u-0126 and ebselen

We found that ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] caused phosphorylation of mitogen-activated protein kinase (MAPK), followed by expression of neurofilament-M, a neuron-specific protein, in cultured PC12 rat pheochromocytoma cells. The ebselen-induced MAPK activation was suppressed by U0126, an inhibitor of MAPK kinase (MEK1/2), but not by K252a, a selective inhibitor of Trk family tyrosine kinases; AG1478, an antagonist of epidermal growth factor receptor (EGFR); pertussis toxin, an inhibitor of Gi/o; or GP antagonist-2A, an inhibitor of Gq. Furthermore, we observed that N-acetyl-L-cysteine, an inhibitor of tyrosine kinases, suppressed ebselen-induced MAPK activation and buthionine sulfoximine, an activator of protein tyrosine phosphatases, enhanced the effect, indicating that ebselen activated MEK1/2 through one or more tyrosine kinases. Based on these results, we propose that ebselen stimulated intracellular tyrosine kinase activity, thus activating a MAPK cascade (tyrosine kinase-MEK1/2-ERK1/2) in PC12 cells and that this activation resulted in their neuronal differentiation.

Topics: Animals; Antioxidants; Azoles; Butadienes; Carbazoles; Enzyme Activation; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gq-G11; Indole Alkaloids; Isoindoles; Mitogen-Activated Protein Kinases; Neurites; Neurofilament Proteins; Nitriles; Organoselenium Compounds; Oxidation-Reduction; PC12 Cells; Pertussis Toxin; Phosphorylation; Quinazolines; Rats; Receptor Protein-Tyrosine Kinases; Selenium; Tyrphostins

Ebselen, a selenium-containing heterocyclic compound, prevents ischemia-induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO-induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO-induced phosphatidyl Serine exposure, cytochrome c release, and caspase-3 activation by ebselen. Analysis of key apoptotic regulators during NO-induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling-regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal protein kinase (JNK). Moreover, ebselen inhibits NO-induced p53 phosphorylation at Ser15 and c-Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol-redox-dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl-2 in SNP-treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.

Topics: Animals; Annexin A5; Apoptosis; Azoles; Blotting, Western; Butadienes; Caspase 3; Caspases; Cell Differentiation; Cell Survival; Chaperonin 60; Cyclooxygenase Inhibitors; Cytochromes c; Dithiothreitol; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; In Vitro Techniques; Isoindoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 5; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; Nerve Growth Factor; Nitric Oxide; Nitric Oxide Donors; Nitriles; Nitroprusside; Organoselenium Compounds; p38 Mitogen-Activated Protein Kinases; PC12 Cells; Propidium; Proto-Oncogene Proteins c-bcl-2; Rats; Serine; Sodium Selenite; Time Factors; Transfection; Tumor Suppressor Protein p53; Vitamin E