Biomedical subjects

Borhane Annabi

Publications and source records attributed to Borhane Annabi.

At least 19 recordsLinked to original sources

PubMed2007

MT1-MMP down-regulates the glucose 6-phosphate transporter expression in marrow stromal cells: a molecular link between pro-MMP-2 activation, chemotaxis, and cell survival.

Bone marrow-derived stromal cells (BMSC) are avidly recruited by experimental vascularizing tumors, which implies that they must respond to tumor-derived growth factor cues. In fact, BMSC chemotaxis and cell survival are regulated, in part, by the membrane type-1 matrix metalloproteinase (MT1-MMP), an MMP also involved in pro-MMP-2 activation and in degradation of the extracellular matrix (ECM). Given that impaired chemotaxis was recently observed in bone marrow cells isolated from a glucose 6-phosphate transporter-deficient (G6PT-/-) mouse model, we sought to investigate the potential MT1-MMP/G6PT signaling axis in BMSC. We show that MT1-MMP-mediated activation of pro-MMP-2 by concanavalin A (ConA) correlated with an increase in the sub-G1 cell cycle phase as well as with cell necrosis, indicative of a decrease in BMSC survival. BMSC isolated from Egr-1-/- mouse or MT1-MMP gene silencing in BMSC with small interfering RNA (siMT1-MMP) antagonized both the ConA-mediated activation of pro-MMP-2 and the induction of cell necrosis. Overexpression of recombinant full-length MT1-MMP triggered necrosis and this was signaled through the cytoplasmic domain of MT1-MMP. ConA inhibited both the gene and protein expression of G6PT, while overexpression of recombinant G6PT inhibited MT1-MMP-mediated pro-MMP-2 activation but could not rescue BMSC from ConA-induced cell necrosis. Cell chemotaxis in response to the tumorigenic growth factor sphingosine 1-phosphate was significantly abrogated in siMT1-MMP BMSC and in chlorogenic acid-treated BMSC. Altogether, we provide evidence for an MT1-MMP/G6PT signaling axis that regulates BMSC survival, ECM degradation, and mobilization. This may lead to optimized clinical applications that use BMSC as a platform for the systemic delivery of therapeutic or anti-cancer recombinant proteins in vivo.

Animals↗

PubMed2006

The response to brain tumor-derived growth factors is altered in radioresistant human brain endothelial cells.

INTRODUCTION: Radioresistant brain tumor vasculature is thought to hamper the efficiency of adjuvant cancer therapies. However, little is known regarding the signalling pathways involved in the angiogenic response to brain tumor-derived growth factors in irradiated human brain microvascular endothelial cells (HBMEC). The goal of this study is to assess the effect of ionizing radiation (IR) on HBMEC survival, migration and tubulogenesis. METHODS: HBMEC were cultured and irradiated at sublethal single doses. Cell survival was assessed by nuclear cell counting and flow cytometry. HBMEC migration in response to brain tumor-derived growth factors (U-87 GF) and tubulogenesis were assayed using modified Boyden chambers and Matrigel, respectively. RESULTS: We observed that single administration of 3-10 Gy IR doses only reduced cell survival by 30%. Radioresistant HBMEC overexpressed RhoA, a small GTPase protein regulating cellular adhesion and migration, and Rho-kinase (ROK), a serine-threonine protein kinase and one of RhoA's major targets. HBMEC migration was induced by vascular endothelial growth factor (VEGF), but even more so in response to sphingo-sine-1-phosphate (S1P) and to U-87 GF. Following IR exposure, HBMEC basal migration increased more than two-fold, whereas the response to S1P and to U-87 GF was significantly diminished. Similarly, the inhibitor of ROK Y-27632 decreased HBMEC migration in response to S1P and U-87 GF. Overexpression of RhoA decreased tubulogenesis, an effect also observed in irradiated HBMEC. CONCLUSION: Our results suggest that radioresistant HBMEC migration response to tumor-secreted growth factors and tubulogenesis are altered following IR. The RhoA/ROK signalling pathway is involved in the IR-altered angiogenic functions and may represent a potential molecular target for enhancing the impact of radiotherapy on tumor-associated endothelial cells.

