Laurent Ramont

Effects of hemolysis and storage condition on neuron-specific enolase (NSE) in cerebrospinal fluid and serum: implications in clinical practice.

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°C for no more than 6 months in the case of CSF and 9months 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.

Implication of interleukin-4 in wound healing.

Interleukin-4 (IL-4) is a pleiotropic cytokine that is able to activate connective tissue cells and stimulate the accumulation of extracellular matrix macromolecules. In this report, the expression of IL-4 in normal wound healing was studied by immunohistochemistry. The effects of exogenous IL-4 or IL-4 antisense oligonucleotides administration were also studied in mouse experimental wounds. IL-4 expression was detected in the lower dermis below the wound as early as Day 1 after wounding. IL-4 expression was maximal by Day 4, then decreased progressively, and completely disappeared by Day 21 after wounding. Topical administration of IL-4 on experimental wounds in mice significantly accelerated the rate of healing, whereas IL-4 antisense oligonucleotides significantly inhibited healing. These results demonstrate that IL-4 may be implicated in normal wound healing.

The Antitumor Properties of the α3(IV)-(185-203) Peptide from the NC1 Domain of Type IV Collagen (Tumstatin) Are Conformation-dependent

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 α3(IV) collagen chain inhibits the in vitro expression of matrix metalloproteinases in human melanoma cells through RGD-independent binding to αvβ3 integrin. In the present paper, we demonstrate that in a mouse melanoma model, the NC1 α3(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 α3(IV)-(185-203) peptides show a β-turn at the YSNS (188-191) sequence level, which is crucial for biological activity. As well, the homologous MNYYSNS heptapeptide keeps the β-turn and the inhibitory activity. In contrast, the DNYYSNS heptapeptide, which does not form the β-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 αvβ3 integrin in melanoma progression.

Expression of lumican, a small leucine-rich proteoglycan with antitumour activity, in human malignant melanoma.

Background.  The family of small leucine‐rich proteoglycans (SLRPs), which includes decorin, lumican, biglycan and fibromodulin, constitutes an abundant component of the skin extracellular matrix. We previously demonstrated that human lumican inhibits melanoma growth and progression in a mouse experimental model, by regulating cell migration, proliferation and apoptosis.

Matrikines from basement membrane collagens: a new anti-cancer strategy.

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.

Transforming growth factor-β1 inhibits tumor growth in a mouse melanoma model by down-regulating the plasminogen activation system

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.

The NC1 domain of type XIX collagen inhibits in vivo melanoma growth

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 YSNSG cyclopeptide derived from tumstatin inhibits tumor angiogenesis by down-regulating endothelial cell migration

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.

Tetrastatin, the NC1 domain of the α4(IV) collagen chain: a novel potent anti-tumor matrikine.

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.

Pleural Effusion in a Patient with Multiple Myeloma

A 62-year-old woman presenting with dyspnea on exertion was admitted to our hospital. Her medical history included type 2 diabetes and κ light chain multiple myeloma (MM) diagnosed 3 years ago. The results of a basic biochemical examination performed 2 weeks earlier were normal. Serum protein electrophoresis revealed hypoproteinemia (65 g/L; reference interval, 68–73 g/L), associated with decreased γ globulins (4 g/L; reference interval, 9–15 g/L) but without a detectable paraprotein band. An assay for serum free light chain showed decreased κ light chains (0.5 mg/L; reference interval, 3.3–19.4 mg/L) and λ light chains (<0.3 mg/L; reference interval, 5.7–26.3 mg/L); the κ/λ ratio could not be accurately determined because of the low concentration of λ light chains. An examination of a bone marrow aspirate taken 3 months earlier showed dystrophic plasma cells accounting for 50% of the nucleated cells. On admission, laboratory tests revealed normal values for hemoglobin (142 g/L; reference interval, 130–180 g/L), white blood cells (6.7 × 109/L; reference interval, 4–10 × 109/L), platelets (211 × 109/L; reference interval, 150–400 × 109/L), and creatinine [38 μmol/L (0.4 mg/dL); reference interval, 45–90 μmol/L (0.5–1.0 mg/dL)], but the tests also revealed mild hypocalcemia [2.14 mmol/L (8.6 mg/dL); reference interval, 2.20–2.60 mmol/L (8.8–10.4 mg/dL)]. A chest radiograph and spiral thoracic computed tomography showed a large right-sided pleural effusion. A sample of the pleural fluid had a total protein concentration of 38 g/L and a white blood cell count of 20.5 × 109/L, with 100% lymphoid cells. The results of bacterial and mycobacterial cultures were negative. Protein electrophoresis evaluations of serum, urine, and the …