Neurol Sci (2014) 35:823–829 DOI 10.1007/s10072-013-1604-5
ORIGINAL ARTICLE
Activation of NMDA receptor of glutamate influences MMP-2 activity and proliferation of glioma cells Palaniswamy Ramaswamy • N. Aditi Devi • K. Hurmath Fathima • Nandakumar Dalavaikodihalli Nanjaiah
Received: 5 August 2013 / Accepted: 10 December 2013 / Published online: 29 December 2013 Ó Springer-Verlag Italia 2013
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-Daspartate) 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 lM and increased the proliferation of both U87MG and U251MG glioma cells at concentrations of 50, 100, 150 and 200 lM. Inhibition of NMDAR using MK801, a non-competitive antagonist of the NMDAR, significantly inhibited the effect of activation of NMDAR on
P. Ramaswamy N. Aditi Devi K. Hurmath Fathima N. Dalavaikodihalli Nanjaiah (&) Department of Neurochemistry, National Institute of Mental Health and NeuroSciences (NIMHANS), Bangalore, Karnataka 560029, India e-mail: [email protected]
MMP-2 activity and on proliferation. We conclude that NMDA receptor activation has role in activity of MMP-2 and proliferation of glioma cells. Keywords NMDA receptor MMP-2 Proliferation Glioblastoma Glutamate
Introduction Glioblastoma multiforme is the most common malignant glioma, which has an extremely invasive phenotype [1]. With the addition of temozolomide to radiotherapy, median overall survival has improved from 12.1 to 14.6 months [2]. However, the prognosis associated with GBM patients remains dismal because GBMs cannot be completely removed surgically because of the highly infiltrative nature of these tumors into the brain parenchyma [3]. The ability of glioblastoma cells to diffusely infiltrate deeply into healthy brain tissue is the major obstacle for successful treatments, including surgery and radiotherapy. Over the years, ample studies have aimed at understanding the molecular mechanisms of invasion and proliferation of glioblastoma cells; however, they still remain elusive. Forty percent of GBM tumors maintain a mutant/nonfunctional p53 [4]. Its prognostic value has not been consistently established in glioma. With respect to p53 gene status, glioma cell lines resemble the original tumors and, therefore, are suitable for studying the biological changes associated with p53 mutations in glial tumors [5]. U87MG glioma cells are wild-type p53 expressing cells, whereas U251MG is mutant p53 expressing glioma cells [6]. Glutamate is one of the major neurotransmitters in the central nervous system. Glioma cells release excitotoxic concentration of glutamate [7]. Downstream effects of
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Glutamate via receptors are thought to be a contributing factor to the malignant behavior of GBM [8]. Excessive glutamate in the brain parenchyma has been associated with brain tumor progression [9]. Glutamate exerts its action by binding to both N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors [10]. Intracellular signaling is thought to be initiated upon glutamate-induced modulation of the ion channels in GBM cells [11]. Invasiveness of glioblastoma is mediated in part by the interaction of glioma cells with the extracellular matrix (ECM), followed by degradation of matrix by tumor cellderived proteases, particularly the matrix metalloproteinases (MMP) [12, 13]. MMPs are a large family of zincdependent neutral endopeptidases, and are involved in the degradation of many different components of the extracellular matrix. It has been reported that malignant gliomas overexpress the gelatinase subfamily of MMPs, MMP-2 and MMP-9 [14, 15]. Establishment of a curative treatment for glioblastoma will require better understanding of the molecular mechanisms underlying the proliferation and invasion of the tumor cells. Although studies have shown that various stimuli promote glioma cell proliferation and invasion, the underlying mechanisms remain largely unknown. Major limitations to the effective treatment of malignant gliomas are the proliferation and propensity of these lesions to infiltrate into the surrounding brain tissue. In this study, we investigated the effect of activation of NMDA receptors of glutamate on gelatinase subfamily MMPs and on proliferation of wild-type and mutant p53 expressing glioma cells.