Apoptosis↗

PubMed2006

A GMCSF and IL-15 fusokine leads to paradoxical immunosuppression in vivo via asymmetrical JAK/STAT signaling through the IL-15 receptor complex.

We hypothesized that a granulocyte macrophage colony-stimulating factor (GMCSF) and interleukin 15 (IL-15) fusokine (GIFT15) would possess greater immune-stimulatory properties than their combined use. Unexpectedly, tumor cells engineered to secrete GIFT15 protein led to suppression of natural killer (NK) and NKT-cell recruitment in vivo, suggesting an unanticipated immune-suppressive effect. We found GIFT15 to have pleiotropic effects on an array of immune-competent cells. Among these, macrophages treated with GIFT15 secrete de novo the tissue inhibitor of metalloproteinase-2 (TIMP-2); activated matrix metalloproteinase-2 (MMP-2); transforming growth factor-beta (TGF-beta); as well as vascular endothelial growth factor (VEGF). We show that the GIFT15 fusokine has increased affinity for the alpha chain component of the IL-15R, leading to aberrant signaling through the beta chain manifested by the hyperphosphorylation of STAT3 both in macrophages and splenocytes. Suppression of common gamma chain-mediated STAT5 phosphorylation and blockade of the IL-15-dependent IFN-gamma response in mouse splenocytes were also observed. We tested GIFT15 as an immunosuppressor and demonstrated that it allowed engraftment of allogeneic B16F0 and human xenograft U87GM glioma cells in immunocompetent mice. Thus, GIFT15 defines a new class of fusokine that mediates proangiogenic and immunosuppressive effects via aberrant signaling by the IL-15R in lymphomyeloid cells.

Amino Acid Sequence↗

PubMed2006

Inhibition of HuR and MMP-9 expression in macrophage-differentiated HL-60 myeloid leukemia cells by green tea polyphenol EGCg.

Matrix metalloproteinase (MMP)-9 expression is linked with myeloid cell differentiation, as well as inflammation and angiogenesis processes related to cancer progression. MMP-9 secretion and macrophage-like HL-60 myeloid leukemia cells differentiation were triggered by the tumor-promoting agent PMA. The chemopreventive effects of green tea catechins epigallocatechin-gallate, catechin-gallate, and epicatechin-gallate, but not those catechins that lack a 3'-galloyl group, inhibited in a time- and dose-dependent manner MMP-9 secretion. The gene and protein expression of MMP-9 and of the mRNA stabilizing factor HuR were also inhibited, while that of the 67 kDa laminin receptor remained unaffected. Specific catechins may help optimize current chemotherapeutic treatment protocols for leukemia.

Anticarcinogenic Agents↗

PubMed2006

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.

Antineoplastic Agents, Phytogenic↗

PubMed2006

Cooperation of matrix metalloproteinases with the RhoA/Rho kinase and mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase signaling pathways is required for the sphingosine-1-phosphate-induced mobilization of marrow-derived stromal cells.

The ease of isolation and ex vivo culture of marrow-derived stromal cells (MSCs) from adult bone marrow renders them a very promising source of adult stem cells for gene transfer and cell therapy. However, little is known about the signaling pathways that control their in vivo mobilization and subsequent biodistribution. Platelet-derived sphingosine-1-phosphate (S1P), a bioactive lipid that acts via G-protein-coupled-receptors, exerts strong chemoattraction upon MSCs through yet-uncharacterized signaling pathways. We show that the S1P-induced migration and morphological changes of MSCs in vitro require the activities of extracellular signal-regulated kinase (ERK), Rho kinase (ROCK), and matrix metalloproteinase (MMP) signaling molecules. Specifically, S1P-induced remodeling of the MSC cytoskeleton led to the rapid (<1 minute) formation of actin stress fibers via activation of the RhoA/ROCK pathway and required the catalytic activity of MMPs. S1P-induced activation of the mitogen-activated protein kinase kinase-1 (MEK1)/ERK pathway also contributed to the induction of the actin stress fibers and to the redistribution of paxillin at the focal adhesions through tyrosine phosphorylation of focal adhesion kinase in an MMP-dependent manner. Moreover, MMP- and ROCK-dependent molecular events are implicated in the regulation of the S1P-induced activation of ERK. Our results demonstrate that MSC mobilization in response to S1P requires cooperation between MMP-mediated signaling events and the RhoA/ROCK and MEK1/ERK intracellular pathways. Therefore, the characterization of the cellular factors and the intracellular signaling pathways underlying MSC mobilization is crucial to achieve high efficacy in therapeutic use.

