Activation of NMDA receptor of glutamate influences MMP-2 activity and proliferation of glioma cells

Abstract

Glioblastoma multiforme (GBM) is the most common malignant glioma, which has high proliferative rate and an extremely invasive phenotype. Major limitations in the effective treatment of malignant gliomas are the proliferation and infiltration into the surrounding brain tissue. Although studies have shown that various stimuli promote glioma cell proliferation and invasion, the underlying mechanisms remain largely unknown. Glioma cells secrete significant amount of glutamate into surrounding tissue and intracellular signaling is thought to be initiated upon glutamate-induced modulation of the ion channels in GBM cells. The objective of the study was to investigate the effect of activation of NMDA (N-methyl-D-aspartate) receptors of glutamate on gelatinase subfamily MMPs and on proliferation of glioma cells. U251MG and U87MG cell lines were maintained in Dulbecco's Modified Eagle's Medium. Proliferation assay was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay. Matrix metalloproteinase (MMP)-2 and MMP-9 activity was investigated by gelatin zymography assay. We demonstrate that activated NMDA receptors (NMDAR) increased the activity of MMP-2 only in U251MG glioma cells at concentrations of 100 and 200 μM and increased the proliferation of both U87MG and U251MG glioma cells at concentrations of 50, 100, 150 and 200 μM. Inhibition of NMDAR using MK-801, a non-competitive antagonist of the NMDAR, significantly inhibited the effect of activation of NMDAR on MMP-2 activity and on proliferation. We conclude that NMDA receptor activation has role in activity of MMP-2 and proliferation of glioma cells.

Survival of glioblastoma cells in response to endogenous and exogenous oxidative challenges: possible implication of NMDA receptor‐mediated regulation of redox homeostasis

“…Studies have reported the presence of functional NMDARs and expression of NMDAR subunits in various cancers including gliomas. Moreover, activation of these receptors by NMDA agonists has been implicated in tumor growth (Stepulak et al, ) and further in this context our study has shown that NMDARs play a role in glioma cell proliferation as well (Ramaswamy et al, ).…”

“…Cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 . Trypsin ethylenediaminetetraacetic acid (EDTA) (Gibco, USA) was used to detach the cells (Ramaswamy et al, ).…”

“…NMDARs in glioma cells were activated by treating these cells with NMDA (100 µM) (Sigma‐Aldrich) and the cofactor glycine (10 µM) (Sigma‐Aldrich). Inhibition of NMDAR activation was done using a highly potent and selective non‐competitive NMDA receptor antagonist, MK‐801 (100 µM) (Sigma‐Aldrich) as used earlier (Ramaswamy et al, ). Glioma cells were incubated in presence or absence of 100 μM MK‐801 for 10 min in serum‐free medium, followed by 100 μM NMDA and 10 μM glycine for 24 h.…”

Transcriptional modulation of calcium‐permeable AMPA receptor subunits in glioblastoma by MEK–ERK1/2 inhibitors and their role in invasion

“…The culture medium was exchanged twice a week. Before use, the cells were detached from the flask with 0.05% trypsin–EDTA (Gibco), centrifuged and the resulting pellet was resuspended in fresh culture medium (Ramaswamy et al, ).…”

“…GBM is known to secrete a significantly high amount of glutamate (Ye and Sontheimer, 1999) that may act as a growth factor in GBM through their specific receptor subtypes in autocrine and paracrine manner (Stepulak et al, 2009;Oh et al, 2012). In addition to a growing body of evidence, including our prior work, revealed the significance of glutamate and its receptors in the biology of glioma growth and progression (Stepulak et al, 2009;Ramaswamy et al, 2014). Glutamate receptors are categorized into α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), N-methyl-D-aspartate (NMDA) receptor, kainate receptor, and metabotropic glutamate receptors based on the pharmacological and physiological property of the receptors.…”