Materials and methods Cell culture Human glioblastoma U251MG and U87MG cells were kindly provided by Prof. Kondaiah (Indian Institute of Sciences, Bangalore, India). The cells were maintained in Dulbecco’s Modified Eagle’s Medium–High glucose (DMEM) (Sigma-Aldrich D5648-1L) supplemented with 10 % fetal bovine serum (FBS) (Gibco), PenStrep (Gibco) [penicillin (100 units/ml) and streptomycin (100 lg/ml)] at 37 °C in a humidified atmosphere containing 5 % CO2. Culture medium was exchanged twice a week. Upon reaching sub-confluence, the cells were detached from the flask with 0.05 % trypsin–EDTA (Gibco). Before use, the cells were rinsed in Dulbecco’s phosphate-buffered saline (DPBS), centrifuged at 2,000 rpm for 1 min and the resulting pellet was resuspended in fresh culture medium. This rinse after the detachment procedure ensured that
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there would be no active trypsin or antibiotics to affect the artificial basement membrane. Proliferation assay Proliferation of glioma cells was investigated by MTT assay [16] with modifications. Cells were washed gently with DPBS, trypsinized and counted in a Neubauer chamber slide using trypan blue dye exclusion method. Viable cells were plated at a density of 1 9 104 cells per well in 96-well plates in a final volume of 0.1 ml media containing 10 % FBS. After 24 h, cells were washed with DPBS and incubated in the presence or absence of 100 lM MK-801 for 30 min and incubated with various concentrations of NMDA (Sigma-Aldrich, USA) along with appropriate concentration of glycine in serum-free media. This experiment was conducted in triplicates. The plates were incubated at 37 °C in a humidified atmosphere containing 5 % CO2 and 95 % air. After 48 h incubation, 100 ll of 2 mg/ml MTT was added to each well and incubated for 4 h at 37 °C. Then, 200 ll of dimethyl sulfoxide (DMSO) was added after removing the used media to each well and mixed. Absorbance was measured at 570 nm using a TECAN Infinite M200 multiwell platereader. Data shown are representative of three independent experiments. Gelatin zymography assay Activity of gelatinase subfamily of MMPs was demonstrated by gelatin zymography assay [17] with modification. Cells (2.5 9 105) were seeded in 6-well plate and grown for 24 h in medium containing 10 % FBS. After 24 h, cells were incubated in the presence or absence of 100 lM MK-801 for 30 min in serum-free medium followed by varying concentration of NMDA and corresponding concentration of glycine for 48 h. The cell conditioned medium was mixed with 2X gel loading buffer and electrophoresed in a 10 % SDS-gel containing 0.1 % gelatin. After electrophoresis, the gel was washed briefly with water and then twice in 2.5 % Triton X-100 at room temperature to remove the SDS, thus allowing the gelatinase to renature. The gel was then incubated at 37 °C in a buffer (50 mM Tris, pH 8.0, 10 mM CaCl2 and 1 lM ZnCl2) for 24 h to facilitate gelatin degradation by gelatinase. The gel was washed with water and subsequently stained with 0.5 % Coomassie brilliant blue R250 and destained in destaining solution (methanol:acetic acid: water in the ratio of 4:1:5). Gelatinase activity was measured densitometrically after scanning gel (Gel-Doc densitometer, BioRad) and bands were quantified in arbitrary units using ImageJ v1.47. Data shown are representative of three independent experiments.
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825 b Fig. 1 Proliferation of glioma cells: Glioma cell lines (a) U251MG
and (b) U87MG were stimulated with indicated concentrations of Nmethyl-D-aspartate (NMDA) ? Glycine for 48 h with or without 100 lM of MK-801 and proliferation was evaluated as described in ‘‘Materials and methods’’. c U251MG and U87MG glioma cell lines were incubated with indicated concentrations of MK-801 for 48 h and proliferation was evaluated. Shown are the averages of minimum three independent experiments performed in triplicate. Error bars indicate the standard deviation. Statistics were performed using twoway ANOVA (in a, and in b) and one-way ANOVA (in c), followed by Tukey’s test. *p \ 0.05 in comparison to corresponding control #p \ 0.01 in comparison to without the inhibitor, MK-801
Statistical analysis The data are presented as mean and standard deviation. The extent of proliferation by activated NMDA receptors in the presence and absence of inhibitor was analyzed by oneway analysis of variance (ANOVA) or two-way ANOVA followed by post hoc Tukey’s test wherever appropriate. Extent of MMP-2 activity in the presence and absence of the inhibitor was analyzed by Student’s t test. A p value of less than 0.05 was considered as significant.
