Generation of hydrogen peroxide-resistant murine neuroblastoma cells: a target discovery platform for novel neuroprotective genes

Abstract

Oxidative stress has been suggested to play an important role in the pathogenesis of various neurodegenerative diseases including Alzheimer's disease (AD). Hydrogen peroxide (H2O2), one of the main reactive oxygen species, is converted into the highly toxic ·OH radical in the presence of redox-active transition metals, which then oxidises nucleic acids, lipids and proteins, leading to neurodegeneration and cell death. There is an urgent need to gain more knowledge about relevant therapeutic targets to combat oxidative stress and it neurotoxic effects, and how this knowledge can be utilized to develop novel neuroprotective therapies for AD. One way to identify new mechanisms combating oxidative stress was via the creation of H2O2-resistant cell lines and identification of the mechanisms responsible for their resistance. However, in most cases catalase overexpression or increased glutathione content was identified as the primary mode of H2O2 resistance in these cell lines. In this study, we have generated six different resistant neuronal cell lines or populations (from the same original murine Neuro2a neuroblastoma line) by exposing cells to increasing concentrations of H2O2 and performing continuous selection for survivors over a period of several months, which appear to have acquired H2O2 resistance based on other, novel mechanisms. These six populations showed a significant, but differential resistance against H2O2 when compared with the parental cell line. Using combinations of catalase-, glutathione synthesis-and glutathione peroxidase-inhibitors it was shown that the increased resistance of Neuro2a-HR cells is not solely based on an increased activity of catalase or the glutathione system, suggesting that their resistance might be based on yet unknown, novel defence mechanisms.

Benzothiazole Aniline Tetra(ethylene glycol) and 3-Amino-1,2,4-triazole Inhibit Neuroprotection against Amyloid Peptides by Catalase Overexpression in Vitro

“…To confirm that the resistance to H 2 O 2 toxicity was due to the catalase activity, and not other endogenous peroxidases, the PVect and PCat SH-SY5Y neuronal cells were treated with 500 μM H 2 O 2 plus a range of concentrations of 3-AT (0−50 mM). 32 Results showed that 3-AT significantly reduced the H 2 O 2 toxicity ( Figure 2B).…”

The Effects of Hydrogen Peroxide and/or Radiation on the Survival of Clinically Relevant Radioresistant Cells

“…Several distinct cell types, such as murine lens, monkey kidney, and hamster ovarian cells, have been found to acquire H 2 O 2 resistance after conditioning with H 2 O 2 . 12 -15 In order to determine whether H 2 O 2 -resistant cells were also resistant to X-rays, we generated H 2 O 2 -resistant cells from HepG2, SAS, and HeLa cells. As shown in Figure 2, we successfully obtained H 2 O 2 -resistant cells, which are referred to by their cell line name followed by H 2 O 2 to indicate their H 2 O 2 resistance.…”

Identification of Nrf2 Activators from the Roots of Valeriana officinalis

“…HepG2 is a human hepatoma cell line that is commonly used in drug metabolism and hepatotoxicity studies. HepG2 cells are nontumorigenic cells with high proliferation rates and an epithelial-like morphology that per- forms many differentiated hepatic functions, including Nrf2-induced expression of detoxification and cytoprotection genes [18].…”

“…H 2 O 2 has been described as an extracellular and intracellular signaling molecule but is also cytotoxic at high concentrations [24]. Cells are protected from H 2 O 2 by two detoxifying enzymes, catalase and GPX [18]. We hypothesized that an increase in GSH concentration (induced by the isolated Nrf2 activators) might increase GPX activity, and therefore protect cells from H 2 O 2 -mediated toxicity.…”

“…Unauthorized distribution is strictly prohibited. forms many differentiated hepatic functions, including Nrf2-induced expression of detoxification and cytoprotection genes [18].…”