dizocilpine-maleate and Glioma

In this review article, we describe the unrecognized roles of glutamate and glutamate receptors in malignant glioma biology. The neurotransmitter glutamate released from malignant glioma cells in the extracellular matrix is responsible for seizure induction and at higher concentration neuronal cell death. This neuronal cell death will create vacated place for tumor growth. Glutamate also stimulates the growth and the migration of glial tumor cells by means of the activation of glutamate receptors on glioma cells in a paracrine and autocrine manner. The multitude of effects of glutamate in glioma biology supports the rationale for pharmacological targeting of glutamate receptors and transporters in the adjuvant treatment of malignant gliomas in neurology and neuro-oncology. Using the website www.clinicaltrials.gov/ as a reference - a service developed by the National Library of Medicine for the National Health Institute in USA - we have evoked the few clinical trials completed and currently ongoing with therapies targeting the glutamate receptors.

Topics: Benzodiazepines; Brain Neoplasms; Cell Death; Cell Movement; Cell Proliferation; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glioma; Glutamic Acid; Humans; Memantine; Neoplasm Invasiveness; Neuroglia; Neurons; Receptors, AMPA; Receptors, Glutamate; Seizures; Sulfasalazine

Autocrine and paracrine factors, including glutamate and epidermal growth factor (EGF), are potent inducers of brain tumor cell invasion, a pathological hallmark of malignant gliomas. System xc(-) consists of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. We previously showed that the EGF receptor (EGFR) interacts with xCT and thereby promotes the activity of system xc(-) in a kinase-independent manner, resulting in enhanced glutamate release in glioma cells. However, the molecular mechanism underlying EGFR-mediated glioma progression in a glutamate-rich microenvironment has remained unclear. Here we show that the GluN2B subunit of the N-methyl-d-aspartate-sensitive glutamate receptor (NMDAR) is a substrate of EGFR in glioma cells. In response to EGF stimulation, EGFR phosphorylated the COOH-terminal domain of GluN2B and thereby enhanced glutamate-NMDAR signaling and consequent cell migration in EGFR-overexpressing glioma cells. Treatment with the NMDAR inhibitor MK-801 or the system xc(-) inhibitor sulfasalazine suppressed EGF-elicited glioma cell migration. The administration of sulfasalazine and MK-801 also synergistically suppressed the growth of subcutaneous tumors formed by EGFR-overexpressing glioma cells. Furthermore, shRNA-mediated knockdown of xCT and GluN2B cooperatively prolonged the survival of mice injected intracerebrally with such glioma cells. Our findings thus establish a central role for EGFR in the signaling crosstalk between xCT and GluN2B-containing NMDAR in glioma cells.

Topics: Amino Acid Transport System y+; Animals; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Disease Progression; Dizocilpine Maleate; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Glioma; Glutamic Acid; Humans; Mice; Neoplasm Transplantation; Phosphorylation; Protein Domains; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Sulfasalazine

N-Methyl-d-aspartate (NMDA) receptors exhibit a dichotomy of signaling with both toxic and plastic responses. Recent reports have shown that exposure to subtoxic concentration of NMDA results in a neuroprotective state that was measured when these neurons were subsequently challenged with toxic doses of glutamate or kainate. Control of polysialylated neural cell adhesion molecule (PSA-NCAM) expression by NMDA receptor activation has been described in several systems, suggesting a functional link between these two proteins. The perception of glial role in CNS function has changed dramatically over the past few years from simple trophic functions to that of cells with important roles in development and maintenance of CNS in cooperation with neurons. We report here the transcriptional regulation of PSA-NCAM expression by subtoxic dose of NMDA in retinoic acid differentiated C6 glioma cell cultures. C6 glioma cell cultures differentiated with retinoic acid (10microM) were exposed to NMDA (100microM) or to antagonist MK-801 (200nM) prior to treatment with NMDA and cells were harvested after 24h of treatment to study the expression of total NCAM, PSA-NCAM, nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) by Western blotting and dual immunocytofluorescence and expression of PST mRNA by fluorescent in situ hybridization (FISH). Significant increase in the levels of PSA-NCAM, NF-kappaB, AP-1 and PST mRNA was observed in NMDA treated cultures. Treatment of cultures with MK-801, a non-competitive NMDA receptor antagonist, prior to NMDA exposure prevented the NMDA-mediated changes indicating the involvement of NMDA receptor activation. The results elucidate the possible cellular and molecular mechanisms of regulation of PSA-NCAM expression in astroglial cultures by extracellular signals.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glioma; JNK Mitogen-Activated Protein Kinases; Neural Cell Adhesion Molecule L1; Neural Cell Adhesion Molecules; Rats; Receptors, N-Methyl-D-Aspartate; Sialic Acids; Transcription Factor AP-1; Transcription Factor RelA; Tretinoin