Actin Cytoskeleton↗

PubMed2006

Silencing of the human microsomal glucose-6-phosphate translocase induces glioma cell death: potential new anticancer target for curcumin.

G6P translocase (G6PT) is thought to play a crucial role in transducing intracellular signaling events in brain tumor-derived cancer cells. In this report, we investigated the contribution of G6PT to the control of U-87 brain tumor-derived glioma cell survival using small interfering RNA (siRNA)-mediated suppression of G6PT. Three siRNA constructs were generated and found to suppress up to 91% G6PT gene expression. Flow cytometry analysis of propidium iodide/annexin-V-stained cells indicated that silencing the G6PT gene induced necrosis and late apoptosis. The anticancer agent curcumin, also inhibited G6PT gene expression by more than 90% and triggered U-87 glioma cells death. Overexpression of recombinant G6PT rescued the cells from curcumin-induced cell death. Targeting G6PT expression may provide a new mechanistic rationale for the action of chemopreventive drugs and lead to the development of new anti-cancer strategies.

Antineoplastic Agents↗

PubMed2006

Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic PSP94-derived peptide PCK3145 cell surface binding.

PURPOSE: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. RESULTS: [(14)C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145 binding but unexpectedly not to its uptake. CONCLUSIONS: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.

Catechin↗

PubMed2006

Combined low dose ionizing radiation and green tea-derived epigallocatechin-3-gallate treatment induces human brain endothelial cells death.

The microvasculature of brain tumors has been proposed as the primary target for ionizing radiation (IR)-induced apoptosis. However, the contribution of low dose IR-induced non-apoptotic cell death pathways has not been investigated. This study aimed to characterize the effect of IR on human brain microvascular endothelial cells (HBMEC) and to assess the combined effect of epigallocatechin-3-gallate (EGCg), a green tea-derived anti-angiogenic molecule. HBMEC were treated with EGCg, irradiated with a sublethal (< or =10 Gy) single dose. Cell survival was assessed 48 h later by nuclear cell counting and Trypan blue exclusion methods. Cell cycle distribution and DNA fragmentation were evaluated by flow cytometry (FC), cell death was assessed by fluorimetric caspase-3 activity, FC and immunoblotting for pro-apoptotic proteins. While low IR doses alone reduced cell survival by 30%, IR treatment was found more effective in EGCg pretreated-cells reaching 70% cell death. Analysis of cell cycle revealed that IR-induced cell accumulation in G2-phase. Expression of cyclin-dependent kinase inhibitors p21(CIP/Waf1) and p27(Kip) were increased by EGCg and IR. Although random DNA fragmentation increased by approximately 40% following combined EGCg/IR treatments, the synergistic reduction of cell survival was not related to increased pro-apoptotic caspase-3, caspase-9 and cytochrome C proteins. Cell necrosis increased 5-fold following combined EGCg/IR treatments while no changes in early or late apoptosis were observed. Our results suggest that the synergistic effects of combined EGCg/IR treatments may be related to necrosis, a non-apoptotic cell death pathway. Strategies sensitizing brain tumor-derived EC to IR may enhance the efficacy of radiotherapy and EGCg may represent such a potential agent.

Apoptosis↗

PubMed2006

A prostate secretory protein94-derived synthetic peptide PCK3145 inhibits VEGF signalling in endothelial cells: implication in tumor angiogenesis.

