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Yvonne Hoehna, Ortrud Uckermann,
Hella Luksch,
Vanya Stefovska,
Jenny Marzahn,
Marlen Theil,
Tomasz Gorkiewicz,
Maciej Gawlak,
Grzegorz M Wilczynski,
Leszek Kaczmarek,
Chrysanthy Ikonomidou
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ABSTRACT: Matrix metalloproteinases (MMPs) are involved in tissue repair, cell death and morphogenesis. We investigated the role of the gelatinases MMP-2 and MMP-9 in the pathogenesis of neuronal death induced by prolonged seizures in the developing brain. Seven-day-old rats, MMP-9 knockout mice and transgenic rats overexpressing MMP-9 received intraperitoneal injections of pilocarpine, 250mg/kg, to induce seizures. After 6-72h pups were sacrificed, tissue from different brain regions was isolated and expression of MMP-9 mRNA and protein was analyzed by real-time PCR or Western blot. Additionally, brains were fixed and processed for TUNEL-staining, immunohistochemistry and in situ zymography. We found increased numbers of TUNEL-positive cells 24h after pilocarpine-induced seizures, most pronounced in cortical areas and the dentate gyrus, and less pronounced in thalamus. At 6-24h, MMP-9 mRNA levels showed significant elevation compared to sham-treated controls; this effect resolved by 48h, whereas MMP-2 mRNA levels remained stable. Cortical gelatinolytic activity, monitored by in situ zymography, was enhanced following pilocarpine-induced seizures. The MMP inhibitor GM 6001 ameliorated cell death following pilocarpine-induced seizures in infant rats. MMP-9 knockout mice were less susceptible to seizure-induced brain injury. Transgenic rats overexpressing MMP-9 were equally susceptible to seizure-induced brain injury as wild type rats. Our results suggest a significant contribution of MMP-9 to cell death after pilocarpine-induced seizures in the developing brain. As indicated by Western blot analysis, MMP-9 activation may be linked to activation of the Erk/CREB-pathway. The findings implicate involvement of MMP-9 in the pathophysiology of brain injury following seizures in the developing brain.
Neurobiology of Disease 07/2012; 48(3):339-47. · 5.40 Impact Factor
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ABSTRACT: Emerging evidence supports a role for glutamate in the biology of cancer. We studied the impact of glutamate receptor subunit silencing on cancer phenotype.
Different fragments of the coding region for ionotropic glutamate receptor AMPA 4 (GLUR4), ionotropic glutamate receptor N-methyl D-aspartate 1 (NR1), ionotropic glutamate receptor kainate 5 (KA2) and ionotropic glutamate receptor N-methyl D-aspartate 2D (NR2D) were stably transfected into human TE671, RPMI8226 and A549 cell lines. Resulting changes in cell proliferation, migration and mRNA expression of genes that determine cancer phenotype were assayed.
Decreased expression of GLUR4 markedly increased cancer cell proliferation, whereas decreased expression of NR1 markedly reduced the propensity of cancer cells to proliferate. Knockdown of KA2 and NR2D did not influence cancer phenotype. Gene silencing of GLUR4 modulated the mRNA expression of various genes in these cancer cell lines, as determined with the Human Cancer PathwayFinder™ PCR Array. Knockdown of GLUR4 influenced the expression and function of genes involved in invasion and metastasis, tumour suppressor genes, oncogenes and adhesion genes.
The findings suggest that glutamate receptor subunits on cancer cells are linked to biochemical pathways that regulate malignant phenotype.
Anticancer research 10/2011; 31(10):3181-92. · 1.73 Impact Factor
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Ortrud Uckermann,
Hella Luksch,
Vanya Stefovska,
Yvonne Hoehna,
Jenny Marzahn,
Marlen Theil,
Mila Pesic,
Tomasz Górkiewicz,
Maciej Gawlak,
Grzegorz M Wilczynski,
Leszek Kaczmarek,
Chrysanthy Ikonomidou
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ABSTRACT: Matrix metalloproteinases (MMPs) play an essential role in tissue repair, cell death, and morphogenesis. The aim of the present study was to investigate potential involvement of selected MMPs in the pathogenesis of neuronal apoptosis induced by the NMDA antagonist MK-801 (dizocilpine) or the GABA(A) agonist phenobarbital in infant rats, transgenic rats overexpressing MMP-9 and MMP-9 knockout mice. Seven-day-old rats or knockout mice received intraperitoneal injections of MK-801, 1 mg/kg, or phenobarbital, 50 mg/kg. At different survival intervals following administration of the compounds (1-72 h), pups were sacrificed, tissue from different brain regions was isolated, and the expression and activity of MMP-2 and MMP-9 were analyzed by real-time PCR, western blot, and zymography. In addition, brains were fixed and processed for TUNEL staining. In all the brain regions analyzed, we found an increased number of TUNEL-positive cells 24 h after administration of MK-801. After treatment, we detected no significant increase in MMP-2 or MMP-9 mRNA expression in cortical areas. No changes in the MMP-9 protein expression or gelatinolytic activity of MMP-2 were observed in conjunction with MK-801 or phenobarbital-induced neuroapoptosis in any brain region analyzed. The extent of neurodegeneration induced by MK-801 or phenobarbital was not altered in MMP-9 transgenic rats and was increased in MMP-9 knockout mice compared to wild-type rats and mice. Treatment with the panmetalloproteinase inhibitor GM6001 did not confer protection against MK-801-induced apoptotic cell death in the developing rat brain. Our results suggest that activation of MMP-9 and MMP-2 does not contribute to pathogenesis of neuronal apoptosis caused by NMDA antagonists or GABA(A) agonists in the developing rat and mouse brain.
