h-89 and Glioblastoma

Dedicator of cytokinesis 1 (Dock1 or Dock180), a bipartite guanine nucleotide exchange factor for Rac1, plays critical roles in receptor tyrosine kinase-stimulated cancer growth and invasion. Dock180 activity is required in cell migration cancer tumorigenesis promoted by platelet derived growth factor receptor (PDGFR) and epidermal growth factor receptor.. To demonstrate whether PDGFRα promotes tumor malignant behavior through protein kinase A (PKA)-dependent serine phosphorylation of Dock180, we performed cell proliferation, viability, migration, immunoprecipitation, immunoblotting, colony formation, and in vivo tumorigenesis assays using established and short-term explant cultures of glioblastoma cell lines.. Stimulation of PDGFRα results in phosphorylation of Dock180 at serine residue 1250 (S1250), whereas PKA inhibitors H-89 and KT5720 oppose this phosphorylation. S1250 locates within the Rac1-binding Dock homology region 2 domain of Dock180, and its phosphorylation activates Rac1, p-Akt, and phosphorylated extracellular signal-regulated kinase 1/2, while promoting cell migration, in vitro. By expressing RNA interference (RNAi)-resistant wild-type Dock180, but not mutant Dock180 S1250L, we were able to rescue PDGFRα-associated signaling and biological activities in cultured glioblastoma multiforme (GBM) cells that had been treated with RNAi for suppression of endogenous Dock180. In addition, expression of the same RNAi-resistant Dock180 rescued an invasive phenotype of GBM cells following intracranial engraftment in immunocompromised mice.. These data describe an important mechanism by which PDGFRα promotes glioma malignant phenotypes through PKA-dependent serine phosphorylation of Dock180, and the data thereby support targeting the PDGFRα-PKA-Dock180-Rac1 axis for treating GBM with molecular profiles indicating PDGFRα signaling dependency.

Topics: Animals; Brain Neoplasms; Carbazoles; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclic AMP-Dependent Protein Kinases; Female; Glioblastoma; HEK293 Cells; Humans; Isoquinolines; Mice; Phosphorylation; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Pyrroles; rac GTP-Binding Proteins; Receptor, Platelet-Derived Growth Factor alpha; Signal Transduction; Sulfonamides

We investigated whether cAMP-mediated protein kinase A(PKA) and Epac1/Rap1 pathways differentially affect brain tumor cell death using 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone(rolipram), specific phosphodiesterase type IV(PDE IV) inhibitor.. A172 and U87MG human glioblastoma cells were used. Percentage of cell survival was determined by MTT assay. PKA and Epac1/Rap1 activation was determined by western blotting and pull-down assay, respectively. Cell cycle and hypodiploid cell formation were assessed by flow cytometry analysis.. Non-specific PDE inhibitors, isobutylmethylxanthine(IBMX) and theophylline reduce survival percentage of A172 and U87MG cells. The expression of PDE4A and PDE4B was detected in A172 and U87MG cells. Rolipram-treated A172 or U87MG cell survival was lower in the presence of forskolin, adenylate cyclase activator, than that in its absence. Co-treatment with rolipram and forskolin also enhanced CREB phosphorylation on serine 133 that was inhibited by H-89, PKA inhibitor and cAMP-responsive guanine nucleotide exchange factor 1(Epac1), a Rap GDP exchange factor-mediated Rap1 activity in A172 cells. When A172 cells were treated with cell-permeable dibutyryl-cAMP(dbcAMP), PKA activator or 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate(CPT), Epac1 activator, basal level of cell death was increased and cell cycle was arrested at the phase of G2/M. Rolipram-induced A172 cell death was also increased by the co-treatment with dbcAMP or CPT, but it was inhibited by the pre-treatment with H-89.. These findings demonstrate that PKA and Epac1/Rap1 pathways could cooperatively play a role in rolipram-induced brain tumor cell death. It suggests that rolipram might regulate glioblastoma cell density through dual pathways of PKA- and Epac1/Rap1-mediated cell death and cell cycle arrest.

Topics: 1-Methyl-3-isobutylxanthine; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Brain Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 4; Dose-Response Relationship, Drug; Glioblastoma; Guanine Nucleotide Exchange Factors; Humans; Isoquinolines; Phosphodiesterase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Rolipram; Shelterin Complex; Signal Transduction; Sulfonamides; Telomere-Binding Proteins; Theophylline

