u-0126 and Glioma

Women's fecundity is known to decrease with the increase in chronologic age. Several biomarkers of the ovarian reserve, including follicle stimulating hormone (FSH), anti Müllerian hormone (AMH), have been proposed as possible predictors for the response to controlled ovarian stimulation (COS). Although there are assumptions indicating that the relationship between age and ovarian reserve is highly variable and the potential different validity of ovarian reserve markers in women in different age groups remains to be demonstrated. The purpose of our study was evaluating FSH and AMH as potential predictors of response to controlled ovarian stimulation and prediction of intracytoplasmic sperm injection (ICSI) outcome according to age. This prospective study has been carried out on 218 women having ICSI cycles. Cases were divided into two groups, group 1 (. We found that below the age of 35 years, the chances of pregnancy are more correlated to FSH levels, while above the age of 35 years, AMH was a more relevant test.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Basigin; beta-Transducin Repeat-Containing Proteins; Brain Neoplasms; Butadienes; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diabetic Cardiomyopathies; Diet, High-Fat; Dietary Supplements; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Endoribonucleases; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Inflammation; Lipopolysaccharides; Male; Mice; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; NF-E2-Related Factor 2; Nitriles; Phosphorylation; Rats; Signal Transduction; Sumoylation; Temozolomide; Unfolded Protein Response; X-Box Binding Protein 1; Xenograft Model Antitumor Assays

We aimed to find out the underlying mechanism of forskolin (Fsk) and 3-isobutyl-1-methylxanthine (IBMX) on glioma stem cells (GSCs).. The expression of cAMP-related protein CREB and pCREB as well as apoptosis-related proteins were detected through Western blot analysis. The level of proliferation and growth rate of human GSCs was measured through thiazolyl blue tetrazolium bromide assay and stem cells forming sphere assay. The apoptosis-related gene expression was measured through reverse transcription-polymerase chain reaction.. cAMP signaling pathway was activated in GSCs with Fsk-IBMX administration. Fsk-IBMX could inhibit the proliferation as well as invasion and promote the apoptosis of U87 cells. Besides, U0126 could inhibit MAPK signaling pathway to increase the sensitivity of GSCs to cAMP signaling pathway. As a result, Fsk-IBMX combined with U0126 had more negative effect on GSCs.. The relationship of cAMP and MAPK signaling pathway in GSCs may provide a potential therapeutic strategy in glioma.

Topics: 1-Methyl-3-isobutylxanthine; Apoptosis; Brain Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Colforsin; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Glioma; Humans; Mitogen-Activated Protein Kinases; Neoplastic Stem Cells; Nitriles; Plant Extracts; Plant Roots; Plectranthus; Signal Transduction

Increasing evidence suggests that ion channel genes play an important role in the progression of gliomas. However, the mechanisms by which ion channel genes influence the progression of glioma are not fully understood. We identified KCNB1 as a novel ion gene, associated with malignant progression and favorable overall survival (OS) and progression-free survival (PFS) in glioma patients from three datasets (CGGA, GSE16011 and REMBRANDT). Moreover, we characterized a novel function of autophagy induction accompanied by increased apoptosis and reduced proliferation and invasion of glioma cells for KCNB1. KEGG pathway analysis and in vitro studies suggested that the ERK pathway is involved in KCNB1-mediated regulation of autophagy, which was confirmed by inhibition of KCNB1-induced autophagy by using a selective ERK1/2 inhibitor (U0126) or siERK1/2. In vivo studies showed that KCNB1 induced autophagy while inhibiting tumor growth and increasing survival. Overall, our studies define KCNB1 as a novel prognostic factor for gliomas that exerts its tumor suppressive function through autophagy induction.