Non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists such as dizocilpine (MK-801) produce schizophrenia-like psychosis in humans and induce the expression of heat shock protein 70 (HSP70) in rats. The present study examines the effects of antipsychotic drugs, haloperidol and risperidone, on the expression of HSP70 produced by MK-801 in rat C6 glioma cells. After pretreating with haloperidol and risperidone for 1 h, 6 h, 24 h and 72 h, respectively, C6 glioma cells were cultivated again in MK-801 for 6 h, and then, the extent of HSP70 expression was measured by immunoblotting using anti-HSP70 monoclonal antibody. The expression of HSP70 induced by MK-801 significantly decreased as the duration of haloperidol pretreatment was extended (p=0.002). Risperidone also increasingly attenuated the expression of HSP70 produced by MK-801 as the duration of pretreatment grew longer (p=0.003). The present findings show that haloperidol and risperidone decrease the HSP70 expression in MK-801-treated rat C6 glioma cells. These results suggest that HSP70 and NMDA receptors may play a significant role in the pathophysiology of schizophrenia.

Topics: Animals; Cell Line, Tumor; Dizocilpine Maleate; Dopamine Antagonists; Excitatory Amino Acid Antagonists; Gene Expression; Glioma; Haloperidol; HSP70 Heat-Shock Proteins; Linear Models; Rats; Risperidone; Time Factors

NMDA class of glutamate receptors plays an important role in regulating toxic and plastic responses in CNS. Astrocytes are the predominant cell type in the adult CNS and recent studies have suggested their role in many aspects of CNS function and dysfunction. We report here the protective effect of a subtoxic dose of NMDA in retinoic acid differentiated C6 glioma cell cultures. C6 glioma cell cultures differentiated with retinoic acid (10 microM) were exposed to NMDA (100 microM) or to antagonist MK-801 (200 nM) alone as well as with NMDA and cells were harvested after 24h of treatment to study the expression of HSP70 and for biochemical assay of free radical scavenger system. The protection imparted by a subtoxic dose of NMDA was checked by challenging the differentiated controls as well as NMDA treated and MK-801 treated cultures with a toxic dose of glutamate and subsequently estimating the free radical scavenger system profile. Biochemical analysis revealed a significant increase in the activities of glutathione peroxidase (GPx), copper zinc-superoxide dismutase (CuZnSOD) and reduced glutathione (GSH) content upon exposure to NMDA. No significant change was observed in the level of lipid peroxidation (LPx). A significant increase was observed in HSP70 expression as seen by Western blotting and immunocytofluorescent studies in NMDA treated cultures. Treatment of cultures with MK-801 alone, a non-competitive NMDA receptor antagonist, or pretreatment with MK-801 prior to NMDA exposure prevented the NMDA mediated changes indicating the involvement of NMDA receptors mediated mechanism. The results illustrate the protective effect of a subtoxic dose of NMDA in RA differentiated C6 glioma cell line.

