tacrolimus and Glioma

This study examined the role of calcineurin, a major calcium-dependent protein phosphatase, in dephosphorylating Ser-9 and activating glycogen synthase kinase-3beta (GSK-3beta). Treatment with calcineurin inhibitors increased phosphorylation of GSK-3beta at Ser-9 in SH-SY5Y human neuroblastoma cells. The over-expression of a constitutively active calcineurin mutant, calcineurin A beta (1-401), led to a significant decrease in phosphorylation at Ser-9, an increase in the activity of GSK-3beta, and an increase in the phosphorylation of tau. K(m) of calcineurin for a GSK-3beta phosphopeptide was 469.3 microM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK-3beta were co-immunoprecipitated in neuron-derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK-3beta. We conclude that in vitro, calcineurin can dephosphorylate GSK-3beta at Ser-9 and form a stable complex with GSK-3beta, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK-3betain vivo.

Topics: Animals; Calcineurin; Calcineurin Inhibitors; Cell Line, Tumor; Cyclosporine; Enzyme Activation; Enzyme Inhibitors; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; Mice; Mutation; Neuroblastoma; Neurons; Phosphorylation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Serine; Tacrolimus; Transfection

After organ transplantation, some patients suffer from mild neurological symptoms, such as tremor, to severe complications, including seizures and encephalopathy. These neurological side effects can be caused by immunosuppressants such as tacrolimus. However, the mechanism of encephalopathy by tacrolimus is not fully understood.. We measured the production of reactive oxygen species (ROS) in glioma cells after tacrolimus treatment. Tacrolimus added to glioma cells was incubated for 60 minutes at 37 degrees C. The production of ROS was evaluated by measuring the fluorescent product from the oxidation of an oxidant-sensitive 2',7'-dichlorofluorescin using VICTOR3TM multilabel counter.. Tacrolimus resulted in the production of the ROS in glioma cells. The production of the ROS was increased in time-dependent fashion.. These findings indicated that the tacrolimus may contribute the neurological side effects by ROS production.

Topics: Animals; Glioma; Kinetics; Neuroglia; Rats; Reactive Oxygen Species; Tacrolimus

After organ transplantation, some patients suffer from mild neurologic symptoms, ranging from tremor to severe complications, including seizures and encephalopathy. Among the immunosuppressants, tacrolimus can cause neurologic side effects. However, the mechanisms of encephalopathy by tacrolimus are not fully understood. We measured the antioxidant status, hydrogen peroxide level, and malondialdehyde level in glioma cells after tacrolimus treatment.. The production of hydrogen peroxide was determined by the modified xylenol orange method. The amount of malondialdehyde was measured by the thiobarbituric acid assay, which is based on malondialdehyde reaction with thiobarbituric acid to give a red species absorbing at 535 nm. Total antioxidant status (TAS) was measured using TAS kits (NX2332).. Tacrolimus resulted in dose- and time-dependent increases in the production of hydrogen peroxide by glioma cells. The antioxidant status decreased in the glioma cells after tacrolimus treatment. Malondialdehyde level was unchanged in the glioma cells after tacrolimus treatment.. Increased production of reactive oxygen species and decreased antioxidant status by tacrolimus in glioma cells may contribute to neurologic side effects.

Topics: Animals; Antioxidants; Glioma; Hydrogen Peroxide; Kinetics; Malondialdehyde; Oxidative Stress; Rats; Reactive Oxygen Species; Tacrolimus

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP(C)) into an altered disease-associated conformation generally designated PrP(Sc), abnormal deposition of PrP(Sc) aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP(C) aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP(C) molecules may trigger its aggregation in neuronal cells. A chimeric PrP(C) composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP(C) were created, and transfected in N2a cells. Similar to PrP(C), Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP(C), both dimerization and oligomerization of PrP(C) resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP(C) molecules may constitute a minimal and sufficient molecular event leading to PrP(C) aggregation and extracellular deposition.