Results Effect of NMDA receptor activation on proliferation of glioma cells To evaluate the effect of activation of NMDA receptor on proliferation of U251MG and U87MG glioma cells, these cells were stimulated with NMDA at concentrations of 50, 100, 150 and 200 lM of NMDA along with 5, 10, 15 and 20 lM of glycine, respectively, for 48 h and the proliferation was demonstrated by MTT assay. NMDA increased proliferation of both U251MG and U87MG cells at all the above concentrations compared to control (Fig. 1a, b). Inhibition of NMDAR activation was done using a highly potent and selective non-competitive NMDA receptor antagonist, MK-801. We optimized the concentration of inhibitor to be used for the reversal of proliferation effect. This was done using a doubling-dose of the inhibitor MK801 (25, 50, 100 and 200 lM). We observed that MK-801 inhibits NMDA receptors in both glioma cell lines at all the above concentrations in a dose-dependent manner except at 25 lM in U87MG glioma cells (Fig. 1c). We chose 100 lM for our further experiments to study inhibitory effect on NMDAR [18–20]. MK-801 at concentration of 100 lM, significantly reduced proliferation of both glioma cells at all concentrations of NMDA (Fig. 1a, b). The reduced proliferation observed was even lower than the control levels. The probable reason is that glioma cells release excitotoxic concentration of glutamate [7], which activates glutamate receptors (NMDAR, AMPAR, kainite
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A
200
D
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MMP - 2 activity (% of control)
MMP-2 activity (% of Control)
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150 125 100 75 50 25 0
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100µM NMDA+10µM Glycine
200µM NMDA+20µM Glycine
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100µM NMDA+10µM Glycine
Control
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Fig. 2 Activity of gelatinase subfamily of MMPs: The gelatinolytic activities from the conditioned media were examined by zymography as described in ‘‘Materials and methods’’. The 92 kDa corresponds to MMP-9 and 72 kDa corresponds to MMP-2. a, b Cells were treated with 100 and 200 lM of N-methyl-D-aspartate (NMDA) along with 10 and 20 lM glycine, respectively, for 48 h. c, d Cells were treated with 100 lM NMDA ? 10 lM Glycine for 48 h with or without
100 lM of MK-801 as described in ‘‘Materials and methods’’. Shown are the averages of three experiments performed in duplicate. Error bars indicate the standard deviation. Data from NMDA ? glycine treated cells were compared statistically with those of control and NMDA ? Glycine ? MK-801 by Student’s t test. *p \ 0.05 in comparison to control, #p \ 0.05 in comparison to activated NMDA receptor
receptor and mGluRs) in autocrine and paracrine manner inherently [21]. MK-801 may inhibit most of the NMDAR activation in glioma cells including inherent activation mediated by secreted glutamate by glioma cells.
801 at concentration of 100 lM significantly inhibited the effect of activated NMDAR on MMP-2 activity in U251MG glioma cells (Fig. 2c, d). The reduced MMP-2 activity observed in the presence of inhibitor was even lower than the control, may be because of inhibition of inherent autocrine and/or paracrine activation of NMDAR by secreted glutamate by glioma cells.
Effect of NMDA receptor activation on gelatinase subfamily of MMPs To evaluate the effect of NMDA receptor on activation of MMP-2 and MMP-9, U251MG and U87MG cells were stimulated with 100 and 200 lM NMDA and the activity of gelatinase subfamily of MMPs was demonstrated by gelatin zymography. NMDA at these concentrations increased the MMP-2 activity only in U251MG cells compared to control and there was no MMP-9 activity (Fig. 2a, b). There was no statistical significance in the extent of MMP2 activity induced in U251MG glioma cells between individual concentrations. We did not observe increase in activity of both MMP-2 and MMP-9 in U87MG glioma cells in any of the above concentrations compared to control (data not shown). Inhibition of NMDAR by MK-
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Discussion Ionotropic NMDA, AMPA and kainate receptors as well as metabotropic glutamate receptors (mGluRs) are expressed in gliomas. NMDAR subunits NR2A, B, and C and NR3A were shown to be present on several glioma cell lines [22, 23]. Glial cells contain NMDAR at dendritic end, near periphery. Functional NMDAR are shown in astrocytes of the mouse neocortex [24]. Krebs et al. [25] have demonstrated for the first time the expression of functional NMDAR in astrocytes of the ischemic hippocampus of the rat and in cultured hippocampal astrocytes after anoxia.
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Gliomas behave exactly opposite to astrocytes, releasing glutamate rather than sequestering it. Co-culturing glioma cells with neurons, the released glutamate activates neuronal NMDA receptors causing a sustained Ca2? influx resulting in excitotoxic cell death. Excitotoxicity is believed to result from the aberrant activation of neuronal NMDA receptors in the peritumoral tissue [21]. Incubation of glioma cells with NMDA receptor agonists NMDA leads to sustained activation of NMDA-type glutamate receptors. In this study, we demonstrated that the activation of NMDA receptors increases activity of MMP-2 in U251MG glioma cells. No MMP-9 activity was detected in these assays suggesting that MMP-9 is not activated by NMDA receptor activation. We did not observe any effect on MMP-2 or MMP-9 activities in U87MG cells. 