ECM degradation leads to the formation of peptides (matricryptins, matrikins) which, in turn, can modulate MMP expression. MMPs (especially gelatinases) can also activate growth factors as pro TGF beta or liberate those factors from matrix sites. Interaction between matrix and gelatinases was shown to influence enzyme activation through several mechanisms. Finally, thrombospondins 1 and 2, matricellular proteins, can regulate gelatinase A by favoring its endocytosis. Those data emphasize the potential interest of certain matrikins or pseudo-matrikins as therapeutic agents to control cell invasion.
Journal de la Société de Biologie 02/2003; 197(1):25-30.
[Show abstract][Hide abstract] ABSTRACT: Human blood monocytes are attracted into connective tissues during early steps of inflammation and wound healing, and locally interact with resident cells and extracellular matrix proteins. We studied the effects of type I collagen on monocyte adhesion and superoxide anion production, using human monocytes elutriated from peripheral blood and type I collagen obtained from rat tail tendon. Both acid-soluble and pepsin-digested type I collagens promoted the adhesion of monocytes, whereas only acid-soluble collagen with intact telopeptides induced the production of superoxide. Adhesion and activation of monocytes on acid-soluble type I collagen depended on the presence of divalent cations. mAbs directed against integrin subunits CD11c and CD18 specifically inhibited adhesion and activation of monocytes on type I collagen. Protein membrane extracts obtained from monocytes were submitted to affinity chromatography on collagen I-Sepharose 4B, and analyzed by Western blotting using specific anti-integrin subunit Abs. In the case of both acid-soluble and pepsin-digested collagens, two bands were revealed with mAbs against CD11c and CD18 integrin subunits. Our results demonstrate that monocytes interact with type I collagen through CD11c-CD18 (αxβ2) integrins, which promote their adhesion and activation. For monocyte activation, specific domains of the type I collagen telopeptides are necessary. Interactions between monocytes and collagen are most likely involved in the cascade of events that characterize the initial phases of inflammation.
The Journal of Immunology 06/2000; 164(11):5928-5934. · 4.92 Impact Factor