Topics: Apoptosis; Autophagy; Brain Neoplasms; Butadienes; Cell Line, Tumor; Datasets as Topic; Disease-Free Survival; Glioma; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; Nitriles; Prognosis; Shab Potassium Channels

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a type of long noncoding RNA. It is associated with metastasis and is a favorable prognostic factor for lung cancer. Recent studies have shown that MALAT1 plays an important role in other malignancies. But, little is known about the role of MALAT1 in glioma. In this study, quantitative reverse transcription PCR (qRT-PCR) was used to demonstrate that the expression of MALAT1 was lower than that in normal brain tissues. Stable RNA interference-mediated knockdown of MALAT1 in human glioma cell lines (U87 and U251) significantly promoted the invasion and proliferation of the glioma cells by in vitro assays. Conversely, overexpression of MALAT1 caused significant reduction in cell proliferation and invasion in vitro, and tumorigenicity in both subcutaneous and intracranial human glioma xenograft models. Furthermore, MALAT1-mediated tumor suppression in glioma cells may be via reduction of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling activity and expression of matrix metalloproteinase 2 (MMP2). In conclusion, overall data demonstrated the tumor-suppressive role of MALAT1 in glioma by attenuating ERK/MAPK-mediated growth and MMP2-mediated invasiveness.

Topics: Adult; Animals; Brain; Brain Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; Matrix Metalloproteinase 2; Mice; Mice, Nude; Middle Aged; Mitogen-Activated Protein Kinases; Nitriles; RNA Interference; RNA, Long Noncoding; Signal Transduction

Increasing body of evidence indicates that neuron-neuroglia interaction may play a key role in determining the progression of neurodegenerative diseases including Parkinson's disease (PD), a chronic pathological condition characterized by selective loss of dopaminergic (DA) neurons in the substantia nigra. We have previously reported that guanosine (GUO) antagonizes MPP(+)-induced cytotoxicity in neuroblastoma cells and exerts neuroprotective effects against 6-hydroxydopamine (6-OHDA) and beta-amyloid-induced apoptosis of SH-SY5Y cells. In the present study we demonstrate that GUO protected C6 glioma cells, taken as a model system for astrocytes, from 6-OHDA-induced neurotoxicity. We show that GUO, either alone or in combination with 6-OHDA activated the cell survival pathways ERK and PI3K/Akt. The involvement of these signaling systems in the mechanism of the nucleoside action was strengthened by a reduction of the protective effect when glial cells were pretreated with U0126 or LY294002, the specific inhibitors of MEK1/2 and PI3K, respectively. Since the protective effect on glial cell death of GUO was not affected by pretreatment with a cocktail of nucleoside transporter blockers, GUO transport and its intracellular accumulation were not at play in our in vitro model of PD. This fits well with our data which pointed to the presence of specific binding sites for GUO on rat brain membranes. On the whole, the results described in the present study, along with our recent evidence showing that GUO when administered to rats via intraperitoneal injection is able to reach the brain and with previous data indicating that it stimulates the release of neurotrophic factors, suggest that GUO, a natural compound, by acting at the glial level could be a promising agent to be tested against neurodegeneration.

Topics: Animals; Apoptosis; Astrocytes; Biological Transport; Butadienes; Cell Line, Tumor; Chromones; DNA Fragmentation; Drug Evaluation, Preclinical; Glioma; Guanosine; In Vitro Techniques; MAP Kinase Signaling System; Morpholines; Neuroprotective Agents; Neurotoxins; Nitriles; Nucleoside Transport Proteins; Oxidopamine; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

Increased PDGFRA signaling is an essential pathogenic factor in many subtypes of gliomas. In this context the cell surface expression of PDGFRA is an important determinant of ligand sensing in the glioma microenvironment. However, the regulation of spatial distribution of PDGFRA in glioma cells remains poorly characterized. Here, we report that cell surface PDGFRA expression in gliomas is negatively regulated by an ERK-dependent mechanism, resulting in reduced proliferation of glioma cells. Glioma tumor tissues and their corresponding cell lines were isolated from 14 patients and analyzed by single-cell imaging and flow cytometry. In both cell lines and their corresponding tumor samples, glioma cell proliferation correlated with the extent of surface expression of PDGFRA. High levels of surface PDGFRA also correlated to high tubulin expression in glioma tumor tissue in vivo. In glioma cell lines, surface PDGFRA declined following treatment with inhibitors of tubulin, actin and dynamin. Screening of a panel of small molecule compounds identified the MEK inhibitor U0126 as a potent inhibitor of surface PDGFRA expression. Importantly, U0126 inhibited surface expression in a reversible, dose- and time-dependent manner, without affecting general PDGFRA expression. Treatment with U0126 resulted in reduced co-localization between PDGFRA and intracellular trafficking molecules e.g. clathrin, RAB11 and early endosomal antigen-1, in parallel with enhanced co-localization between PDGFRA and the Golgi cisternae maker, Giantin, suggesting a deviation of PDGFRA from the endosomal trafficking and recycling compartment, to the Golgi network. Furthermore, U0126 treatment in glioma cells induced an initial inhibition of ERK1/2 phosphorylation, followed by up-regulated ERK1/2 phosphorylation concomitant with diminished surface expression of PDGFRA. Finally, down-regulation of surface PDGFRA expression by U0126 is concordant with reduced glioma cell proliferation. These findings suggest that manipulation of spatial expression of PDGFRA can potentially be used to combat gliomas.