Topics: Animals; Astrocytes; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Survival; Cytoprotection; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Fluorescent Antibody Technique; Glioma; HSP70 Heat-Shock Proteins; N-Methylaspartate; Neuroprotective Agents; Oxidative Stress; Rats; Receptors, N-Methyl-D-Aspartate; Tretinoin

Clozapine is an atypical antipsychotic with particular efficacy in schizophrenia, possibly related to potentiation of brain N-methyl-D-aspartate receptor (NMDAR) -mediated neurotransmission. NMDARs are regulated in vivo by glycine, which is regulated in turn by glycine transporters. The present study investigates transport processes regulating glycine uptake into rat brain synaptosomes, along with effects of clozapine on synaptosomal glycine transport. Amino-acid uptake of amino acids was assessed in rat brain P2 synaptosomal preparations using a radiotransport assay. Synaptosomal glycine transport was inhibited by a series of amino acids and by the selective System A antagonist MeAIB (2-methyl-aminoisobutyric acid). Clozapine inhibited transport of both glycine and MeAIB, but not other amino acids, at concentrations associated with preferential clinical response (0.5-1 microg/ml). By contrast, other antipsychotics studied were ineffective. The novel glycine transport inhibitor N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS) produced biphasic inhibition of [(3)H]glycine transport, with IC(50) values of approximately 25 nM and 25 microM, respectively. NFPS inhibition of [(3)H]MeAIB was monophasic with a single IC(50) value of 31 microM. Clozapine significantly inhibited [(3)H]glycine binding even in the presence of 100 nM NFPS. In conclusion, this study suggests first that System A transporters, or a subset thereof, may play a critical role in regulation of synaptic glycine levels and by extension of NMDA receptor regulation, and second that System A antagonism may contribute to the differential clinical efficacy of clozapine compared with other typical or atypical antipsychotics.

Topics: Amino Acid Transport System A; Amino Acids; Animals; Antipsychotic Agents; beta-Alanine; Cell Line, Tumor; Cerebral Cortex; Clozapine; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glioma; Glycine; Hippocampus; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sarcosine; Synaptosomes; Tritium

Rapid growth and diffuse brain infiltration are hallmarks of malignant gliomas. The underlying molecular pathomechanisms of these tumors, however, remain to be determined. The authors present a novel glioma invasion model that allows researchers to monitor consecutively tumor cell proliferation and migration in an organotypic brain environment. Enhanced green fluorescent protein-labeled F98 rat glioma cells were implanted into slice cultures obtained from a rat hippocampus, and tumor growth was microscopically documented up to 20 days in vitro. Invasion along radially oriented migratory streams could be observed 5 days after implantation of rat F98, human U87MG, and mouse GL261 glioma cells, whereas human Be(2)c neuroblastoma cells and mouse HT22 hippocampal neurons failed to invade the brain parenchyma. Following implantation of F98 glioma cells into the entorhinal cortex, cell death was observed within the infiltrated brain parenchyma as well as in the neuroanatomically connected dentate gyrus. Application of the N-methyl-D-aspartate receptor antagonist MK801 to the culture medium significantly reduced neuronal degeneration in the dentate gyrus, whereas the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonist GYKI 52466 inhibited peritumoral cytotoxicity. This new model allows researchers to address in a systematic manner the molecular pathways of brain invasion as well as specific tumor-host interactions such as necrosis.

Topics: Animals; Benzodiazepines; Brain Neoplasms; Cell Death; Cell Movement; Cell Proliferation; Dentate Gyrus; Disease Models, Animal; Dizocilpine Maleate; Glioma; Hippocampus; Humans; Mice; Neuroprotective Agents; Rats; Transplantation, Heterologous; Tumor Cells, Cultured

Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative diseases. Although recent data show that cultured glioma cells secrete glutamate, the growth potential of brain tumors has not yet been linked to an excitotoxic mechanism. Using bioluminescence detection of glutamate release from freshly prepared brain slices, we show that implanted glioma cells continue to secrete glutamate. Moreover, gliomas with high glutamate release have a distinct growth advantage in host brain that is not present in vitro. Treatment with the NMDA receptor antagonists MK801 or memantine slowed the growth of glutamate-secreting tumors in situ, suggesting that activation of NMDA receptors facilitates tumor expansion. These findings support a new approach for therapy of brain tumors, based upon antagonizing glutamate secretion or its target receptors.

