diamide and Neuroblastoma

Redox changes are often reported as causative of neoplastic transformation and chemoresistance, but are also exploited as clinical tools to selectively kill tumor cells. We previously demonstrated that gastrointestinal-derived tumor histotypes are resistant to ROS-based treatments by means of the redox activation of Nrf2, but highly sensitive to disulfide stressors triggering apoptosis via the redox induction of Trx1/p38(MAPK)/p53 signaling pathway. Here, we provide evidence that neuroblastoma SH-SY5Y has a complete opposite behavior, being sensitive to H₂O₂, but resistant to the glutathione (GSH)-oxidizing molecule diamide. Consistent with these observations, the apoptotic pathway activated upon H₂O₂ treatment relies upon Trx1 oxidation, and is mediated by the p38(MAPK)/p53 signaling axis. Pre-treatment with different antioxidants, pharmacological inhibitor of p38(MAPK), or small interfering RNA against p53 rescue cell viability. On the contrary, cell survival to diamide relies upon redox activation of Nrf2, in a way independent on Keap1 oxidation, but responsive to ERK1/2 activation. Chemical inhibition of GSH neo-synthesis or ERK1/2 phosphorylation, as well as overexpression of the dominant-negative form of Nrf2 sensitizes cells to diamide toxicity. In the searching for the molecular determinant(s) unifying these phenomena, we found that SH-SY5Y cells show high GSH levels, but exhibit very low GPx activity. This feature allows to efficiently buffer disulfide stress, but leaves them being vulnerable to H₂O₂-mediated insult. The increase of GPx activity by means of selenium supplementation or GPx1 ectopic expression completely reverses death phenotype, indicating that the response of tumor cells to diverse oxidative stimuli deeply involves the entire GSH redox system.

Topics: Cell Death; Cell Line, Tumor; Cell Survival; Diamide; Glutathione; Glutathione Peroxidase; Humans; Hydrogen Peroxide; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroblastoma; NF-E2-Related Factor 2; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Tumor Suppressor Protein p53

Oxidative stress appears to contribute to neuronal dysfunction associated with Alzheimer's disease and other CNS neurodegenerative disorders. This investigation examined if oxidative stress might contribute to impairments in cholinergic receptor-linked signaling systems and if intracellular glutathione levels modulated responses to oxidative stress. To do this the activation of the AP-1 and NF-kappaB transcription factors and of the phosphoinositide second-messenger system was measured in human neuroblastoma SH-SY5Y cells after exposure to the oxidants H2O2 or diamide, with or without prior depletion of cellular glutathione. H2O2 concentration-dependently inhibited carbachol-stimulated AP-1 activation and this inhibition was potentiated in glutathione-depleted cells. Carbachol-stimulated NF-kappaB activation was unaffected by H2O2 unless glutathione was depleted, in which case there was a H2O2 concentration-dependent inhibition. Glutathione depletion also potentiated the inhibition by H2O2 of carbachol- or G-protein (NaF)-stimulated phosphoinositide hydrolysis, whereas phospholipase C activated by the calcium ionophore ionomycin was not inhibited. The thiol-oxidizing agent diamide also inhibited phosphoinositide hydrolysis stimulated by carbachol or NaF, and glutathione depletion potentiated the diamide concentration-dependent inhibition. Unlike H2O2, diamide also inhibited ionomycin-stimulated phosphoinositide hydrolysis. Activation of both AP-1 and NF-kappaB stimulated by carbachol was inhibited by diamide, and glutathione depletion potentiated the inhibitory effects of diamide. Thus, diamide inhibited a wider range of signaling processes than did H2O2, but glutathione depletion increased the susceptibility of phosphoinositide hydrolysis and of transcription factor activation to inhibition by both H2O2 and diamide. These results demonstrate that the vulnerability of signaling systems to oxidative stress is influenced by intracellular glutathione levels, indicating that cell-selective susceptibility to inhibition of signal transduction systems by oxidative stress can arise from cellular variations in antioxidant capacity.

Topics: Analysis of Variance; Base Sequence; Binding Sites; Buthionine Sulfoximine; Carbachol; Consensus Sequence; Diamide; Glutathione; GTP-Binding Proteins; Humans; Hydrogen Peroxide; Ionomycin; Neuroblastoma; NF-kappa B; Oligodeoxyribonucleotides; Oxidative Stress; Phosphatidylinositols; Second Messenger Systems; Sodium Fluoride; Transcription Factor AP-1; Tumor Cells, Cultured; Vanadates