concanavalin-a and epigallocatechin-gallate

Epithelial-to-mesenchymal transition (EMT) recapitulates metastasis and can be induced in vitro through transforming growth factor (TGF)-β signaling. A role for MMP activity in glioblastoma multiforme has been ascribed to EMT, but the molecular crosstalk between TGF-β signaling and membrane type 1 MMP (MT1-MMP) remains poorly understood. Here, the expression of common EMT biomarkers, induced through TGF-β and the MT1-MMP inducer concanavalin A (ConA), was explored using RNA-seq analysis and differential gene arrays in human U87 glioblastoma cells. TGF-β triggered SNAIL and fibronectin expressions in 2D-adherent and 3D-spheroid U87 glioblastoma cell models. Those inductions were antagonized by the TGF-β receptor kinase inhibitor galunisertib, the JAK/STAT inhibitors AG490 and tofacitinib, and by the diet-derived epigallocatechin gallate (EGCG). Transient gene silencing of MT1-MMP prevented the induction of SNAIL by ConA and abrogated TGF-β-induced cell chemotaxis. Moreover, ConA induced STAT3 and Src phosphorylation, suggesting these pathways to be involved in the MT1-MMP-mediated signaling axis that led to SNAIL induction. Our findings highlight a new signaling axis linking MT1-MMP to TGF-β-mediated EMT-like induction in glioblastoma cells, the process of which can be prevented by the diet-derived EGCG.

Topics: Brain Neoplasms; Catechin; Cell Line, Tumor; Concanavalin A; Epithelial-Mesenchymal Transition; Fibronectins; Glioblastoma; Humans; Matrix Metalloproteinase 14; Piperidines; Pyrazoles; Pyrimidines; Quinolines; Receptors, Transforming Growth Factor beta; Signal Transduction; Snail Family Transcription Factors; STAT3 Transcription Factor; Transforming Growth Factor beta1; Tyrphostins

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP which triggers intracellular signaling and regulates extracellular matrix proteolysis, two functions that are critical for tumor-associated angiogenesis and inflammation. While green tea catechins, particularly epigallocatechin gallate (EGCG), are considered very effective in preventing MT1-MMP-mediated functions, lack of structure-function studies and evidence regarding their direct interaction with MT1-MMP-mediated biological activities remain. Here, we assessed the impact in both cellular and biophysical assays of four ungallated catechins along with their gallated counterparts on MT1-MMP-mediated functions and molecular binding partners. Concanavalin-A (ConA) was used to trigger MT1-MMP-mediated proMMP-2 activation, expression of MT1-MMP and of endoplasmic reticulum stress biomarker GRP78 in U87 glioblastoma cells. We found that ConA-mediated MT1-MMP induction was inhibited by EGCG and catechin gallate (CG), that GRP78 induction was inhibited by EGCG, CG, and gallocatechin gallate (GCG), whereas proMMP-2 activation was inhibited by EGCG and GCG. Surface plasmon resonance was used to assess direct interaction between catechins and MT1-MMP interactors. We found that gallated catechins interacted better than their ungallated analogs with MT1-MMP as well as with MT1-MMP binding partners MMP-2, TIMP-2, MTCBP-1 and LRP1-clusterIV. Overall, current structure-function evidence supports a role for the galloyl moiety in both direct and indirect interactions of green tea catechins with MT1-MMP-mediated oncogenic processes.

Topics: Carcinogenesis; Catechin; Cell Line, Tumor; Concanavalin A; Endoplasmic Reticulum Chaperone BiP; Enzyme Precursors; Gelatinases; Glioblastoma; Heat-Shock Proteins; Humans; Matrix Metalloproteinase 14; Matrix Metalloproteinase Inhibitors; Protein Binding; Structure-Activity Relationship; Tea

Epigallocatechin-3-gallate (EGCG) is the most effective compound in green tea, and possesses a wide range of beneficial effects, including anti-inflammatory, antioxidant, antiobesity, and anticancer effects. In this study, we investigated the protective effects of EGCG in concanavalin A (ConA)-induced hepatitis in mice and explored the possible mechanisms involved in these effects.. Balb/C mice were injected with ConA (25 mg/kg) to induce acute autoimmune hepatitis, and EGCG (10 or 30 mg/kg) was administered orally twice daily for 10 days before ConA injection. Serum liver enzymes, proinflammatory cytokines, and other marker proteins were determined 2, 8, and 24 hours after the ConA administration.. BNIP3 mediated cell apoptosis and autophagy in ConA-induced hepatitis. EGCG decreased the immunoreaction and pathological damage by reducing inflammatory factors, such as TNF-α, IL-6, IFN-γ, and IL-1β. EGCG also exhibited an antiapoptotic and antiautophagic effect by inhibiting BNIP3 via the IL-6/JAKs/STAT3 pathway.. EGCG attenuated liver injury in ConA-induced hepatitis by downregulating IL-6/JAKs/STAT3/BNIP3-mediated apoptosis and autophagy.

