Activation of c-Jun-N-terminal kinase is required for apoptosis triggered by glutathione disulfide in neuroblastoma cells

Changes in intracellular redox status are crucial events that trigger downstream proliferation or death responses through activation of specific signaling pathways. Moreover, cell responses to oxidative challenge may depend on the pattern of redox-sensitive molecular factors. The stress-activated protein kinases c-Jun-N-terminal kinase (JNK) and p38 MAP kinase (p38(MAPK)) are implicated in different forms of apoptotic neuronal cell death. Here, we investigated the effects, on neuroblastoma cells, of the prooxidant molecule GSSG, which we previously demonstrated to be an efficient proapoptotic compound able to activate the p38(MAPK) death pathway in promonocytic cells. We found that neuroblastoma cells are not prone to GSSG-induced apoptosis, although the treatment slightly induced growth arrest through the accumulation of p53 and its downstream target gene, p21. However, GSSG treatment became cytotoxic when cells were previously depleted of intracellular GSH content. Under this condition, apoptosis was triggered by an increased production of superoxide that led to a specific activation of the JNK-dependent pathway. The involvement of superoxide and JNK was demonstrated by cell death inhibition in experiments carried out in the presence of Cu,Zn superoxide dismutase or with specific inhibitors of JNK activity. Our data give support to the studies that indicate preferential requirements for the involvement of stress-activated kinases in apoptotic neuronal cells. (c) 2005 Elsevier Inc. All rights reserved

Oxidative Assets Toward Biomolecules and Cytotoxicity of New Oxindolimine-Copper(II) and Zinc(II) Complexes

A new oxindolimine ligand derived from isatin (1H-indole-2,3-dione) and 2-aminomethylbenzimidazole was synthesized, leading to two novel complexes after metalation with copper(II) perchlorate or zinc(II) chloride, [Cu(isambz)2](ClO4)2 (complex 1) and [Zn(isambz)Cl2] (complex 2). This new ligand was designed as a more lipophilic compound, in a series of oxindolimine⁻metal complexes with antitumor properties, having DNA, mitochondria, and some proteins, such as CDK1 kinase and topoisomerase IB, as key targets. The new complexes had their reactivity to human serum albumin (HSA) and DNA, and their cytotoxicity toward tumor cells investigated. The binding to CT-DNA was monitored by circular dichroism (CD) spectroscopy and fluorescence measurements using ethidium bromide in a competitive assay. Consequent DNA cleavage was verified by gel electrophoresis with complex 1, in nmolar concentrations, with formation of linear DNA (form III) after 60 min incubation at 37 °C, in the presence of hydrogen peroxide, which acts as a reducing agent. Formation of reactive oxygen species (ROS) was observed, monitored by spin trapping EPR. Interaction with HSA lead to α-helix structure disturbance, and formation of a stable radical species (HSA⁻Tyr·) and carbonyl groups in the protein. Despite showing oxidative ability to damage vital biomolecules such as HSA and DNA, these new complexes showed moderate cytotoxicity against hepatocellular carcinoma (HepG2) and neuroblastoma (SHSY5Y) cells, similarly to previous compounds in this series. These results confirm DNA as an important target for these compounds, and additionally indicate that oxidative damage is not the leading mechanism responsible for their cytotoxicity. Additionally, this work emphasizes the importance of ligand characteristics and of speciation in activity of metal complexes