Modulation of neurotrophic factors to protect neurons from damage is proposed as a novel mechanism for the action of antidepressants. However, the effect of antidepressants on modulation of glial cell line-derived neurotrophic factor (GDNF), which has potent and widespread effects, remains unknown. Here, we demonstrated that long-term use of antidepressant treatment significantly increased GDNF mRNA expression and GDNF release in time- and concentration-dependent manners in rat C6 glioblastoma cells. Amitriptyline treatment also increased GDNF mRNA expression in rat astrocytes. GDNF release continued for 24 h following withdrawal of amitriptyline. Furthermore, following treatment with antidepressants belonging to several different classes (amitriptyline, clomipramine, mianserin, fluoxetine and paroxetine) significantly increased GDNF release, but which did not occur after treatment with non-antidepressant psychotropic drugs (haloperidol, diazepam and diphenhydramine). Amitriptyline-induced GDNF release was inhibited by U0126 (10 microM), a mitogen-activated protein kinase (MAPK)-extracellular signal-related kinase (ERK) kinase (MEK) inhibitor, but was not inhibited by H-89 (1 microM), a protein kinase A inhibitor, calphostin C (100 nM), a protein kinase C inhibitor and PD 169316 (10 microM), a p38 mitogen-activated protein kinase inhibitor. These results suggested that amitriptyline-induced GDNF synthesis and release occurred at the transcriptional level, and may be regulated by MEK/MAPK signalling. The enhanced and prolonged induction of GDNF by antidepressants could promote neuronal survival, and protect neurons from the damaging effects of stress. This may contribute to explain therapeutic action of antidepressants and suggest new strategies of pharmacological intervention.

Topics: Amitriptyline; Animals; Antidepressive Agents; Astrocytes; Butadienes; Cell Line; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glial Cell Line-Derived Neurotrophic Factor; Glioblastoma; Imidazoles; Isoquinolines; Kinetics; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Naphthalenes; Nerve Growth Factors; Nerve Tissue Proteins; Nitriles; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Psychotropic Drugs; Rats; Sulfonamides

In human glioblastoma A172 cells, interleukin-6 (IL-6) production was induced by interleukin-1 beta (IL-1 beta) and dibutyryl cyclic AMP. These cells have been shown to induce IL-6 production via a cAMP-protein kinase A system. Since calcitonin (CT) and calcitonin gene-related peptide (CGRP) are known to increase cAMP accumulation in murine and rat astrocytes, we examined whether these neuropeptides induced IL-6 production in A172 cells. Human CT and human CGRP increased IL-6 production and cAMP accumulation in a dose-dependent manner. A specific protein kinase A inhibitor, H-89, inhibited both CT- and CGRP-induced IL-6 production. CT and CGRP have been shown to cross-react with each other. To exclude the possibility of this cross-reactivity, we studied the additive effects of CT and CGRP and the inhibitory effects of specific inhibitors. When 100 nM CT was added, cAMP accumulation stimulated by 10 nM CGRP (the maximal dose) was increased. CGRP (8-37), a specific CGRP receptor inhibitor, inhibited cAMP accumulation and IL-6 production induced by CGRP, but did not inhibit these effects when they were induced by CT. Salmon CT (8-32), a specific inhibitor of the CT receptor, inhibited cAMP accumulation induced by CT, but did not inhibit the effect induced by CGRP. These results demonstrated that CT can induce IL-6 production via cAMP accumulation and the effects of CT are mediated via its own receptors.

Topics: Calcitonin; Calcitonin Gene-Related Peptide; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Glioblastoma; Humans; Interleukin-6; Isoquinolines; Sulfonamides; Tumor Cells, Cultured

The brain creatine kinase (CKB) gene is expressed in a variety of tissues with highest expression seen in the brain. We have previously shown in primary rat brain cell cultures that CKB mRNA levels are high in oligodendrocytes and astrocytes and low in neurons (Molloy et al.: J Neurochem 59:1925-1932, 1992). In this report we show that treatment of human U87 glioblastoma cells with forskolin and IBMX, to elevate intracellular cAMP, induces expression of CKB mRNA from the transiently transfected rat CKB gene by 14-fold and also increases expression from the endogenous human CKB gene. This induction of CKB mRNA i) is due to increased transcription; ii) occurs rapidly (with maximal induction after 6 hr; iii) requires the activity of protein kinase A (PKA), but iv) does not require de novo protein synthesis and, in fact, is superinduced in the presence of cycloheximide. Given the role of oligodendrocytes in the energy-demanding process of myelination and of astrocytes in ion transport, these results have physiological significance, since they suggest that changes in cellular energy requirements in the brain during events, such as glial cell differentiation and increased neuronal activity, may in part be met by a cAMP-mediated modulation of CKB gene expression. Of particular importance is the possible modulation of CKB gene expression during myelinogenesis, since oligodendrocyte differentiation has been shown previously to be stimulated by increases in cAMP.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Animals; Brain; Brain Neoplasms; Cloning, Molecular; Colforsin; Creatine Kinase; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Energy Metabolism; Enzyme Activation; Enzyme Induction; Glioblastoma; Glioma; HeLa Cells; Humans; Isoenzymes; Isoquinolines; Myelin Sheath; Nerve Tissue Proteins; Neuroglia; Protein Synthesis Inhibitors; Rats; Recombinant Fusion Proteins; RNA, Antisense; Sulfonamides; Transcription, Genetic; Transfection; Tumor Cells, Cultured