We have previously observed that the synthetic peptide corresponding to amino acids 31-45 (PCK3145) of PSP94 can reduce prostate tumor growth in vivo. Moreover, a recently concluded phase IIa clinical trial with patients with hormone refractory prostate cancer indicated that PCK3145 down-regulates the levels of plasma matrix metalloproteinase (MMP)-9, a MMP involved in metastasis and tumor angiogenesis. The purpose of our study was to investigate the molecular mechanisms of action of PCK3145 and whether this peptide could antagonize tumor neovascularization. We show that, in a syngeneic in vivo model of rat prostate cancer, the expression of endothelial cell (EC) specific CD31, a marker of tumor vessel density, was decreased by 43% in PCK3145-treated animals. In vitro, PCK3145 specifically antagonized in a dose-dependent manner the VEGF-induced ERK phosphorylation as well as the phosphorylation of the VEGFR-2 in cultured EC (HUVEC). These anti-VEGF effects were partly reproduced by pharmacological inhibitors such as PD98059 and PTK787, suggesting that PCK3145 inhibits the tyrosine kinase activity associated to VEGFR-2, which in turn prevents intracellular signalling through the MAPK cascade. Moreover, PCK3145 was also found to inhibit the PDGF-induced phosphorylation of PDGFR in smooth muscle cells. Finally, PCK3145 inhibited in vitro EC tubulogenesis and VEGF-induced MMP-2 secretion suggesting its potential implication as an antiangiogenic agent. Our study demonstrates that PCK3145 interferes with the tyrosine kinase activity associated with VEGF signalling axis in EC. The antiangiogenic properties of this peptide could be highly beneficial and exploited in novel antiangiogenic therapies, for patients with various cancers.

Animals↗

PubMed2006

The chemopreventive properties of chlorogenic acid reveal a potential new role for the microsomal glucose-6-phosphate translocase in brain tumor progression.

BACKGROUND: Chlorogenic acid (CHL), the most potent functional inhibitor of the microsomal glucose-6-phosphate translocase (G6PT), is thought to possess cancer chemopreventive properties. It is not known, however, whether any G6PT functions are involved in tumorigenesis. We investigated the effects of CHL and the potential role of G6PT in regulating the invasive phenotype of brain tumor-derived glioma cells. RESULTS: RT-PCR was used to show that, among the adult and pediatric brain tumor-derived cells tested, U-87 glioma cells expressed the highest levels of G6PT mRNA. U-87 cells lacked the microsomal catalytic subunit glucose-6-phosphatase (G6Pase)-alpha but expressed G6Pase-beta which, when coupled to G6PT, allows G6P hydrolysis into glucose to occur in non-glyconeogenic tissues such as brain. CHL inhibited U-87 cell migration and matrix metalloproteinase (MMP)-2 secretion, two prerequisites for tumor cell invasion. Moreover, CHL also inhibited cell migration induced by sphingosine-1-phosphate (S1P), a potent mitogen for glioblastoma multiform cells, as well as the rapid, S1P-induced extracellular signal-regulated protein kinase phosphorylation potentially mediated through intracellular calcium mobilization, suggesting that G6PT may also perform crucial functions in regulating intracellular signalling. Overexpression of the recombinant G6PT protein induced U-87 glioma cell migration that was, in turn, antagonized by CHL. MMP-2 secretion was also inhibited by the adenosine triphosphate (ATP)-depleting agents 2-deoxyglucose and 5-thioglucose, a mechanism that may inhibit ATP-mediated calcium sequestration by G6PT. CONCLUSION: We illustrate a new G6PT function in glioma cells that could regulate the intracellular signalling and invasive phenotype of brain tumor cells, and that can be targeted by the anticancer properties of CHL.

Journal Article↗

PubMed2006

The Survivin-mediated radioresistant phenotype of glioblastomas is regulated by RhoA and inhibited by the green tea polyphenol (-)-epigallocatechin-3-gallate.