Topics: Amino Acid Transport System X-AG; Animals; Brain Neoplasms; Carrier Proteins; Cell Division; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glioma; Glutamate Plasma Membrane Transport Proteins; Glutamic Acid; Luminescent Measurements; Male; Memantine; Neoplasm Transplantation; Neurons; Rats; Rats, Inbred F344; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Symporters; Tumor Cells, Cultured

We found in cultured glioma (C6BU-1) cells that excitatory amino acids (EAAs) such as glutamate, N-methyl-D-aspartate (NMDA), aspartate, and metabotropic glutamate receptor agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylate caused an increase in the inositol 1,4,5-trisphosphate formation and the intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Mg2+ and Ca2+. Pertussis toxin treatment abolished this glutamate-induced [Ca2+]i increase. Various antagonists against NMDA receptor-ion channel complex, such as Mg2+, D-2-amino-5-phosphonovalerate (D-APV), HA-966, and MK-801, also inhibited the increase in [Ca2+]i induced by glutamate. These results indicate that these metabotropic EAA receptors coupled to pertussis toxin-susceptible GTP-binding protein and phospholipase C system in C6BU-1 glioma cells have the pharmacological properties of NMDA receptor-ion channel complexes. We also found that in the presence of Mg2+ these metabotropic receptors resemble the NMDA receptor-ion channel complex interacted with 5-hydroxytryptamine2 (5-HT2) receptor signaling. EAAs inhibited 5-HT2 receptor-mediated intracellular Ca2+ mobilization and inositol 1,4,5-trisphosphate formation in a concentration-dependent manner. The inhibitory effect of glutamate was reversed by various NMDA receptor antagonists (D-APV, MK-801, phencyclidine, and HA-966), but L-APV failed to block the inhibitory effect of glutamate. The same result was observed in the absence of extracellular Ca2+. In addition, this inhibitory effect on 5-HT2 receptor-mediated signal transduction was abolished by treatment of C6BU-1 cells with pertussis toxin, whereas 5-HT2 receptor-mediated [Ca2+]i increase was not abolished by pertussis toxin treatment. We can, therefore, conclude that the inhibitory effect of glutamate is not a result of the influx of Ca2+ through the ion channel and that it operates via metabotropic glutamate receptors, having NMDA receptor-ion channel complex-like properties and being coupled with pertussis toxin-sensitive GTP-binding protein and phospholipase C.

Topics: 2-Amino-5-phosphonovalerate; Animals; Aspartic Acid; Calcium; Cell Line; Cycloleucine; Dizocilpine Maleate; Glioma; Glutamic Acid; Inositol 1,4,5-Trisphosphate; Ion Channels; Kainic Acid; Kinetics; Magnesium Chloride; N-Methylaspartate; Neurotoxins; Pyrrolidinones; Rats; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Receptors, Serotonin; Signal Transduction; Tumor Cells, Cultured

In the present study, we have attempted to clarify whether neuroblastoma glioma hybrid NG 108-15 cells (NG cells) possess the NMDA receptor complex using [45Ca2+]influx and [3H]MK-801 binding as functional measures. Glutamate and NMDA dose-dependently increased [45Ca2+]influx and these increases were further enhanced by glycine. Scatchard analysis revealed the presence of a high-affinity binding site for [3H]MK-801 with a KD of 18.8 nM and a Bmax of 0.328 pmol/mg protein. This [3H]MK-801 binding was also increased by NMDA in a dose-dependent manner and this increase was further enhanced by glycine. Both ketamine and MK-801 inhibited glutamate- and NMDA-induced [45Ca2+]influx as well as the increase of [3H]MK-801 binding in a dose-dependent manner. Similarly, Mg2+ and Zn2+ dose-dependently reduced both glutamate-induced [45Ca2+]influx and [3H]MK-801 binding. Spermine, one of the polyamines, showed a biphasic stimulatory effects on glutamate-induced [45Ca2+]influx and [3H]MK-801 binding. These results indicate that NG cells possess a pharmacologically distinct NMDA receptor complex and suggest that these cells may be useful for the analyses on pharmacological and biochemical characteristics of the NMDA receptor complex.

Topics: Calcium Radioisotopes; Cations, Divalent; Dizocilpine Maleate; Glioma; Glutamates; Glutamic Acid; Glycine; Hybrid Cells; N-Methylaspartate; Neuroblastoma; Radioligand Assay; Receptors, N-Methyl-D-Aspartate; Spermine; Tritium