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 in U251MG cells. Prevention of NMDA receptormediated changes indicates the involvement of NMDA receptor activation. Autocrine/paracrine activation of NMDA receptors leads to an increased activity of MMP2. This suggests that MMP-2 activity is also mediated through NMDA receptors. We observed an increase in MMP-2 activity by activation of NMDA receptors only in U251MG glioma cells which express mutant p53 and not in U87MG glioma cells which express wild-type p53. Muller et al. [26] have reported that loss of p53 function in migration is associated with an increase in MMP-2 activity. Earlier study showed that gamma linolenic acid significantly decreased MMP-2 activity (measured by zymography) but significantly increased p53 expression in C6 glioma cells [27]. p53 upregulated MMP-2 expression in SK-OV-3 cells (ovarian cancer), but downregulated it in PC-3 (prostate cancer) and NCI-H460 cells (lung cancer) [28]. These findings suggest that activity of MMP-2 is related to p53 status and we infer that MMP-2 activity which is mediated by activation of NMDAR is probably dependent on p53 status of glioma cells. Further studies using U87MG cells engineered to express dominant-negative p53 mutant p53V143A will improve our understanding in this direction. Calcium entry through NMDA receptors is a potent regulator of the signaling pathways mediated by this receptor. Glutamatergic stimulation of the NMDA receptor can lead to activation of Akt and MAPK pathways [29]. Autocrine glutamate activation of AMPA receptors is required for invasion by regulating extracellular levels of MMP-2 by influencing the function of invadopodia [30]. In our study, we have shown NMDAR-mediated increase in activity of MMP-2 as measured by zymography. It is worth investigating whether MMP activity regulation by NMDAR activation is a qualitative effect at the protein
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level or a quantitative effect on transcriptional or translational level. We demonstrated that activation of NMDA receptors increased the proliferation of both U251MG and U87MG glioma cells. Non-competitive inhibitor of NMDAR reversed the proliferation mediated by activation of these receptors. This suggests that NMDAR activation has a role in proliferation of glioma cells. Since proliferative effect is seen in both wild-type and mutant p53 expressing glioma cells, it appears that the proliferation mediated by the activation of NMDAR is probably independent of p53 status. Elucidation of these mechanisms should further our understanding of the role of NMDA receptors in the growth and proliferation of astrocytic neoplasms. Rzeski et al. [18] have reported that in WHO grade III human brain astrocytoma cell line MOGGCCM, the NMDA receptor antagonist (?)-dizocilpine was able to decrease proliferation. MMP-2 plays a significant role in the proteolytic degradation of extracellular matrix and plays an important role in migration, invasion of cancer cells [31–34] and proliferation of glioma cells [35]. Also MMP-2 activity has been shown to have role in angiogenesis in gliomas [36]. MMP2 expression is shown to have prognostic importance in pediatric gliomas [37]. Increasing the activity of MMP-2 leads to degradation of ECM and thereby contributes to invasion. Glioma cells that were treated only with p-MMP2 showed a significant downregulation of tumorigenic properties and were capable of acquiring an apoptotic phenotype. The administration of p-MMP-2 prior to radiotherapy has been reported to be a potent adjuvant therapeutic approach to improve the efficacy of radiotherapy for glioma [38]. Inhibiting NMDA receptors is synergistic with chemotherapy which emphasizes the need to pursue combination therapy strategies when targeting glutamate receptors [21]. Hence inhibition of NMDAR can be considered in the combination therapy in management of glioblastoma. Our study gives additional evidence of role of NMDA receptors in glioma biology. In this study, we have shown MMP-2 activity and proliferation of glioma cells by activation of NMDAR. NMDAR for its activation requires glutamate/NMDA as agonist and glycine as co-agonist [39]. We have used increasing concentrations (50, 100, 150 and 200 lM) of NMDA along with 5, 10, 15 and 20 lM of glycine, respectively. Nong et al. [40] have reported that stimulation of the glycine site initiates signaling through the NMDAR complex, priming the receptors for clathrin-dependent endocytosis, but requires pre-treatment with glycine for this internalization of NMDAR. Whether glycine functions more than simply a co-agonist for NMDAR or has a divergent function needs to be evaluated in the context of glioma. To activate NMDAR in our experiments, glycine was used up to concentration of 20 lM, whereas the
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effector concentration for half-maximum response (EC50) for the priming effect of glycine is reported to be 39 ± 2 lM and glycine site activation is required to prime NMDAR internalization [40]. Activating NMDAR with higher concentrations of exogenous glycine (100 lM) to understand the divergent functions in glioma biology is worth investigating.
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Conclusion Activation of NMDA receptor influences the activity of MMP-2 only in U251MG glioma cells and proliferation of both U87MG and U251MG glioma cells. Blocking the activation of NMDAR abrogates both stimulatory effect on MMP-2 activity and proliferative effect on glioma cells. Inhibiting the activity of MMP-2 reduces the degradation of many different components of the extracellular matrix and thereby on growth and progress of glioma. We conclude that NMDA receptor activation has role in activity of MMP-2 and proliferation of glioma cells. We propose that activity of MMP-2 is regulated also by NMDA receptors. Further studies are necessary to identify the intermediary signaling molecules linking NMDA receptors, proliferation and activation of MMP-2. Acknowledgments This work was supported by research grant (No.BT/PR3431/MED/30/648/2011) to Nandakumar Dalavaikodihalli Nanjaiah from Department of Biotechnology (DBT), New Delhi, India. Palaniswamy R gratefully acknowledges the junior research fellowship from Council of Scientific and Industrial Research (CSIR), India. Conflict of interest
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The authors declare no conflict of interest.