INTRODUCTION: Glioblastoma multiforme's (GBM) aggressiveness is potentiated in radioresistant tumor cells. The combination of radiotherapy and chemotherapy has been envisioned as a therapeutic approach for GBM. The goal of this study is to determine if epigallocatechin-3-gallate (EGCg), a green tea-derived anti-cancer molecule, can modulate GBMs' response to ionizing radiation (IR) and whether this involves mediators of intracellular signaling and inhibitors of apoptosis proteins. MATERIAL AND METHODS: U-87 human GBM cells were cultured and transfected with cDNAs encoding for Survivin, RhoA or Caveolin-1. Mock and transfected cells were irradiated at sublethal single doses. Cell proliferation was analyzed by nuclear cell counting. Apoptosis was detected using a fluorometric caspase-3 assay. Analysis of protein expression was accomplished by Western immunoblotting. RESULTS: IR (10 Gy) reduced control U-87 cell proliferation by 40% through a caspase-independent mechanism. The overexpression of Survivin induced a cytoprotective effect against IR, while the overexpression of RhoA conferred a cytosensitizing effect upon IR. Control U-87 cells pretreated with EGCg exhibited a dose-dependent decrease in their proliferation rate. The growth inhibitory effect of EGCg was not antagonized by overexpressed Survivin. However, Survivin -transfected cells pretreated with EGCg became sensitive to IR, and their RhoA expression was downregulated. A potential therapeutic effect of EGCg targeting the prosurvival intracellular pathways of cancer cells is suggested to act synergistically with IR. CONCLUSION: The radioresistance of GBM is possibly mediated by a mechanism dependent on Survivin in conjunction with RhoA. The combination of natural anti-cancerous molecules such as EGCg with radiotherapy could improve the efficacy of IR treatments.

Anticarcinogenic Agents↗

PubMed2006

Decrease in LDL receptor-related protein expression and function correlates with advanced stages of Wilms tumors.

BACKGROUND: The molecular processes responsible for the invasive phenotype of pediatric Wilms tumors (WT) are poorly understood. A candidate WT suppressor gene (WT1) has been found mutated in a number of these pediatric kidney tumors. However, the disruption of normal WT1 protein function cannot solely explain WT growth. The aim of the present study is to identify new molecular players that regulate the invasive character of WT. PROCEDURE: Fresh frozen samples from 45 renal tumors of Wilms were obtained from the National Wilms Tumor Study Group's Biological Samples Bank. Gelatin zymography, Western blotting, and immunodetection were used to compare tissue biopsies originating from the infiltrating (stage III), metastatic (stage IV), and anaplastic phenotype of Wilms tumors (WT). RESULTS: The expression of the low-density lipoprotein receptor-related protein (LRP) diminished in stage IV and anaplastic WT. Moreover, the expression of RAP, an LRP intracellular chaperone, was also decreased. The diminished expression of LRP and RAP correlated with increased levels of several known extracellular ligands that LRP usually recycles from the extracellular matrix (ECM) environment, including PAI-1, MMP-9, and TIMP-1. The proteolytic processing of MT1-MMP, a functional regulator of LRP, also correlated with the WT invasive phenotype. CONCLUSIONS: The low expression of LRP, whose function is regulated by MT1-MMP and whose activity in recycling ECM-associated proteolytic enzymes becomes drastically diminished in advanced stages of WT, may in part explain the acquired invasive potential of the developing WT pediatric cancer.

Analysis of Variance↗

PubMed2006

Inhibition of MMP-9 secretion by the anti-metastatic PSP94-derived peptide PCK3145 requires cell surface laminin receptor signaling.

