Hyaluronan cell surface binding is induced by type I collagen and regulated by caveolae in glioma cells

Hyaluronan (HA) is a component of the brain extracellular matrix environment that is synthesized and secreted by glioma cells. The primary cell surface receptor for HA is CD44, a membrane glycoprotein that is functionally regulated by a membrane type 1 matrix metalloproteinase (MT1-MMP). Both CD44 and MT1-MMP are partially located in Triton X-100-insoluble domains, but no functional link has yet been established between them. In the present study, we studied the regulation of HA cell surface binding in U-87 glioma cells. We show that an MMP-dependent mechanism regulates the intrinsic cell surface binding of HA as ilomastat, a broad MMP inhibitor, increased HA binding to glioma cells. HA binding was also rapidly and specifically up-regulated by 3-fold by type I collagen in U-87 cells, which also induced a significant morphological reorganization associated with the activation of a latent form of MMP-2 through a MT1-MMP-mediated mechanism. Interestingly, caveolae depletion with a cell surface cholesterol-depleting agent beta-cyclodextrin triggered an additional increase (9-fold) in the binding of HA, in synergy with type I collagen. On the other hand, HA cell surface binding was diminished by the MEK inhibitor PD98059 and by the overexpression of a recombinant, wild type MT1-MMP, whereas its cytoplasmic-deleted form had no effect. Taken together, our results suggest that MT1-MMP regulates, through its cytoplasmic domain, the cell surface functions of CD44 in a collagen-rich pericellular environment. Additionally, we describe a new molecular mechanism regulating the invasive potential of glioma cells involving a MT1-MMP/CD44/caveolin interaction, which could represent a potential target for anti-cancer therapies.     

Melanoma Cell Adhesion Molecule (MCAM) expression in the myogenic lineage during early chick embryonic development

We describe the expression pattern of cMCAM, a cell adhesion molecule of the immunoglobulin superfamily, in early chick embryonic development by in situ hybridisation. An initial ectodermal domain of expression is subsequently expanded, and cMCAM is expressed in the neural crest cells, otic vesicle, heart primordium, notochord and endoderm. In addition, cMCAM expression localises in the myotome once the somite cells have been specified. An in vitro murine cellular system allowed us to confirm that MCAM expression coincides with the onset of myogenic cell determination.     

Silencing of the MT1-MMP/ G6PT axis suppresses calcium mobilization by sphingosine-1-phosphate in glioblastoma cells

The contributions of membrane type-1 matrix metalloproteinase (MT1-MMP) and of the glucose-6-phosphate transporter (G6PT) in sphingosine-1-phosphate (S1P)-mediated Ca(2+) mobilization were assessed in glioblastoma cells. We show that gene silencing of MT1-MMP or G6PT decreased the extent of S1P-induced Ca(2+) mobilization, chemotaxis, and extracellular signal-related kinase phosphorylation. Chlorogenic acid and (-)-epigallocatechin-3-gallate, two diet-derived inhibitors of G6PT and of MT1-MMP, respectively, reduced S1P-mediated Ca(2+) mobilization. An intact MT1-MMP/G6PT signaling axis is thus required for efficient Ca(2+) mobilization in response to bioactive lipids such as S1P. Targeted inhibition of either MT1-MMP or G6PT may lead to reduced infiltrative and invasive properties of brain tumor cells.     

Inhibition of MMP-2 secretion from brain tumor cells suggests chemopreventive properties of a furanocoumarin glycoside and of chalcones isolated from the twigs of Dorstenia turbinata

A furanocoumarin glycoside new named turbinatocoumarin (1) was isolated from the twigs of Dorstenia turbinata. The structure of turbinatocoumarin (1) was assigned as 5-methoxy-3-[3-(beta-glucopyranosyloxy)-2-hydroxy-3-methylbutyl]psoralen by means of spectroscopic analysis. Known compounds have also been isolated from this genus and identified as (2'S, 3'R)-3'-hydroxymarmesin (2), 5-methoxy-3-(3-methyl-2,3-dihydroxybutyl)psoralen (3), psoralen (4), kanzonol C (5) which was isolated for the first time from this genus, 4-hydroxylonchocarpin (6), umbelliferone, 4-hydroxy-3-methoxybenzaldehyde and 4-methoxyphenol. As part of our continuing search for potential naturally-occurring antitumor drug candidates, the inhibition of matrix metalloproteinase (MMP)-2 secretion from brain tumor-derived glioblastoma cells by the isolated compounds 1, 3, 5, and 6 was evaluated by zymography and compared to the documented naturally-occurring MMP secretion inhibitors chlorogenic acid (CHL) and epigallocatechin-3-gallate (EGCg). Among the compounds tested, the inhibiting MMP secretion concentrations ranged from 0.025 to 250 microM with up to 80% inhibition. The inhibitory activities of compounds 5 and 6 were found comparable to the common reference compounds CHL and EGCg. This suggests that alternate sources can be explored and exploited for the availability of chemopreventive molecules.     