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ORIGINAL ARTICLE
Activation of NMDA receptor of glutamate influences MMP-2 activity and proliferation of glioma cells Palaniswamy Ramaswamy • N. Aditi Devi • K. Hurmath Fathima • Nandakumar Dalavaikodihalli Nanjaiah
Received: 5 August 2013 / Accepted: 10 December 2013 / Published online: 29 December 2013 Ó Springer-Verlag Italia 2013
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-Daspartate) 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 lM and increased the proliferation of both U87MG and U251MG glioma cells at concentrations of 50, 100, 150 and 200 lM. Inhibition of NMDAR using MK801, a non-competitive antagonist of the NMDAR, significantly inhibited the effect of activation of NMDAR on
P. Ramaswamy N. Aditi Devi K. Hurmath Fathima N. Dalavaikodihalli Nanjaiah (&) Department of Neurochemistry, National Institute of Mental Health and NeuroSciences (NIMHANS), Bangalore, Karnataka 560029, India e-mail: [email protected]
MMP-2 activity and on proliferation. We conclude that NMDA receptor activation has role in activity of MMP-2 and proliferation of glioma cells. Keywords NMDA receptor MMP-2 Proliferation Glioblastoma Glutamate
Introduction Glioblastoma multiforme is the most common malignant glioma, which has an extremely invasive phenotype [1]. With the addition of temozolomide to radiotherapy, median overall survival has improved from 12.1 to 14.6 months [2]. However, the prognosis associated with GBM patients remains dismal because GBMs cannot be completely removed surgically because of the highly infiltrative nature of these tumors into the brain parenchyma [3]. The ability of glioblastoma cells to diffusely infiltrate deeply into healthy brain tissue is the major obstacle for successful treatments, including surgery and radiotherapy. Over the years, ample studies have aimed at understanding the molecular mechanisms of invasion and proliferation of glioblastoma cells; however, they still remain elusive. Forty percent of GBM tumors maintain a mutant/nonfunctional p53 [4]. Its prognostic value has not been consistently established in glioma. With respect to p53 gene status, glioma cell lines resemble the original tumors and, therefore, are suitable for studying the biological changes associated with p53 mutations in glial tumors [5]. U87MG glioma cells are wild-type p53 expressing cells, whereas U251MG is mutant p53 expressing glioma cells [6]. Glutamate is one of the major neurotransmitters in the central nervous system. Glioma cells release excitotoxic concentration of glutamate [7]. Downstream effects of
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Glutamate via receptors are thought to be a contributing factor to the malignant behavior of GBM [8]. Excessive glutamate in the brain parenchyma has been associated with brain tumor progression [9]. Glutamate exerts its action by binding to both N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors [10]. Intracellular signaling is thought to be initiated upon glutamate-induced modulation of the ion channels in GBM cells [11]. Invasiveness of glioblastoma is mediated in part by the interaction of glioma cells with the extracellular matrix (ECM), followed by degradation of matrix by tumor cellderived proteases, particularly the matrix metalloproteinases (MMP) [12, 13]. MMPs are a large family of zincdependent neutral endopeptidases, and are involved in the degradation of many different components of the extracellular matrix. It has been reported that malignant gliomas overexpress the gelatinase subfamily of MMPs, MMP-2 and MMP-9 [14, 15]. Establishment of a curative treatment for glioblastoma will require better understanding of the molecular mechanisms underlying the proliferation and invasion of the tumor cells. Although studies have shown that various stimuli promote glioma cell proliferation and invasion, the underlying mechanisms remain largely unknown. Major limitations to the effective treatment of malignant gliomas are the proliferation and propensity of these lesions to infiltrate into the surrounding brain tissue. In this study, we investigated the effect of activation of NMDA receptors of glutamate on gelatinase subfamily MMPs and on proliferation of wild-type and mutant p53 expressing glioma cells.