Topics: Animals; Apoptosis; Autophagy; Catechin; Chemical and Drug Induced Liver Injury; Concanavalin A; Cytokines; Janus Kinase 1; Janus Kinase 2; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mitochondrial Proteins; Signal Transduction; STAT3 Transcription Factor

(-)-Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenolic compound present in green tea and has been shown to possess anti-inflammatory and anti-oxidative properties. In this study, we investigated the protective effects of EGCG against concanavalin A (ConA)-induced liver injury and the underlying mechanisms. EGCG (5 mg/kg) was administered orally by gavage to mice twice daily for 10 days before an intravenous injection of ConA. We found that EGCG effectively rescued lethality, improved hepatic pathological damage, and decreased serum levels of alanine aminotransaminase (ALT) in ConA-challenged mice. Furthermore, EGCG also significantly prevented the release of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4, and IL-6 in serum, reduced malondialdehyde (MDA) levels, and restored glutathione (GSH) content and superoxide dismutase (SOD) activity in liver tissues from ConA-challenged mice. Finally, nuclear factor (NF)-κB activation and expression levels of Toll-like receptor (TLR) 2, TLR4 and TLR9 protein in liver tissues were significantly inhibited by EGCG pretreatment. Taken together, our data suggest that EGCG possesses hepatoprotective properties against ConA-induced liver injury through its anti-inflammatory and anti-oxidant actions.

Topics: Animals; Catechin; Chemical and Drug Induced Liver Injury; Concanavalin A; Disease Models, Animal; Male; Mice, Inbred C57BL; Oxidative Stress; Toll-Like Receptors; Tumor Necrosis Factor-alpha

CSF-2 and CSF-3 confer proangiogenic and immunomodulatory properties to mesenchymal stromal cells (MSCs).. Transcriptional regulation of CSF-2 and CSF-3 in concanavalin A-activated MSCs requires MT1-MMP signaling and is inhibited by EGCG.. The chemopreventive properties of diet-derived EGCG alter MT1-MMP-mediated intracellular signaling.. Pharmacological targeting of MSCs proangiogenic functions may prevent their contribution to tumor development. Epigallocatechin gallate (EGCG), a major form of tea catechins, possesses immunomodulatory and antiangiogenic effects, both of which contribute to its chemopreventive properties. In this study, we evaluated the impact of EGCG treatment on the expression of colony-stimulating factors (CSF) secreted from human bone marrow-derived mesenchymal stromal cells (MSCs), all of which also contribute to the immunomodulatory and angiogenic properties of these cells. MSCs were activated with concanavalin A (ConA), a Toll-like receptor (TLR)-2 and TLR-6 agonist as well as a membrane type 1 matrix metalloproteinase (MT1-MMP) inducer, which increased granulocyte macrophage-CSF (GM-CSF, CSF-2), granulocyte CSF (G-CSF, CSF-3), and MT1-MMP gene expression. EGCG antagonized the ConA-induced CSF-2 and CSF-3 gene expression, and this process required an MT1-MMP-mediated sequential activation of the Src and JAK/STAT pathways. Gene silencing of MT1-MMP expression further demonstrated its requirement in the phosphorylation of Src and STAT3, whereas overexpression of a nonphosphorylatable MT1-MMP mutant (Y573F) abrogated CSF-2 and CSF-3 transcriptional increases. Given that MSCs are recruited within vascularizing tumors and are believed to contribute to tumor angiogenesis, possibly through secretion of CSF-2 and CSF-3, our study suggests that diet-derived polyphenols such as EGCG may exert chemopreventive action through pharmacological targeting of the MT1-MMP intracellular signaling.

Topics: Angiogenesis Inhibitors; Catechin; Cells, Cultured; Concanavalin A; Gene Silencing; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Janus Kinases; Macrophage Colony-Stimulating Factor; Matrix Metalloproteinase 14; Mesenchymal Stem Cells; Phosphorylation; Protein Processing, Post-Translational; Signal Transduction; src-Family Kinases; STAT3 Transcription Factor; Toll-Like Receptor 2; Toll-Like Receptor 6; Transcription, Genetic