Topics: Animals; Binding Sites; Cell Line, Tumor; Dimerization; Dose-Response Relationship, Drug; Endopeptidase K; Gene Expression Regulation; Glioma; Humans; Ligands; Mice; Microscopy, Electron, Transmission; Mutation; Neuroblastoma; Prions; Protein Binding; Tacrolimus; Transfection

To clarify the molecular basis of the cytoprotective properties of immunophilin ligands (IPLs), the anti-apoptotic effects of IPLs were determined in human glioma U251 cells. GPI1046 and V10367, non-immunosuppressive IPLs (NI-IPLs), as well as FK506, an immunosuppressive IPL (I-IPL), had cytoprotective effects against hydrogen peroxide (H20O)-induced apoptotic cell death in U251 cells. H2O2 increased both the ratio of bax/bcl-2 and the p53 mRNA expression. However, pre-treatment with FK506 and V10367 significantly prevented any increase in this ratio or p53 mRNA expression. GPI1046 also reduced the ratio of bax/bcl-2 to the normal level. In addition, H2O2 significantly increased activities of all three caspases, caspase-3, caspase-8, and caspase-9, in comparison with non-H2O2 controls. However, FK506 prevented the increase of these caspase activities. On the other hand, it is well-known that glutathione (GSH) and neurotrophic factor (NTF) is related to the induction of apoptosis in neuronal cells. In U251 cells, FK506, GPI1046 and V10367 had GSH-activating and NTF-activating effects. Thus, the immunosuppressive effect is not essential for the cytoprotective properties of IPLs, and IPLs have multiple beneficial properties such as the anti-apoptotic effect, GSH-activating effect, and NTF-activating effect, although the anti-apoptotic effect of NI-IPLs is independent of the regulation of apoptotic activators such as caspase-3.

Topics: Apoptosis; Brain Neoplasms; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Cell Survival; Glial Cell Line-Derived Neurotrophic Factor; Glioma; Glutathione; Humans; Hydrogen Peroxide; Immunophilins; Ligands; Nerve Growth Factors; Pyridines; Pyrrolidines; Tacrolimus

Topics: Animals; Apoptosis; Benzimidazoles; Brain; Cell Survival; Fluorescent Dyes; Free Radicals; Glioma; Glutathione; Hydrogen Peroxide; Immunophilins; Immunosuppressive Agents; Ligands; Neurodegenerative Diseases; Neuroglia; Neuroprotective Agents; Oxidative Stress; Pyrrolidines; Rats; Tacrolimus; Tumor Cells, Cultured; Up-Regulation

The Ca2+/calmodulin-dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+-dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+-channel activity, phosphatase expression was altered in NG108-15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2Bbeta3. Relative to mock-transfected (wild-type) controls, cells overexpressing calcineurin showed dramatically reduced high-voltage-activated Ca2+ currents which were recoverable by the inclusion of 1 microM FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high-voltage-activated Ca2+ currents were larger relative to controls. Additionally in these cells, low-voltage-activated currents were significantly reduced. Analysis of high-voltage-activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large-scale Ca2+ influx across the cell membrane, Ca2+-dependent inactivation of high-voltage-activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 mM BAPTA or 1 microM FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage-operated Ca2+ channels.

Topics: Animals; Antisense Elements (Genetics); Calcineurin; Calcium; Calcium Channels; Chelating Agents; Egtazic Acid; Gene Expression; Glioma; Humans; Immunoblotting; Immunosuppressive Agents; Membrane Potentials; Neuroblastoma; Neurons; Patch-Clamp Techniques; Tacrolimus; Transfection; Tumor Cells, Cultured

The effects of immunosuppressant cyclosporin A (CsA) on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in rat C6 glioma cell line were investigated. CsA applied simultaneously with iNOS activator IFN-gamma caused dose-dependent reduction of NO synthesis in confluent C6 cells, as determined by measuring accumulation of nitrite, an indicator of NO production, in 48 h culture supernatants. IFN-gamma-induced expression of iNOS, but not interferon regulatory factor-1 (IRF-1) mRNA was reduced in CsA-treated cells. The enzymatic activity of iNOS was not changed by CsA, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-gamma and any further induction was blocked by protein synthesis inhibitor cycloheximide (CHX). FK506 was not able to mimic inhibitory effect of CsA on NO production in C6 cells, suggesting calcineurin-independent mechanism of CsA action.

Topics: Animals; Calcineurin Inhibitors; Cyclosporine; DNA-Binding Proteins; Enzyme Activation; Glioma; Interferon Regulatory Factor-1; Interferon-gamma; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phosphoproteins; Rats; RNA, Messenger; Tacrolimus; Tumor Cells, Cultured