concanavalin-a and Glioblastoma

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

Glioma is the most common brain tumor. C6 rat glioblastoma cells provide the possibility to the scientist to study brain cancer. Concanavalin A (Con A) has a lot of antitumoral effects, especially over oxidative stress. In the present study, it was aimed to decide the impacts of various doses of Con A on C6 glioblastoma cells regarding cytotoxicity, thiol/disulfide homeostasis, apoptosis, and inflammation. We detected the cytotoxic activity of Con A (from 7.8 to 500 µg/ml) in C6 cells by utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and determined the toxic concentration of Con A. Once the optimal doses were found, the thiol-disulfide homeostasis, levels of total antioxidant and oxidant status (TAS and TOS), malondialdehyde (MDA) and glutathione (GSH), pro-inflammatory cytokines as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), apoptotic proteins as cytochrome c (CYCS), and caspase 3 (CASP3) were measured. Apoptotic and morphological changes in the C6 cells were examined with an inverted microscope and flow cytometry technique. Dose-dependent Con A triggered oxidative damage in the C6 cells, affecting the inflammatory pathway, so reducing proliferation with apoptotic proteins and morphological changes. But especially, Con A increased disulfide formation by disrupting the thiol/disulfide balance in C6 cells. This study revealed that Con A, known as carbohydrate-binding protein, generated oxidative damage, inflammation, and apoptosis in a dose-dependent manner by modulating thiol/disulfide homeostasis in C6 glioblastoma cells.

Topics: Apoptosis; Cell Line, Tumor; Concanavalin A; Disulfides; Dose-Response Relationship, Drug; Glioblastoma; Humans; Sulfhydryl Compounds

Membrane type-1 matrix metalloproteinase (MT1-MMP) possesses both extracellular proteolytic and intracellular signal-transducing functions in tumorigenesis. An imbalance in MT1-MMP expression and/or function triggers a metastatic, invasive, and therapy resistance phenotype. MT1-MMP is involved in extracellular matrix (ECM) proteolysis, activation of latent MMPs, as well as in autophagy signaling in human hepatoma and glioblastoma cells. A low autophagy index in tumorigenesis has been inferred by recent studies where autophagic capacity was decreased during tumor progression. Here, we establish ARP101 as a dual-function small-molecule inhibitor against MT1-MMP ECM hydrolysis and autophagy signal-transducing functions in a model of grade IV glioblastoma cells. ARP101 inhibited concanavalin-A-mediated proMMP-2 activation into MMP-2, as well as MT1-MMP auto-proteolytic processing. When overexpressing recombinant Wt MT1-MMP, ARP101 inhibited proMMP-2 activation and triggered the formation of MT1-MMP oligomers that required trafficking to the plasma membrane. ARP101 further induced cell autophagy as reflected by increased formation of acidic vacuole organelles, LC3 puncta, and autophagy-related protein ATG9 transcription. These were all significantly reversed upon siRNA-mediated gene silencing of MT1-MMP. ARP101 can thus concomitantly inhibit MT1-MMP extracellular catalytic function and exploit its intracellular transducing signal function to trigger autophagy-mediated cell death in U87 glioblastoma cancer cells.

Topics: Autophagy; Cell Line, Tumor; Cell Movement; Concanavalin A; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; RNA Interference; RNA, Small Interfering; Signal Transduction; Sulfonamides

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

Plant lectins have been considered as possible anti-tumor drugs because of their property to induce autophagic cell death. Given that expression of membrane type-1 matrix metalloproteinase (MT1-MMP) has been found to regulate expression of the autophagy biomarker Bcl-2/adenovirus E1B 19kDa interacting protein 3 (BNIP3), we sought to investigate possible signaling interplay mechanisms between MT1-MMP and BNIP3 in Concanavalin-A (ConA) lectin-activated U87 glioblastoma cells. ConA induced acidic vacuole organelle formation as well as BNIP3 and MT1-MMP gene and protein expressions, whereas only BNIP3 expression was dose-dependently inhibited by the JAK2 tyrosine kinase inhibitor AG490 suggesting a requirement for some STAT-mediated signaling. Gene silencing of MT1-MMP and of STAT3 abrogated ConA-induced STAT3 phosphorylation and BNIP3 expression. Correlative analysis shows that STAT3 signaling events occur downstream from MT1-MMP induction. Overexpression of a full length MT1-MMP recombinant protein led to increased BNIP3 gene and protein expressions. The cytoplasmic domain of MT1-MMP was also found necessary for transducing STAT3 phosphorylation. Among JAK1, JAK2, JAK3, and TYK2, only JAK2 gene silencing abrogated ConA's effects on MT1-MMP and BNIP3 gene and protein expressions. Our study elucidates how MT1-MMP signals autophagy, a process which could contribute to the chemoresistance phenotype in brain cancer cells.

Topics: Autophagy; Biomarkers; Cell Line, Tumor; Concanavalin A; Glioblastoma; Humans; Janus Kinase 2; Matrix Metalloproteinase 14; Membrane Proteins; Phosphorylation; Protein Structure, Tertiary; Proto-Oncogene Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Transcription, Genetic; Tyrphostins; Up-Regulation; Vacuoles

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.