(-)-Epigallocatechin-3-gallate (EGCG) is the major active component of green tea. Increasing evidence has suggested that EGCG exhibits anti-inflammatory, anti-oxidant and immunosuppressive effects. In this study, we investigated the effect of EGCG on concanavalin A (ConA)-induced hepatitis (CIH) in mice, a model of immune-mediated liver injury in humans. We pretreated mice with EGCG before ConA injection, and then measured alanine aminotransferase (ALT) levels in plasma, inflammatory infiltration and hepatocyte apoptosis in liver. Potential therapeutic mechanisms were elucidated further by measuring several inflammatory mediators. Mice pretreated with EGCG exhibited much less increased ALT levels in plasma, reduced inflammatory infiltration and hepatocyte apoptosis in liver compared with control mice pretreated with vehicle solutions. We further investigated the mechanisms of the protective effects of EGCG. In EGCG-pretreated mice, we found abrogated tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma at both protein levels in plasma and mRNA levels in liver. At the same time, the concentration of nitrite in plasma and inducible nitric oxide synthase production in liver were both down-regulated in these mice. Moreover, IFN-inducible protein-10 and macrophage inflammatory protein-1alpha expressions in liver were decreased significantly. Therefore, EGCG is capable of regulating immune-mediated liver injury in vivo. The protective effect depended on its suppressive effect on the production of important inflammatory mediators.

Topics: Alanine Transaminase; Animals; Apoptosis; Catechin; Chemical and Drug Induced Liver Injury; Chemokine CCL4; Chemokine CXCL10; Chemokines, CXC; Concanavalin A; Hepatocytes; Immunohistochemistry; Interferon-gamma; Liver; Macrophage Inflammatory Proteins; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Nitrites; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha

EGCG [(-)-epigallocatechin gallate] and (+)-catechin hydrate are flavanoids, which are known as anticancer and healthy drugs. To test the immune modulatory effects of EGCG and catechin, various concentrations were tested on primary white blood cells (WBC) in cell cultures stimulated with the T-cell mitogen concanavalin A (ConA). WBC from dairy cows (1 x 10(6) cells/mL) were cultivated using RPMI medium with FCS and gentamycin. First, WBC were stimulated with ConA, and 6 h later the flavanoid treatment was started. Cultivated WBC were treated with various physiological flavanol concentrations (0-100 microM) in cross-combination with various ConA concentrations (0-1 microg/mL). After 24 h, cells were harvested, cell viability was verified, and total RNA was isolated. Relative mRNA expression levels of proinflammatory cytokines TNFalpha, IL1beta, IL6, and transcription factor cFos and of nucleosome component histon H3 were quantified with real-time qRT-PCR. High EGCG and catechin concentrations had inhibitory effects on total RNA expression. Low EGCG concentration can induce total RNA expression in WBC. EGCG reduced cFos mRNA expression, which can be abolished by high ConA concentrations in a reverse dose-dependent manner. TNFalpha showed a flavanoid-specific expression pattern. EGCG acts in blood physiological concentrations (micromolar range), and catechin acts in higher gut-relevant concentrations (millimolar range) and has the potential to influence the proinflammatory TNFalpha expression. Higher flavanoid concentration had more pronounced effects than lower, whereas EGCG showed a more potent suppression of gene expression than catechin (toward TNFalpha). EGCG and catechin had no significant effects in primary WBC on the expression pattern of the proinflammatory cytokines IL1beta and IL6 and on the expression of the housekeeping genes GAPDH and histon H3. It is presumed that both flavanoids have the potential to regulate total RNA expression and gene-specific expression in WBC.

Topics: Animals; Catechin; Cattle; Cells, Cultured; Concanavalin A; Cytokines; Gene Expression; Leukocytes; RNA, Messenger; Tumor Necrosis Factor-alpha

We have investigated the effects of different biologically active components from natural products, including green tea polyphenols (GTP), resveratrol, genistein and organosulfur compounds from garlic, on matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 activities. GTP caused the strongest inhibition of the three enzymes, as measured by fluorescence assays using gelatin or elastin as substrates. The inhibition of MMP-2 and MMP-9 caused by GTP was confirmed by gelatin zymography and was observed for MMPs associated with both various rat tissues and human brain tumors (glioblastoma and pituitary tumors). The activities of MMPs were also measured in the presence of various catechins isolated from green tea including (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate(ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC) and (+)-catechin (C). The most potent inhibitors of these activities, as measured by fluorescence and by gelatin or casein zymography, were EGCG and ECG. GTP and the different catechins had no effect on pancreatic elastase, suggesting that the effects of these molecules on MMP activities are specific. Furthermore, in vitro activation of proMMP-2 secreted from the glioblastomas cell line U-87 by the lectin concanavalin A was completely inhibited by GTP and specifically by EGCG. These results indicate that catechins from green tea inhibit MMP activities and proMMP-2 activation.

Topics: Animals; Catechin; Cell Line; Concanavalin A; Cricetinae; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; Gelatinases; Guanosine Triphosphate; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Mice; Molecular Structure; Swine; Tea