Effects of In Vitro Low Oxygen Tension Preconditioning of Adipose Stromal Cells on Their In Vivo Chondrogenic Potential: Application in Cartilage Tissue Repair

Purpose

Multipotent stromal cell (MSC)-based regenerative strategy has shown promise for the repair of cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to promote the early chondrogenic differentiation of MSC. The aim of our study was therefore to determine whether low oxygen tension could be used to enhance the regenerative potential of MSC for cartilage repair.

Methods

MSC from rabbit or human adipose stromal cells (ASC) were preconditioned in vitro in control or chondrogenic (ITS and TGF-β) medium and in 21 or 5% O₂. Chondrogenic commitment was monitored by measuring COL2A1 and ACAN expression (real-time PCR). Preconditioned rabbit and human ASC were then incorporated into an Si-HPMC hydrogel and injected (i) into rabbit articular cartilage defects for 18 weeks or (ii) subcutaneously into nude mice for five weeks. The newly formed tissue was qualitatively and quantitatively evaluated by cartilage-specific immunohistological staining and scoring. The phenotype of ASC cultured in a monolayer or within Si-HPMC in control or chondrogenic medium and in 21 or 5% O₂ was finally evaluated using real-time PCR.

Results/Conclusions

5% O₂ increased the in vitro expression of chondrogenic markers in ASC cultured in induction medium. Cells implanted within Si-HPMC hydrogel and preconditioned in chondrogenic medium formed a cartilaginous tissue, regardless of the level of oxygen. In addition, the 3D in vitro culture of ASC within Si-HPMC hydrogel was found to reinforce the pro-chondrogenic effects of the induction medium and 5% O₂. These data together indicate that although 5% O₂ enhances the in vitro chondrogenic differentiation of ASC, it does not enhance their in vivo chondrogenesis. These results also highlight the in vivo chondrogenic potential of ASC and their potential value in cartilage repair.     

Concanavalin-A-induced autophagy biomarkers requires membrane type-1 matrix metalloproteinase intracellular signaling in glioblastoma cells

Pre-clinical trials for cancer therapeutics support the anti-neoplastic properties of the lectin from Canavalia ensiformis (Concanavalin-A, ConA) in targeting apoptosis and autophagy in a variety of cancer cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP), a plasma membrane-anchored matrix metalloproteinase, is a glycoprotein strongly expressed in radioresistant and chemoresistant glioblastoma that mediates pro-apoptotic signalling in brain cancer cells, we investigated whether MT1-MMP could also signal autophagy. Among the four lectins tested, we found that the mannopyranoside/glucopyranoside-binding ConA, which is also well documented to trigger MT1-MMP expression, increases autophagic acidic vacuoles formation as demonstrated by Acridine Orange cell staining. Although siRNA-mediated MT1-MMP gene silencing effectively reversed ConA-induced autophagy, inhibition of the MT1-MMP extracellular catalytic function with Actinonin or Ilomastat did not. Conversely, direct overexpression of the recombinant Wt-MT1-MMP protein triggered proMMP-2 activation and green fluorescent protein-microtubule-associated protein light chain 3 puncta indicative of autophagosomes formation, while deletion of MT1-MMP's cytoplasmic domain disabled such autophagy induction. ConA-treated U87 cells also showed an upregulation of BNIP3 and of autophagy-related gene members autophagy-related protein 3, autophagy-related protein 12 and autophagy-related protein 16-like 1, where respective inductions were reversed when MT1-MMP gene expression was silenced. Altogether, we provide molecular evidence supporting the pro-autophagic mechanism of action of ConA in glioblastoma cells. We also highlight new signal transduction functions of MT1-MMP within apoptotic and autophagic pathways that often characterize cancer cell responses to chemotherapeutic drugs.     

Bioaccumulation du Cd et du Zn chez les plants de tomates (Lycopersicon esculentum L.)

This work aims at evaluating the accumulation of cadmium (Cd) and zinc (Zn) (trace elements) in the organs of young tomato plants (Lycopersicon esculentum L. var. Rio Grande) and their effects on the rate of chlorophyll and enzyme activities involved in the antioxidant system: catalase (CAT), glutathion-S-transferase (GST) and peroxysase ascorbate (APX). Plants previously grown on a basic nutrient solution were undergoing treatment for 7 days, either by increasing concentrations of CdCl₂ or ZnSO₄ (0, 50, 100, 250, 500 μM) or by the combined concentrations of Cd and Zn (100/50, 100/100, 100/250, 100/500 μM). The results concerning the determination of metals in the various compartments of tomato plants as a function of increasing concentrations of Cd or Zn, suggest a greater accumulation of Cd and Zn in the roots compared to leaves. The combined treatment (Cd/Zn) interferes with the absorption of the two elements according to their concentrations in the culture medium. The presence of Zn at low concentrations (50 μM of Zn/100 μM Cd) has little influence on the accumulation of Cd in the roots and leaves, while the absorption of these two elements in the leaves increases and decreases in roots when their concentrations are equivalent (100/100 μM) compared to treatment alone. When the concentration of Zn is higher than that of Cd (500 μM of Zn/100 μM Cd) absorption of the latter is inhibited in the roots while increasing their translocation to the leaves. Meanwhile, the dosage of chlorophylls shows that they tend to decrease in a dose-dependent for both treatments (Cd or Cd/Zn), however, treatment with low concentrations of Zn (50 and 100 μM) stimulates chlorophyll synthesis. However, treatment with different concentrations of Cd seems to induce the activity of the enzymes studied (CAT, APX, GST). It is the same for treatment with different concentrations of Zn and this particularly for the highest concentrations. Finally, the combined treatment (Zn/Cd) also appears to cause enzyme inductions: CAT, APX and GST.     