Materials and methods Cell culture Human glioblastoma U251MG and U87MG cells were kindly provided by Prof. Kondaiah (Indian Institute of Sciences, Bangalore, India). The cells were maintained in Dulbecco’s Modified Eagle’s Medium–High glucose (DMEM) (Sigma-Aldrich D5648-1L) supplemented with 10 % fetal bovine serum (FBS) (Gibco), PenStrep (Gibco) [penicillin (100 units/ml) and streptomycin (100 lg/ml)] at 37 °C in a humidified atmosphere containing 5 % CO2. Culture medium was exchanged twice a week. Upon reaching sub-confluence, the cells were detached from the flask with 0.05 % trypsin–EDTA (Gibco). Before use, the cells were rinsed in Dulbecco’s phosphate-buffered saline (DPBS), centrifuged at 2,000 rpm for 1 min and the resulting pellet was resuspended in fresh culture medium. This rinse after the detachment procedure ensured that
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there would be no active trypsin or antibiotics to affect the artificial basement membrane. Proliferation assay Proliferation of glioma cells was investigated by MTT assay [16] with modifications. Cells were washed gently with DPBS, trypsinized and counted in a Neubauer chamber slide using trypan blue dye exclusion method. Viable cells were plated at a density of 1 9 104 cells per well in 96-well plates in a final volume of 0.1 ml media containing 10 % FBS. After 24 h, cells were washed with DPBS and incubated in the presence or absence of 100 lM MK-801 for 30 min and incubated with various concentrations of NMDA (Sigma-Aldrich, USA) along with appropriate concentration of glycine in serum-free media. This experiment was conducted in triplicates. The plates were incubated at 37 °C in a humidified atmosphere containing 5 % CO2 and 95 % air. After 48 h incubation, 100 ll of 2 mg/ml MTT was added to each well and incubated for 4 h at 37 °C. Then, 200 ll of dimethyl sulfoxide (DMSO) was added after removing the used media to each well and mixed. Absorbance was measured at 570 nm using a TECAN Infinite M200 multiwell platereader. Data shown are representative of three independent experiments. Gelatin zymography assay Activity of gelatinase subfamily of MMPs was demonstrated by gelatin zymography assay [17] with modification. Cells (2.5 9 105) were seeded in 6-well plate and grown for 24 h in medium containing 10 % FBS. After 24 h, cells were incubated in the presence or absence of 100 lM MK-801 for 30 min in serum-free medium followed by varying concentration of NMDA and corresponding concentration of glycine for 48 h. The cell conditioned medium was mixed with 2X gel loading buffer and electrophoresed in a 10 % SDS-gel containing 0.1 % gelatin. After electrophoresis, the gel was washed briefly with water and then twice in 2.5 % Triton X-100 at room temperature to remove the SDS, thus allowing the gelatinase to renature. The gel was then incubated at 37 °C in a buffer (50 mM Tris, pH 8.0, 10 mM CaCl2 and 1 lM ZnCl2) for 24 h to facilitate gelatin degradation by gelatinase. The gel was washed with water and subsequently stained with 0.5 % Coomassie brilliant blue R250 and destained in destaining solution (methanol:acetic acid: water in the ratio of 4:1:5). Gelatinase activity was measured densitometrically after scanning gel (Gel-Doc densitometer, BioRad) and bands were quantified in arbitrary units using ImageJ v1.47. Data shown are representative of three independent experiments.
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825 b Fig. 1 Proliferation of glioma cells: Glioma cell lines (a) U251MG
and (b) U87MG were stimulated with indicated concentrations of Nmethyl-D-aspartate (NMDA) ? Glycine for 48 h with or without 100 lM of MK-801 and proliferation was evaluated as described in ‘‘Materials and methods’’. c U251MG and U87MG glioma cell lines were incubated with indicated concentrations of MK-801 for 48 h and proliferation was evaluated. Shown are the averages of minimum three independent experiments performed in triplicate. Error bars indicate the standard deviation. Statistics were performed using twoway ANOVA (in a, and in b) and one-way ANOVA (in c), followed by Tukey’s test. *p \ 0.05 in comparison to corresponding control #p \ 0.01 in comparison to without the inhibitor, MK-801
Statistical analysis The data are presented as mean and standard deviation. The extent of proliferation by activated NMDA receptors in the presence and absence of inhibitor was analyzed by oneway analysis of variance (ANOVA) or two-way ANOVA followed by post hoc Tukey’s test wherever appropriate. Extent of MMP-2 activity in the presence and absence of the inhibitor was analyzed by Student’s t test. A p value of less than 0.05 was considered as significant.