Topics: Butadienes; Cell Membrane; Cell Proliferation; Cytoskeleton; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glioma; Humans; Nitriles; Protein Kinase Inhibitors; Protein Transport; rab GTP-Binding Proteins; Receptor, Platelet-Derived Growth Factor alpha; Tumor Cells, Cultured; Vesicular Transport Proteins

Conditioned mediums (CMs) from glioma cells U87, GBM-8401, and C6 significantly induced iNOS protein and NO production by microglial cells BV-2 but without altering the cell viability or cell-cycle progression of BV2 microglia. Significant increases in intracellular peroxide by U87-CM and C6-CM were detected by a DCHF-DA assay, and vitamin (Vit) C and N-acetyl cysteine (NAC)-reduced intracellular peroxide levels elicited by CMs lead to inhibition of iNOS/NO production The extracellular signal-regulated kinase (ERK) inhibitor, U0126, and c-Jun N-terminal kinase (JNK) inhibitor, SP600125, suppressed U87-CM- and C6-CM-induced iNOS/NO production by respectively blocking phosphorylated ERK (pERK) and JNK (pJNK) protein expressions stimulated by U87-CM and C6-CM. Increased migration of U87 and C6 glioma cells by a co-culture with BV-2 microglial cells or adding the nitric oxide (NO) donor, sodium nitroprusside (SNP) was observed, and that was blocked by adding an NO synthase (NOS) inhibitor, N-nitro L-arginine methyl ester (NAME). Contributions of ROS, pERK, and pJNK to the migration of glioma cells was further demonstrated in a transwell coculture system of U87 and C6 gliomas with BV-2 microglial cells. Furthermore, expressions of tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 messenger (m)RNA in U87 and C6 cells were detected by an RT-PCR, and TNF-α and MCP-1 induced iNOS protein expression in time- and concentration-dependent manners. Neutralization of TNF-α or MCP-1 in U87-CM and C6-CM using a TNF-α or MCP-1 antibody inhibited iNOS protein expression, and increased intracellular peroxide by TNF-α or MCP-1 was identified in BV-2 cells. The reciprocal activation of glioma cells and microglia via ROS-dependent iNOS/NO elevation at least partially mediated by TNF-α and MCP-1 is elucidated.

Topics: Anthracenes; Butadienes; Cell Line, Tumor; Cell Survival; Chemokine CCL2; Coculture Techniques; Culture Media, Conditioned; Gene Expression Regulation, Neoplastic; Glioma; Humans; JNK Mitogen-Activated Protein Kinases; Microglia; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Nitriles; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

Dioxins are the most concerned environmental pollutants. Recent studies have shown that these compounds could disrupt the proper functioning of our endocrine system. Estrogen is synthesized in glial cells of the brain. The hormone has been linked to the maintenance of normal brain operation, ranging from neurotransmission to synapse formation. Aromatase or CYP19 is the enzyme responsible for estrogen synthesis. In the present study, we demonstrated that 2,3,7,8-tetrachloro-dibenzo-para-dioxin (TCDD) stimulated the enzyme activity in human brain cells as low as 1pM. Increased brain-specific CYP19 mRNA species was also observed in these cells. Since the brain-specific promoter I.f of CYP19 contains two binding motifs for CCAAT/enhancer binding protein, electrophoretic mobility shift assay was performed to validate the activation. We further traced the triggering signal and found that the mitogen-activated protein kinases ERK-1/2 were activated. In summary, TCDD could induce CYP19 transcription in brain cells. Exposure to the pollutant might perturb the hormonal balance in the brain.