PCK3145 is a synthetic peptide corresponding to amino acids 31-45 of prostate secretory protein 94 which can reduce experimental skeletal metastases and prostate tumor growth. These anti-metastatic and anti-tumoral effects of PCK3145 are partially explained by the in-vivo and in-vitro decrease in matrix metalloproteinase (MMP)-9 extracellular levels through as yet unidentified molecular mechanisms of action. Gelatin zymography and immunoblots were used to monitor the levels of secreted MMP-9 from HT-1080 cells. Flow cytometry was used to monitor HT-1080 cell surface binding of FITC-labeled PCK3145 and biotin-labeled laminin. PCK3145-coated cell culture dishes were used to monitor cell adhesion. HT-1080 cell lysates were used for immunoblotting of HuR, extracellular signal-regulated protein kinase (ERK) and phospho-ERK. Total RNA was isolated and RT-PCR used to monitor HuR gene expression. We found that PCK3145 bound to the HT-1080 cell surface and that this binding rapidly triggered ERK phosphorylation that, ultimately, led to a reduction of secreted MMP-9. Laminin inhibited both cell surface binding and ERK phosphorylation by PCK3145. Overexpression of the 67-kDa laminin receptor led to an increased binding of the cells to PCK3145. HuR, a protein that can bind to and stabilize MMP-9 mRNA, was found to be downregulated by PCK3145. The mitogen-activated protein kinase/ERK (MEK) inhibitor PD98059 as well as native laminin and SIKVAV laminin-derived peptide prevented that downregulation. Our data suggest that PCK3145 rapidly triggers intracellular signaling through cell surface laminin receptors. This leads to decreased HuR expression and subsequent destabilization of MMP-9 transcripts. This is the first molecular evidence demonstrating the intracellular signaling and anti-metastatic mechanism of action of PCK3145 that leads to the inhibition of MMP-9 secretion.

Antigens, Surface↗

PubMed2005

Plasminogen kringle 5-engineered glioma cells block migration of tumor-associated macrophages and suppress tumor vascularization and progression.

Angiostatin, a well-characterized angiostatic agent, is a proteolytic cleavage product of human plasminogen encompassing the first four kringle structures. The fifth kringle domain (K5) of human plasminogen is distinct from angiostatin and has been shown, on its own, to act as a potent endothelial cell inhibitor. We propose that tumor-targeted K5 cDNA expression may act as an effective therapeutic intervention as part of a cancer gene therapy strategy. In this study, we provide evidence that eukaryotically expressed His-tagged human K5 cDNA (hK5His) is exported extracellularly and maintains predicted disulfide bridging conformation in solution. Functionally, hK5His protein produced by retrovirally engineered human U87MG glioma cells suppresses in vitro migration of both human umbilical vein endothelial cells and human macrophages. Subcutaneous implantation of Matrigel-embedded hK5His-producing glioma cells in nonobese diabetic/severe combined immunodeficient mice reveals that hK5His induces a marked reduction in blood vessel formation and significantly suppresses the recruitment of tumor-infiltrating CD45+ Mac3+ Gr1- macrophages. Therapeutically, we show in a nude mouse orthotopic brain cancer model that tumor-targeted K5 expression is capable of effectively suppressing glioma growth and promotes significant long-term survival (>120 days) of test animals. These data suggest that plasminogen K5 acts as a novel two-pronged anticancer agent, mediating its inhibitory effect via its action on host-derived endothelial cells and tumor-associated macrophages, resulting in a potent, clinically relevant antitumor effect.

Amino Acid Sequence↗

PubMed2005

Probing the infiltrating character of brain tumors: inhibition of RhoA/ROK-mediated CD44 cell surface shedding from glioma cells by the green tea catechin EGCg.

Glioma cell-surface binding to hyaluronan (HA), a major constituent of the brain extracellular matrix (ECM) environment, is regulated through a complex membrane type-1 matrix metalloproteinase (MT1-MMP)/CD44/caveolin interaction that takes place at the leading edges of invading cells. In the present study, intracellular transduction pathways required for the HA-mediated recognition by infiltrating glioma cells in brain was investigated. We show that the overexpression of the GTPase RhoA up-regulated MT1-MMP expression and triggered CD44 shedding from the U-87 glioma cell surface. This potential implication in cerebral metastatic processes was also observed in cells overexpressing the full-length recombinant MT1-MMP, while the overexpression of a cytoplasmic domain truncated from of MT1-MMP failed to do so. This suggests that the cytoplasmic domain of MT1-MMP transduces intracellular signaling leading to RhoA-mediated CD44 shedding. Treatment of glioma cells with the Rho-kinase (ROK) inhibitor Y27632, or with EGCg, a green tea catechin with anti-MMP and anti-angiogenesis activities, antagonized both RhoA- and MT1-MMP-induced CD44 shedding. Conversely, overexpression of recombinant ROK stimulated CD44 release. Taken together, our results suggest that RhoA/ROK intracellular signaling regulates MT1-MMP-mediated CD44 recognition of HA. These molecular processes may partly explain the diffuse brain-infiltrating character of glioma cells within the surrounding parenchyma and thus be a target for new approaches to anti-tumor therapy.

