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 cap‘n’collar (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 µМ–100 µМ 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 µМ. 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 µМ–10 µМ for 24 hours and 10 µМ for 72 hours, the activity of SOD increased. At concentrations of Н₂О₂ of 50 µМ and 100 µМ (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.

Keywords

nuclear factor E2-related factor 2 Nrf2 / superoxide dismutase / oxidative stress / Caco-2 line cells

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Abalenikhina Y.V., Erokhina P.D., Seidkuliyeva A.A., Zav’yalova O.A., Shchul’kin A.V., Yakusheva E.N.