Topics: Aromatase; Butadienes; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Environmental Pollutants; Enzyme Induction; Extracellular Signal-Regulated MAP Kinases; Glioma; Humans; MAP Kinase Signaling System; Neuroglia; Nitriles; Organ Specificity; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Protein Binding

Sphingosine kinase (SphK), which is regulated by hypoxia, catalyses phosphorylation of sphingosine to produce sphingosine-1-phosphate, which stimulates invasiveness of gliomas. However, whether SphK is involved in proliferation of glioma cells under hypoxic conditions is not clearly understood. In this study, we have investigated the role of SphK in of proliferation glioma cells under hypoxia..   Effects of small interfering RNA (siRNA) on SphKs, SKI (inhibitor of SphK) and U0126 (inhibitor of ERK) on proliferation of glioma cells under hypoxia were studied using CCK-8 assay and flow cytometry. Protein expression profiles were evaluated by Western blot analysis.. SKI suppressed proliferation of glioma cells under hypoxia. Similarly, downregulation of SphKs by siRNA inhibited glioma cell proliferation, and the cell cycle was arrested in G(2) /M phase when SphK1 was inhibited. In addition, inhibition of SphK1 attenuated phosphorylation of ERK in hypoxic conditions. Furthermore, U0126 markedly inhibited cell population growth and arrested cells in G(2) /M as effectively as SKI. However, silencing SphK2 induced cell cycle arrest in the S phase and it showed little effect on hypoxia-induced activation of ERK..   SphK1 and SphK2 are involved in proliferation of glioma cells in hypoxic conditions through distinct signalling pathways. SphK1, but not SphK2, promotes cell population expansion in hypoxic conditions by activating ERK.

Topics: Base Sequence; Butadienes; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; G2 Phase Cell Cycle Checkpoints; Glioblastoma; Glioma; Humans; Nitriles; Phosphotransferases (Alcohol Group Acceptor); Receptors, G-Protein-Coupled; RNA, Small Interfering; S Phase Cell Cycle Checkpoints

Neurotrophins play essential roles in the development, differentiation, and survival of neuronal and nonneuronal cells. Alterations in neurotrophin expression have been implicated in a variety of neurodegenerative disorders. Dysregulation of brain-derived neurotrophic factor (BDNF) has been implicated in deficits of long-term potentiation and cognition and may contribute to the development of Alzheimer's disease (AD). In this study, we used complementary pharmacological and molecular approaches to evaluate the role of ERK1/2 and ERK5, two members of the MAPK pathway associated with neuroprotection, in regulating BDNF expression in C6 glial cells and primary astrocytes. Our data revealed that U0126, an inhibitor of both ERK5 and ERK1/2, increased the levels of BDNF mRNA, whereas the MEK1/2-specific inhibitor PD184352 did not, suggesting that ERK5 exerts negative control over BDNF expression. This was supported by experiments in which RNAi-mediated depletion of ERK5 led to an increase in BDNF. In contrast, transfection with constitutively active MEK5 resulted in an inhibition of BDNF expression, confirming the inhibitory role of ERK5 in the regulation of BDNF. Interestingly, transfection with the dominant active mutant of MEK1 (MEKR4F), the upstream activator of ERK1/2, resulted in a modest increase in BDNF levels. Collectively, our data suggest that ERK5 and ERK1/2 exert opposite effects on BDNF expression and support the hypothesis that an imbalance of these two signaling pathways may contribute to the pathology of diseases in which neurotrophin dysregulation is noted.

Topics: Animals; Animals, Newborn; Astrocytes; Benzamides; Brain-Derived Neurotrophic Factor; Butadienes; Cell Line, Tumor; Glioma; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase 7; Neurons; Nitriles; Primary Cell Culture; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley

Activated mitogen-activated protein kinase MAPK cascade leading to ERK1/2 phosphorylation is expressed in the majority of glial neoplasms and negatively correlates with survival time of patients. Here we show that ERK1/2 kinases are constitutively activated in glioma cell lines and stem cell-enriched primary cultures of glioblastoma. Pharmacological targeting of the activated MEK/ERK1/2 module with the MEK inhibitor U0126 attenuates cell cycle progression (11 out of 11 cell lines), impairs single (7 out of 10) and collective cell migration (9 out of 11) and abolishes single cell emigration from monolayers (4 out of 9). Attacking the activated MEK/ERK1/2 module thus partially blocks the tumorigenic potential of glial cancer cells on different levels and strongly suggests the application of combination molecularly targeted therapies to interfere more efficiently with glial tumor development and progression.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Enzyme Inhibitors; Glioma; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Neoplastic Stem Cells; Nitriles; Rats