Brain Neoplasms↗

PubMed2005

A PSP94-derived peptide PCK3145 inhibits MMP-9 secretion and triggers CD44 cell surface shedding: implication in tumor metastasis.

PURPOSE: PCK3145 is a synthetic peptide corresponding to amino acids 31-45 of prostate secretory protein 94, which can reduce experimental skeletal metastases and prostate tumor growth in vivo. Part of its biological action involves the reduction of circulating plasma matrix metalloproteinase (MMP)-9, a crucial mediator in extracellular matrix (ECM) degradation during tumor metastasis and cancer cell invasion. The antimetastatic mechanism of action of PCK3145 is however, not understood. EXPERIMENTAL DESIGN: HT-1080 fibrosarcoma cells were treated with PCK3145, and cell lysates used for immunoblot analysis of small GTPase RhoA and membrane type (MT)1-MMP protein expression. Conditioned media was used to monitor soluble MMP-9 gelatinolytic activity by zymography and protein expression by immunoblotting. RT-PCR was used to assess RhoA, MT1-MMP, MMP-9, RECK, and CD44 gene expression. Flow cytometry was used to monitor cell surface expression of CD44 and of membrane-bound MMP-9. Cell adhesion was performed on different purified ECM proteins, while cell migration was specifically performed on hyaluronic acid (HA). RESULTS: We found that PCK3145 inhibited HT-1080 cell adhesion onto HA, laminin-1, and type-I collagen suggesting the common implication of the cell surface receptor CD44. In fact, PCK3145 triggered the shedding of CD44 from the cell surface into the conditioned media. PCK3145 also inhibited MMP-9 secretion and binding to the cell surface. This effect was correlated to increased RhoA and MT1-MMP gene and protein expression. CONCLUSIONS: Our data suggest that PCK3145 may antagonize tumor cell metastatic processes by inhibiting both MMP-9 secretion and its potential binding to its cell surface docking receptor CD44. Such mechanism may involve RhoA signaling and increase in MT1-MMP-mediated CD44 shedding. Together with its beneficial effects in clinical trials, this is the first demonstration of PCK3145 acting as a MMP secretion inhibitor.

Fibrosarcoma↗

PubMed2005

Inhibition of angiogenic properties of brain endothelial cells by platelet-derived sphingosine-1-phosphate.

The platelet-derived lysophospholipid sphingosine-1-phosphate (S1P) is present in blood plasma and is one of the most potent growth factors displaying proangiogenic activity towards endothelial cells (EC) derived from various tissues. The paracrine regulation of brain angiogenesis by platelet-derived growth factors is, however, poorly understood. In the present study, we assessed the role of S1P on brain EC migration and tubulogenesis, using rat brain-derived (RBE4) EC as an in vitro model. We show that S1P inhibits brain EC migration and tubulogenesis, while it displays proangiogenic activity towards noncerebral EC. Overexpression of the S1P receptor S1P-1 in RBE4 cells potentiated all of the S1P-mediated events. We also show that the lack of expression of MT1-MMP, a membrane-bound matrix metalloproteinase that is thought to cooperate with S1P in tubulogenic processes, may explain the antiangiogenic activity of S1P on brain vasculature. Altogether our results support the hypothesis of a tissue-specific, antiangiogenic role of S1P in the brain, which may help to stabilize the cerebral vasculature and thus have crucial impact on the setting and regulation of normal brain vascularization.

Animals↗