cytochrome-c-t and ebselen

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.

Topics: Apoptosis; Azoles; bcl-2-Associated X Protein; Buthionine Sulfoximine; Cell Line, Tumor; Cell Survival; Coculture Techniques; Cytochromes c; Cytosol; Ethacrynic Acid; Glioblastoma; Glutathione; Humans; Isoindoles; Mitochondria; Neuroblastoma; Neurons; Organoselenium Compounds; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

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