Neuropilin-1 (NRP1) functions as a coreceptor through interaction with plexin A1 or vascular endothelial growth factor (VEGF) receptor during neuronal development and angiogenesis. NRP1 potentiates the signaling pathways stimulated by semaphorin 3A and VEGF-A in neuronal and endothelial cells, respectively. In this study, we investigate the role of tumor cell-expressed NRP1 in glioma progression. Analyses of human glioma specimens (WHO grade I-IV tumors) revealed a significant correlation of NRP1 expression with glioma progression. In tumor xenografts, overexpression of NRP1 by U87MG gliomas strongly promoted tumor growth and angiogenesis. Overexpression of NRP1 by U87MG cells stimulated cell survival through the enhancement of autocrine hepatocyte growth factor/scatter factor (HGF/SF)/c-Met signaling. NRP1 not only potentiated the activity of endogenous HGF/SF on glioma cell survival but also enhanced HGF/SF-promoted cell proliferation. Inhibition of HGF/SF, c-Met and NRP1 abrogated NRP1-potentiated autocrine HGF/SF stimulation. Furthermore, increased phosphorylation of c-Met correlated with glioma progression in human glioma biopsies in which NRP1 is upregulated and in U87MG NRP1-overexpressing tumors. Together, these data suggest that tumor cell-expressed NRP1 promotes glioma progression through potentiating the activity of the HGF/SF autocrine c-Met signaling pathway, in addition to enhancing angiogenesis, suggesting a novel mechanism of NRP1 in promoting human glioma progression.

Topics: Animals; Butadienes; Cell Line; Cell Line, Tumor; Cell Proliferation; Disease Progression; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glioma; Hepatocyte Growth Factor; Humans; Immunoblotting; Mice; Neoplasms, Experimental; Neovascularization, Pathologic; Neuropilin-1; Nitriles; Phosphorylation; Proto-Oncogene Proteins c-met; RNA, Small Interfering; Signal Transduction; Transfection; Transplantation, Heterologous; Tumor Burden

The extracellular signal-regulated kinase (ERK) signaling pathway has been implicated in diverse cellular functions. ERK and its activating kinase, mitogen-activated/extracellular signal-regulated kinase kinase (MEK), are downstream of cell surface receptors known to be up-regulated in many malignant gliomas. We sought to investigate the role of ERK in glioma cell migration, proliferation and differentiation using the rat-derived C6 glioma cell line and the MEK inhibitor, U0126. Treatment of C6 cells with U0126 caused a significant concentration-dependent reduction in cell proliferation and migration and also induced expression of glial fibrillary acidic protein, a marker of astrocytic differentiation. These results suggest that the ERK pathway regulates glioma cell proliferation, migration and differentiation.

Topics: Analysis of Variance; Animals; Blotting, Western; Bromodeoxyuridine; Butadienes; Caspase 3; Cell Count; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression; Glial Fibrillary Acidic Protein; Glioma; Immunohistochemistry; Mice; Nitriles; Tetrazolium Salts; Thiazoles

Malignant gliomas are highly invasive tumors which are lethal despite aggressive therapy. The motility behavior of two human glioma cell lines i.e. T98G and U87-MG cells was analysed. The glioma cells showed a high degree of basal motility (especially U87-MG cells) that may be related to the considerable local invasiveness of such tumours even in the absence of exogenous factors. The two cell lines responded equally well to platelet-derived growth factor (PDGF) as chemoattractant factor. The phosphatidylinositol 3-kinase (PI3-K) signaling, but not the extracellular signal-related kinase (ERK) signaling, was strongly involved in the PDGF-stimulated glioma cell motility. Somatostatin was capable of inhibiting the migration in both glioma cell lines without affecting crucial targets for motility control like PI3-K and Rac activity. These data suggest that somatostatin, by interfering with a target further downstream to Rac, negatively affects glioma cell motility, and may thus offer a pharmacological approach to controlling the deregulated motility of these aggressive tumoral cells.