Results Effect of NMDA receptor activation on proliferation of glioma cells To evaluate the effect of activation of NMDA receptor on proliferation of U251MG and U87MG glioma cells, these cells were stimulated with NMDA at concentrations of 50, 100, 150 and 200 lM of NMDA along with 5, 10, 15 and 20 lM of glycine, respectively, for 48 h and the proliferation was demonstrated by MTT assay. NMDA increased proliferation of both U251MG and U87MG cells at all the above concentrations compared to control (Fig. 1a, b). Inhibition of NMDAR activation was done using a highly potent and selective non-competitive NMDA receptor antagonist, MK-801. We optimized the concentration of inhibitor to be used for the reversal of proliferation effect. This was done using a doubling-dose of the inhibitor MK801 (25, 50, 100 and 200 lM). We observed that MK-801 inhibits NMDA receptors in both glioma cell lines at all the above concentrations in a dose-dependent manner except at 25 lM in U87MG glioma cells (Fig. 1c). We chose 100 lM for our further experiments to study inhibitory effect on NMDAR [18–20]. MK-801 at concentration of 100 lM, significantly reduced proliferation of both glioma cells at all concentrations of NMDA (Fig. 1a, b). The reduced proliferation observed was even lower than the control levels. The probable reason is that glioma cells release excitotoxic concentration of glutamate [7], which activates glutamate receptors (NMDAR, AMPAR, kainite
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A
200
D
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MMP - 2 activity (% of control)
MMP-2 activity (% of Control)
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150 125 100 75 50 25 0
Control
100µM NMDA+10µM Glycine
200µM NMDA+20µM Glycine
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100µM NMDA+10µM Glycine
Control
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Fig. 2 Activity of gelatinase subfamily of MMPs: The gelatinolytic activities from the conditioned media were examined by zymography as described in ‘‘Materials and methods’’. The 92 kDa corresponds to MMP-9 and 72 kDa corresponds to MMP-2. a, b Cells were treated with 100 and 200 lM of N-methyl-D-aspartate (NMDA) along with 10 and 20 lM glycine, respectively, for 48 h. c, d Cells were treated with 100 lM NMDA ? 10 lM Glycine for 48 h with or without
100 lM of MK-801 as described in ‘‘Materials and methods’’. Shown are the averages of three experiments performed in duplicate. Error bars indicate the standard deviation. Data from NMDA ? glycine treated cells were compared statistically with those of control and NMDA ? Glycine ? MK-801 by Student’s t test. *p \ 0.05 in comparison to control, #p \ 0.05 in comparison to activated NMDA receptor
receptor and mGluRs) in autocrine and paracrine manner inherently [21]. MK-801 may inhibit most of the NMDAR activation in glioma cells including inherent activation mediated by secreted glutamate by glioma cells.
801 at concentration of 100 lM significantly inhibited the effect of activated NMDAR on MMP-2 activity in U251MG glioma cells (Fig. 2c, d). The reduced MMP-2 activity observed in the presence of inhibitor was even lower than the control, may be because of inhibition of inherent autocrine and/or paracrine activation of NMDAR by secreted glutamate by glioma cells.
Effect of NMDA receptor activation on gelatinase subfamily of MMPs To evaluate the effect of NMDA receptor on activation of MMP-2 and MMP-9, U251MG and U87MG cells were stimulated with 100 and 200 lM NMDA and the activity of gelatinase subfamily of MMPs was demonstrated by gelatin zymography. NMDA at these concentrations increased the MMP-2 activity only in U251MG cells compared to control and there was no MMP-9 activity (Fig. 2a, b). There was no statistical significance in the extent of MMP2 activity induced in U251MG glioma cells between individual concentrations. We did not observe increase in activity of both MMP-2 and MMP-9 in U87MG glioma cells in any of the above concentrations compared to control (data not shown). Inhibition of NMDAR by MK-
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Discussion Ionotropic NMDA, AMPA and kainate receptors as well as metabotropic glutamate receptors (mGluRs) are expressed in gliomas. NMDAR subunits NR2A, B, and C and NR3A were shown to be present on several glioma cell lines [22, 23]. Glial cells contain NMDAR at dendritic end, near periphery. Functional NMDAR are shown in astrocytes of the mouse neocortex [24]. Krebs et al. [25] have demonstrated for the first time the expression of functional NMDAR in astrocytes of the ischemic hippocampus of the rat and in cultured hippocampal astrocytes after anoxia.
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Gliomas behave exactly opposite to astrocytes, releasing glutamate rather than sequestering it. Co-culturing glioma cells with neurons, the released glutamate activates neuronal NMDA receptors causing a sustained Ca2? influx resulting in excitotoxic cell death. Excitotoxicity is believed to result from the aberrant activation of neuronal NMDA receptors in the peritumoral tissue [21]. Incubation of glioma cells with NMDA receptor agonists NMDA leads to sustained activation of NMDA-type glutamate receptors. In this study, we demonstrated that the activation of NMDA receptors increases activity of MMP-2 in U251MG glioma cells. No MMP-9 activity was detected in these assays suggesting that MMP-9 is not activated by NMDA receptor activation. We did not observe any effect on MMP-2 or MMP-9 activities in U87MG cells. 