Tumor environment dictates medulloblastoma cancer stem cell expression and invasive phenotype

The neural precursor surface marker CD133 is thought to be enriched in brain cancer stem cells and in radioresistant DAOY medulloblastoma-derived tumor cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP) expression is a hallmark of highly invasive, radioresistant, and hypoxic brain tumor cells, we sought to determine whether MT1-MMP and other MMPs could regulate the invasive phenotype of CD133(+) DAOY cells. We found that when DAOY medulloblastoma or U87 glioblastoma cells were implanted in nude mice, only those cells specifically implanted in the brain environment generated CD133(+) brain tumors. Vascular endothelial growth factor and basic fibroblast growth factor gene expression increases in correlation with CD133 expression in those tumors. When DAOY cultures were induced to generate in vitro neurosphere-like cells, gene expression of CD133, MT1-MMP, MMP-9, and MDR-1 was induced and correlated with an increase in neurosphere invasiveness. Specific small interfering RNA gene silencing of either MT1-MMP or MMP-9 reduced the capacity of the DAOY monolayers to generate neurospheres and concomitantly abrogated their invasive capacity. On the other hand, overexpression of MT1-MMP in DAOY triggered neurosphere-like formation which was further amplified when cells were cultured in neurosphere medium. Collectively, we show that both MT1-MMP and MMP-9 contribute to the invasive phenotype during CD133(+) neurosphere-like formation in medulloblastoma cells. Increases in MMP-9 may contribute to the opening of the blood-brain barrier, whereas increased MT1-MMP would promote brain tumor infiltration. Our study suggests that MMP-9 or MT1-MMP targeting may reduce the formation of brain tumor stem cells.     

Tetra- and hexavalent mannosides inhibit the pro-apoptotic, antiproliferative and cell surface clustering effects of concanavalin-A: impact on MT1-MMP functions in marrow-derived mesenchymal stromal cells

Mesenchymal stromal cells (MSC) mobilization and recruitment by experimental vascularizing tumors involves membrane type 1-matrix metalloproteinase (MT1-MMP) functions. Given that the mannose-specific lectin Concanavalin-A (ConA) induces MT1-MMP expression and mimics biological lectins/carbohydrate interactions, we synthesized and tested the potential of 11 mannoside clusters to block ConA activities on MSC. We found that tetra- and hexavalent mannosides reversed ConA-mediated changes in MSC morphology and antagonized ConA-induced caspase-3 activity and proMMP-2 activation. Tetra- and hexavalent mannosides also inhibited ConA- but not the cytoskeleton disrupting agent Cytochalasin-d-induced MT1-MMP cell surface proteolytic processing mechanisms, and effects on cell cycle phase progression. The antiproliferative and pro-apoptotic impact of ConA on the MT1-MMP/glucose-6-phosphate transporter signaling axis was also reversed by these mannosides. In conclusion, we designed and identified glycocluster constructions that efficiently interfered with carbohydrate-binding proteins (lectins) interaction with oligosaccharide moieties of glycoproteins at the cell surface of MSC. These glycoclusters may serve in carbohydrate-based anticancer strategies through their ability to specifically target MT1-MMP pleiotropic functions in cell survival, proliferation, and extracellular matrix degradation.     

Brain endothelial cells as pharmacological targets in brain tumors

The blood-brain barrier contributes to brain homeostasis by controlling the access of nutrients and toxic substances to the central nervous system (CNS). The acquired brain endothelial cells phenotype results from their sustained interactions with their microenvironment. The endothelial component is involved in the development and progression of most CNS diseases such as brain tumors, Alzheimer’s disease, or stroke, for which efficient treatments remain to be discovered. The endothelium constitutes an attractive therapeutical target, particularly in the case of brain tumors, because of the high level of angiogenesis associated with this disease. Drug development based on targeting differential protein expression in the vasculature associated with normal tissues or with disease states holds great potential. This article highlights some of the growing body of evidence showing molecular differences between the vascular bed phenotype of normal and pathological endothelium, with a particular focus on brain tumor endothelium targets, which may play crucial roles in the development of brain cancers. Finally, an overview is presented of the emerging therapies for brain tumors that take the endothelial component into consideration. Equal first authors