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Among the primary brain tumors, glioblastoma is the most common and severe. Glioblastoma have poor prognosis because of their highly diffusive growth pattern and invasion into surrounding brain tissue. Human glioblastoma cells overexpress interleukin-1β (IL-1β) and also the levels of IL-1β in U87MG glioma cells are significantly higher than in U373, T98G, A172 glioma cell lines. Malignant tumors are characterized by unlimited proliferation, migration and invasion. This study examines the effect of IL-1β microenvironment on proliferation, migration and invasion in human glioma cell line U87MG that expresses wild-type p53 protein, and U251MG which expresses mutant p53. Proliferation was investigated by MTT assay, migration by wound-healing migration assay and invasion by in vitro transwell Matrigel invasion assay. An IL-1β microenvironment significantly increased migration and invasion of both wild-type and mutant p53 expressing glioma cells, but significantly increased proliferation only in U87MG glioma cells. These effects were inhibited by IL-1 receptor antagonist (IL-1Ra), thus giving evidence that an IL-1β milieu promotes glioma cell migration, invasion, and proliferation.

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.

Glioblastoma is the most common malignant primary brain tumor with poor prognosis. Invasion involves pro-inflammatory cytokines and major signaling hubs. Tumor necrosis factor-α (TNF-α) acts as a master switch in establishing an intricate link between inflammation and cancer. The present study attempted to explore the possible implication of MAPK extracellular signaling-regulated kinase kinase (MEK)-extracellular signaling-regulated kinase (ERK) signaling pathway and expression of nuclear factor-κB (NF-κB), signal transducers and activators of transcription-6 (STAT-6), ERK, and phosphorylated-ERK (p-ERK) signaling proteins in TNF-α microenvironment. U0126 and PD98059 were used to inhibit the MEK-ERK1/2 pathway. TNF-α stimulation enhanced invasion in U87MG, U251MG and patient-derived primary glioma cells, whereas cell viability was not altered. Matrix metalloproteinase-2 (MMP-2) activity was increased only in U251MG glioma cells. These data suggest that TNF-α microenvironment plays an important role in the invasion of U251MG, U87MG, and patient-derived primary glioma cells, without any cytotoxic effect. The MMP-2 activity is differentially regulated by TNF-α stimulation in these cells. TNF-α stimulation upregulated the protein expression of ERK-1, ERK-2 and also increased the level of p-ERK1/2. TNF-α stimulation further upregulated the expression of NF-κB1, STAT-6 in tandem with Ras-MEK signaling system in U87MG cells, which emphasized the possible involvement of these signaling hubs in the glioma microenvironment. MEK-ERK inhibitors significantly attenuated the invasion of U87MG cells mediated by the TNF-α stimulation, probably through their inhibitory impact on p-ERK1/2 and ERK-2. This study provides the possible rationale of invasion by glioma cells in a TNF-α-induced proinflammatory milieu, which involves direct role of MEK-ERK signaling, with possible implication of NF-κB and STAT-6.

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A AN NN NA AL LS S RES ARTICLE a key role in tumor progression [13]. Over the years, several studies have aimed at understanding the mechanisms governing the aggressive behavior of GBM, but it still remains elusive [14-17]. One of the most common signaling cascades dysregulated in cancer is MEK-ERK pathway, which is involved in cancer cell survival [18]. Similarly, in malignant gliomas the RAS/ RAF/MEK/ERK pathway is aberrantly activated [19]. An earlier study has shown that the ablation of the MEK1/2 kinases and/or ERK1/2 kinases in mice model of non-small cell carcinoma effectively prevented K-RAS-driven tumor development [20]. There are conflicting reports on the role of ERK1/2 pathway on glioma cells. One study demonstrated that transient activation of ERK1/2 by human chorionic gonadotropin β resulted in migration and invasion [21], while the other study reported that sustained activation of ERK1/2 by Sulforaphane inhibits migration and invasion of glioma cells [17]. Nonetheless, these findings suggest the importance of ERK signaling in growth of glioma necessitating the need for further studies to decipher ERK pathway in glioma biology. With this viewpoint the current study was designed to investigate the effect of inhibition of MEK-ERK1/2 signaling by PD98059 and U0126 on the growth and migration of glioma cells as well as their adhesion to ECM.

Purpose Glioblastoma cells create glutamate-rich tumor microenvironment, which initiates activation of ion channels and modulates downstream intracellular signaling. N-methyl-D-aspartate receptors (NMDARs; a type of glutamate receptors) have a high affinity for glutamate. The role of NMDAR activation on invasion of glioblastoma cells and the crosstalk with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is yet to be explored. Main methods LN18, U251MG, and patient-derived glioblastoma cells were stimulated with NMDA to activate NMDAR glutamate receptors. The role of NMDAR activation on invasion and migration and its crosstalk with AMPAR were evaluated. Invasion and migration of glioblastoma cells were investigated by in vitro trans-well Matrigel invasion and trans-well migration assays, respectively. Expression of NMDARs and AMPARs at transcript level was evaluated by quantitative real-time polymerase chain reaction. Results We determined that NMDA stimulation leads to enhanced invasion in LN18, U251MG, and patient-derived glioblastoma cells, whereas inhibition of NMDAR using MK-801, a non-competitive antagonist of the NMDAR, significantly decreased the invasive capacity. Concordant with these findings, migration was significantly augmented by NMDAR in both cell lines. Furthermore, NMDA stimulation upregulated the expression of GluN2 and GluA1 subunits at the transcript level. Conclusions This study demonstrated the previously unexplored role of NMDAR in invasion of glioblastoma cells. Furthermore, the expression of the GluN2 subunit of NMDAR and the differential overexpression of the GluA1 subunit of AMPAR in both cell lines provide a plausible rationale of crosstalk between these calcium-permeable subunits in the glutamate-rich microenvironment of glioblastoma.

Glioblastoma is the most common primary brain tumor. Glioblastoma cells secrete a significant amount of glutamate, which serve as a potential growth factor in glioma pathobiology through their specific receptor subtypes including α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR). Glioblastoma express AMPAR subunits; however, its regulation and activation with downstream intracellular signaling are not well‐understood. Phosphorylated‐extracellular signaling‐regulated kinase (ERK)1/2 is known to regulate the ionotropic glutamate receptors in cortical neurons. The mitogen‐activated protein kinase cascade is frequently activated in several tumors, including glioma. Nonetheless, the association of ERK signaling with AMPAR subunits in glioblastoma is undetermined. Here, we demonstrated potential role of AMPAR in invasion, and the modulation of AMPAR subunits at transcript level by ERK signaling in glioblastoma cells. Inhibition of ERK signaling specifically downregulated the expression of calcium‐permeable AMPAR subunits, GluA1 and GluA4, and upregulated calcium‐impermeable AMPAR subunit GluA2 implying differential regulation of the expression of calcium‐permeable AMPAR subunits of glioblastoma. Concomitantly, it significantly decreased the invasion of U87MG cells. Taken together, these findings suggest that the AMPAR enhances invasion of glioblastoma, and ERK signaling modulates the differential expression of calcium‐permeable AMPAR phenotype that might play a crucial role in the invasive propensity of glioblastoma cells.