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 in U251MG cells. Prevention of NMDA receptormediated changes indicates the involvement of NMDA receptor activation. Autocrine/paracrine activation of NMDA receptors leads to an increased activity of MMP2. This suggests that MMP-2 activity is also mediated through NMDA receptors. We observed an increase in MMP-2 activity by activation of NMDA receptors only in U251MG glioma cells which express mutant p53 and not in U87MG glioma cells which express wild-type p53. Muller et al. [26] have reported that loss of p53 function in migration is associated with an increase in MMP-2 activity. Earlier study showed that gamma linolenic acid significantly decreased MMP-2 activity (measured by zymography) but significantly increased p53 expression in C6 glioma cells [27]. p53 upregulated MMP-2 expression in SK-OV-3 cells (ovarian cancer), but downregulated it in PC-3 (prostate cancer) and NCI-H460 cells (lung cancer) [28]. These findings suggest that activity of MMP-2 is related to p53 status and we infer that MMP-2 activity which is mediated by activation of NMDAR is probably dependent on p53 status of glioma cells. Further studies using U87MG cells engineered to express dominant-negative p53 mutant p53V143A will improve our understanding in this direction. Calcium entry through NMDA receptors is a potent regulator of the signaling pathways mediated by this receptor. Glutamatergic stimulation of the NMDA receptor can lead to activation of Akt and MAPK pathways [29]. Autocrine glutamate activation of AMPA receptors is required for invasion by regulating extracellular levels of MMP-2 by influencing the function of invadopodia [30]. In our study, we have shown NMDAR-mediated increase in activity of MMP-2 as measured by zymography. It is worth investigating whether MMP activity regulation by NMDAR activation is a qualitative effect at the protein
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level or a quantitative effect on transcriptional or translational level. We demonstrated that activation of NMDA receptors increased the proliferation of both U251MG and U87MG glioma cells. Non-competitive inhibitor of NMDAR reversed the proliferation mediated by activation of these receptors. This suggests that NMDAR activation has a role in proliferation of glioma cells. Since proliferative effect is seen in both wild-type and mutant p53 expressing glioma cells, it appears that the proliferation mediated by the activation of NMDAR is probably independent of p53 status. Elucidation of these mechanisms should further our understanding of the role of NMDA receptors in the growth and proliferation of astrocytic neoplasms. Rzeski et al. [18] have reported that in WHO grade III human brain astrocytoma cell line MOGGCCM, the NMDA receptor antagonist (?)-dizocilpine was able to decrease proliferation. MMP-2 plays a significant role in the proteolytic degradation of extracellular matrix and plays an important role in migration, invasion of cancer cells [31–34] and proliferation of glioma cells [35]. Also MMP-2 activity has been shown to have role in angiogenesis in gliomas [36]. MMP2 expression is shown to have prognostic importance in pediatric gliomas [37]. Increasing the activity of MMP-2 leads to degradation of ECM and thereby contributes to invasion. Glioma cells that were treated only with p-MMP2 showed a significant downregulation of tumorigenic properties and were capable of acquiring an apoptotic phenotype. The administration of p-MMP-2 prior to radiotherapy has been reported to be a potent adjuvant therapeutic approach to improve the efficacy of radiotherapy for glioma [38]. Inhibiting NMDA receptors is synergistic with chemotherapy which emphasizes the need to pursue combination therapy strategies when targeting glutamate receptors [21]. Hence inhibition of NMDAR can be considered in the combination therapy in management of glioblastoma. Our study gives additional evidence of role of NMDA receptors in glioma biology. In this study, we have shown MMP-2 activity and proliferation of glioma cells by activation of NMDAR. NMDAR for its activation requires glutamate/NMDA as agonist and glycine as co-agonist [39]. We have used increasing concentrations (50, 100, 150 and 200 lM) of NMDA along with 5, 10, 15 and 20 lM of glycine, respectively. Nong et al. [40] have reported that stimulation of the glycine site initiates signaling through the NMDAR complex, priming the receptors for clathrin-dependent endocytosis, but requires pre-treatment with glycine for this internalization of NMDAR. Whether glycine functions more than simply a co-agonist for NMDAR or has a divergent function needs to be evaluated in the context of glioma. To activate NMDAR in our experiments, glycine was used up to concentration of 20 lM, whereas the
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effector concentration for half-maximum response (EC50) for the priming effect of glycine is reported to be 39 ± 2 lM and glycine site activation is required to prime NMDAR internalization [40]. Activating NMDAR with higher concentrations of exogenous glycine (100 lM) to understand the divergent functions in glioma biology is worth investigating.
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Conclusion Activation of NMDA receptor influences the activity of MMP-2 only in U251MG glioma cells and proliferation of both U87MG and U251MG glioma cells. Blocking the activation of NMDAR abrogates both stimulatory effect on MMP-2 activity and proliferative effect on glioma cells. Inhibiting the activity of MMP-2 reduces the degradation of many different components of the extracellular matrix and thereby on growth and progress of glioma. We conclude that NMDA receptor activation has role in activity of MMP-2 and proliferation of glioma cells. We propose that activity of MMP-2 is regulated also by NMDA receptors. Further studies are necessary to identify the intermediary signaling molecules linking NMDA receptors, proliferation and activation of MMP-2. Acknowledgments This work was supported by research grant (No.BT/PR3431/MED/30/648/2011) to Nandakumar Dalavaikodihalli Nanjaiah from Department of Biotechnology (DBT), New Delhi, India. Palaniswamy R gratefully acknowledges the junior research fellowship from Council of Scientific and Industrial Research (CSIR), India. Conflict of interest
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The authors declare no conflict of interest.
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