Topics: Acridine Orange; Autophagy; Brain Neoplasms; Cell Line, Tumor; Concanavalin A; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioblastoma; Humans; Hydroxamic Acids; Indoles; Mannose; Matrix Metalloproteinase 14; Neoplasm Proteins; Protein Structure, Tertiary; RNA, Small Interfering; Signal Transduction; Vacuoles

Recent findings in cell death signalling show that membrane type 1 matrix metalloproteinase (MT1-MMP), an MMP known for its involvement in cancer cell invasion and metastasis, can act as a "bioswitch" in the invasion versus cell death decision in brain tumour cells. Given that the endoplasmic reticulum (ER) is a subcellular compartment involved in metabolic control and cell death signalling and that cytoskeleton disruption, as encountered during cancer cell invasion, can lead to ER stress, we questioned whether MT1-MMP contributes to ER stress. We found that MT1-MMP gene silencing or pharmacological inhibition of vesicular trafficking with Brefeldin-A abrogated MT1-MMP cell surface-mediated proMMP-2 activation by the lectin Concanavalin-A (ConA) in U87 glioblastoma cells. ConA, also known to trigger the expression of pro-inflammatory cyclooxygenase (COX)-2 through MT1-MMP signalling from the plasma membrane, failed to do so when MT1-MMP was prevented from reaching the cell surface by Brefeldin-A. Gene silencing of MT1-MMP antagonized the expression of ConA-induced COX-2 and of the ER stress marker glucose-related protein 78 (GRP78), further suggesting that plasma membrane localization of MT1-MMP contributes to signalling ER stress. MT1-MMP maturation, which partially occurs during its trafficking from the ER to the plasma membrane, showed correlation of the 60 kDa MT1-MMP with GRP78 expression. Finally, Brefeldin-A treatment of glioblastoma cells led to Akt dephosphorylation; this effect was reversed when MT1-MMP was silenced. Collectively, our results provide a molecular rationale for a new role for MT1-MMP in the regulation of cancer cell death processes through ER stress signalling.

Topics: Brefeldin A; Cell Death; Cell Line, Tumor; Cell Membrane; Concanavalin A; Cyclooxygenase 2; Drug Interactions; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Glioblastoma; Heat-Shock Proteins; Humans; Matrix Metalloproteinase 14; Protein Synthesis Inhibitors; RNA Interference; Signal Transduction; Time Factors; Transfection

Recent profiling has identified the aminopeptidase N/CD13 inhibitor actinonin as a selective soluble secreted matrix metalloproteinase (MMP) inhibitor. Given that actinonin's effects against membrane-bound MMPs remain unknown and that MT1-MMP has been linked to chemo- and radio-therapy resistance in brain tumor development, we therefore assessed MT1-MMP functional inhibition by actinonin in U87 glioblastoma cells. We show that actinonin inhibits concanavalin-A (ConA)-induced proMMP-2 activation, while it does not inhibit ConA-induced MT1-MMP gene expression suggesting post-transcriptional effects of the drug possibly mediated through the membrane-anchored protease regulator RECK. Specific gene silencing of MT1-MMP with siRNA abrogated the ability of ConA to activate proMMP-2. Functional recombinant MT1-MMP whose constitutive expression led to proMMP-2 activation was also efficiently antagonized by actinonin. We provide evidence for actinonin's new therapeutic application in the direct targeting of MT1-MMP-mediated proMMP-2 activation, an essential step in both brain tumor infiltration and in brain tumor-associated angiogenesis.

Topics: Antineoplastic Agents; CD13 Antigens; Cell Line, Tumor; Concanavalin A; Enzyme Activation; Enzyme Precursors; Glioblastoma; Humans; Hydroxamic Acids; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; PHEX Phosphate Regulating Neutral Endopeptidase; Protein Binding

Human glioblastoma cell lines showed profound suppression of both DNA and RNA synthesis when exposed to supernatants (SNs) of mitogen-activated blood mononuclear cells. Cloning efficiency of these glioma cells also decreased 10- to 500-fold. In monolayer cultures, growth inhibition was evident within 12 h of adding SN and peaked at 24 h. A decrease in absolute cell number was evident by 72 h. The inhibitory effect of SNs, however, was not permanent as more cells entered S-phase when SN-treated cultures were refed with fresh medium (without SN). The factor(s) responsible for this inhibitory activity was a product of lymphocytes and was produced in comparable amounts by cells of normal blood donors and patients with glioma. The compromised immunological status of glioblastoma patients did not influence their capacity to produce cytostatic lymphokines.

Topics: Adult; Aged; Animals; Cell Line; Colorimetry; Concanavalin A; DNA; Female; Glioblastoma; Glioma; Humans; In Vitro Techniques; Lymphocyte Activation; Lymphocytes; Lymphokines; Male; Middle Aged; Phytohemagglutinins; Rats; RNA