Neurotoxicity Research 05/2011; 19(4):638-48. · 3.51 Impact Factor
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Andrzej Stepulak,
Hella Luksch, Ortrud Uckermann,
Marco Sifringer,
Wojciech Rzeski,
Krzysztof Polberg,
Krzysztof Kupisz,
Janusz Klatka,
Michal Kielbus,
Aneta Grabarska,
Jenny Marzahn,
Lechoslaw Turski,
Chrysanthy Ikonomidou
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ABSTRACT: Despite recent improvements in treatment strategies, the results of chemotherapy in patients with advanced squamous cell carcinoma of the larynx are not satisfactory. Thus, the development of new approaches which influence specific metabolic pathways are needed. In the last decade, evidence has emerged implicating a role for glutamate as a signal mediator in tumors.
The presence of glutamate receptor subunits in two laryngeal cancer cell lines (RK33 and RK45) was evaluated by means of end-point PCR, real-time PCR, and immunocytochemistry.
Glutamate receptor subunits are differentially expressed in laryngeal cancer cell lines. In addition, we show that selected ionotropic glutamate receptor antagonists and metabotropic glutamate receptor 5 antagonist inhibit proliferation of laryngeal cancer cells. Glutamate antagonists also affected activity of extracellular signal-regulated kinases 1/2 in tumor cells.
Signaling through glutamate receptors may influence growth of laryngeal cancer cells and may constitute an adjunctive therapeutic target.
Anticancer research 02/2011; 31(2):565-73. · 1.73 Impact Factor
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Andrzej Stepulak,
Hella Luksch,
Christine Gebhardt, Ortrud Uckermann,
Jenny Marzahn,
Marco Sifringer,
Wojciech Rzeski,
Christian Staufner,
Katja S Brocke,
Lechoslaw Turski,
Chrysanthy Ikonomidou
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ABSTRACT: Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.
Histochemie 07/2009; 132(4):435-45. · 2.59 Impact Factor
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ABSTRACT: Sedative and anticonvulsant drugs, which inhibit N-methyl-D-aspartate receptor-mediated excitation or enhance GABA-mediated action, may cause apoptotic neurodegeneration in the developing mammalian brain. Here we explored whether such agents influence early postnatal neurogenesis.
The N-methyl-D-aspartate antagonist MK801 and the GABA subtype A agonists phenobarbital and diazepam were administered to infant rats, and cell proliferation and neurogenesis were studied in the brain using 5-bromo-2'-deoxyuridine and doublecortin immunohistochemistry and stereology. Using confocal microscopy, we quantified neurogenesis in the dentate gyrus on postnatal day 15 (P15) after treatment with MK801 or phenobarbital on P6 to P10. Learning and memory were assessed at the age of 6 months after early postnatal treatment with phenobarbital.
MK801, phenobarbital, and diazepam reduced numbers of newly born cells in the brain. We found no evidence that these agents caused apoptosis of 5-bromo-2'-deoxyuridine-positive cells. In the dentate gyrus, many of the newly formed cells differentiated toward a neuronal phenotype. Phenobarbital and MK801 reduced numbers of newly formed neurons in the dentate gyrus. At the age of 6 months, phenobarbital-treated rats had fewer neurons in the dentate gyrus and performed worse than saline-treated littermates in water maze learning and memory task.
These findings show that blockade of N-methyl-D-aspartate receptor-mediated excitation and enhancement of GABA subtype A receptor activation impair cell proliferation and inhibit neurogenesis in the immature rat brain. Because many sedative and antiepileptic drugs used in pediatric medicine act via these mechanisms, our findings raise concerns about their potential impact on human brain development.
Annals of Neurology 11/2008; 64(4):434-45. · 11.09 Impact Factor
