Intracellular Location and Function of Nuclear Factor of Erythroid Origin 2 (Nrf2) in Modeling Oxidative Stress in vitro

Abstract

INTRODUCTION: Nuclear factor E2-related factor 2 (Nrf2) is a member of capncollar (CNC) family of subfamily of leucine zipper transcription factors that regulates cell protection against toxic substances and oxidants. AIM: To determine location, mechanism of activation and role of Nrf2 in conditions of oxidative stress in vitro. MATERIALS AND METHODS: The study was performed on human colon adenocarcinoma cell line (Caco-2). Oxidative stress (OS) was modeled by adding hydrogen peroxide (Н2О2) at concentrations of 0.1 М100 М to the nutritive medium and incubation for 24 and 72 hours. In assessment of Nrf2 function, its inhibitor ― AEM1 ― was added to cells at a concentration of 5 М. The extent of OS development was determined using photometric methods by the concentration of protein SH-groups and carbonyl derivatives of protein, and the activity of superoxide dismutase (SOD). Viability of cells was assessed by the results of cytotoxic test (MTT assay), the amount of Nrf2 in the cytoplasm and nucleus was determined by heterogenous ELISA method. RESULTS: Incubation of Caco-2 cells with Н2О2 resulted in decrease in the level of protein SH-groups and increase in the concentration of carbonyl derivatives of protein. In incubation with H2O2 at concentrations of 0.1 М10 М for 24 hours and 10 М for 72 hours, the activity of SOD increased. At concentrations of Н2О2 of 50 М and 100 М (24 hour and 72 hour), SOD activity and viability of cells decreased. Exposure to Н2О2 led to translocation of Nrf2 from the cytoplasm into nucleus. Direct correlation dependence was revealed between concentration of protein SH-groups and the amount of Nrf2 in the cytoplasm in incubation with H2O2 for 24 hour (r = 0.44, р = 0.03), 72 hour (r = 0.34, р = 0.05). The amount of Nrf2 in the nucleus positively correlated with SOD activity in the cytoplasm on exposure to H2O2 for 24 hour (r = 0.77, р = 0.0001) and 72 hour (r = 0.36, р = 0.06). In inhibition of Nrf2 in conditions of exposure to H2O2, the viability of cells decreased to a larger extent. CONCLUSION: Hydrogen peroxide induces the nuclear translocation of Nrf2, which promotes activation of antioxidant enzyme SOD and preserves viability of cells of OS conditions in vitro.