Topics: Butadienes; Cell Line, Tumor; Cell Movement; Chemotactic Factors; Chromones; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Glioma; Hormones; Humans; Morpholines; Neoplasm Metastasis; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-akt; rac GTP-Binding Proteins; Somatostatin

In this study the contribution of the ERK1/2 pathway to sphingosine-induced death and morphological changes of the actin cytoskeleton in glioma C6 cells was investigated. Surprisingly, the level of ERK1/2 phosphorylation does not change after incubation of cells with sphingosine. Despite this, sphingosine induces rounding and detachment of cells without formation of apoptotic bodies. To shed light on this process, a specific inhibitor of ERK1/2 phosphorylation, U0126, was used. Cells incubated simultaneously with sphingosine and U0126 not only detached, but also exhibited formation of apoptotic-like blebs. These data suggest that during sphingosine-induced glioma C6 cell death apoptotic blebbing is dependent on ERK1/2 signalling and occurs only when ERK1/2 activity is decreased or abolished.

Topics: Animals; Butadienes; Cell Death; Cell Line, Tumor; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Glioma; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Rats; Sphingosine

We previously showed that the aggregated form of Hsp27 in cultured cells becomes dissociated as a result of phosphorylation with various types of stress. In order to clarify the signal transduction cascade involved, the effects of various inhibitors of protein kinases and dithiothreitol on the dissociation of Hsp27 were here examined by means of an immunoassay after fractionation of cell extracts by sucrose density gradient centrifugation. The dissociation of Hsp27 induced by exposure of U251 MG human glioma cells to metals (NaAsO2 and CdCl2), hypertonic stress (sorbitol and NaCI), or anisomycin, an activator of p38 mitogen-activated protein (MAP) kinase, was completely suppressed by the presence of SB 203580 or PD 169316, inhibitors of p38 MAP kinase, but not by PD 98059 and Uo 126, inhibitors of MAP kinase kinase (MEK), nor by staurosporine, Go 6983, and bisindolylmaleimide I, inhibitors of protein kinase C. Phorbol ester (PMA)-induced dissociation of Hsp27 was completely suppressed by staurosporine, Go 6983, or bisindolylmaleimide I and partially suppressed by SB 203580, or PD 169316 but not by PD 98059 or Uo 126, indicating mediation by 2 cascades. The presence of 1 mM dithiothreitol in the culture medium during exposure to chemicals suppressed the dissociation of Hsp27 induced by arsenite and CdCl2 but not by other chemicals. These results suggest that the phosphorylation of Hsp27 is catalyzed by 2 protein kinases, p38 MAP kinase-activated protein (MAPKAP) kinase-2/3 and protein kinase C. In addition, metal-induced signals are sensitive to reducing power.

Topics: Anisomycin; Arsenites; Butadienes; Cadmium Chloride; Carcinogens; Dithiothreitol; Enzyme Inhibitors; Flavonoids; Glioma; Heat-Shock Proteins; Humans; Hydrogen Peroxide; Imidazoles; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Osmotic Pressure; Oxidants; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Protein Synthesis Inhibitors; Pyridines; Sodium Compounds; Sorbitol; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Protein kinase C (PKC) is a family of serine/threonine kinases involved in the transduction of a variety of signals. There is increasing evidence to indicate that specific PKC isoforms are involved in the regulation of distinct cellular processes. In glioma cells, PKC alpha was found to be a critical regulator of proliferation and cell cycle progression, while PKC epsilon was found to regulate adhesion and migration. Herein, we report that specific PKC isoforms are able to differentially activate extracellular-signal regulated kinase (ERK) in distinct cellular locations: while PKC alpha induces the activation of nuclear ERK, PKC epsilon induces the activation of ERK at focal adhesions. Inhibition of the ERK pathway completely abolished the PKC-induced integrin-mediated adhesion and migration. Thus, we present the first evidence that PKC epsilon is able to activate ERK at focal adhesions to mediate glioma cell adhesion and motility, providing a molecular mechanism to explain the different biological functions of PKC alpha and epsilon in glioma cells.

Topics: Androstadienes; Brain Neoplasms; Butadienes; Cell Adhesion; Cell Movement; Enzyme Activation; Enzyme Inhibitors; Focal Adhesions; Glioma; Humans; Isoenzymes; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Nitriles; Phosphorylation; Protein Kinase C; Protein Kinase C-epsilon; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyrimidines; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Wortmannin