The effect of S-Nitrosoglutathione on the amount and activity of erythroid nuclear factor Nrf2 in human hepatocellular carcinoma cells

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Abstract

S-nitrosoglutathione (GSNO) is an endogenous donor of nitric oxide (NO), which, at the same time, can act both as a signaling molecule and a toxic agent, forming active forms of nitrogen. The purpose of this work was to study the mechanism of NO participation in the regulation of erythroid nuclear factor 2 (Nrf2) functioning, which is a redox-sensitive transcription factor. It was shown that when GSNO was exposed to human hepatocellular carcinoma cells (HepG2), the level of intracellular NO increased dose-dependently during incubation for 24 and 72 hours. The maximum increase of NO level at 100 mM concentration led to decrease of the amount of non-protein SH groups, to maximum increase of 3-nitrothyrosine and bityrosine levels, which contributed to the decline of cell viability. The NO donor — S-nitrosoglutation activated Nrf2 during exposure for 24 hours, most likely due to nitrosylation of Keap1 protein, and at 72 hours not only activated Nrf2, but also led to an increase in its amount. This process was carried out through NO-cGMP signaling pathway. Activation of Nrf2 is a key factor in protecting cells from the toxic effects of nitrosative stress products.

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About the authors

Yu. V. Abalenikhina

Ryazan State Medical University named after Academician I.P. Pavlov

Author for correspondence.
Email: abalenihina88@mail.ru
Russian Federation, Ryazan, 390026

O. N. Suchkova

Ryazan State Medical University named after Academician I.P. Pavlov

Email: abalenihina88@mail.ru
Russian Federation, Ryazan, 390026

E. V. Kostyukova

Ryazan State Medical University named after Academician I.P. Pavlov

Email: abalenihina88@mail.ru
Russian Federation, Ryazan, 390026

A. V. Shchulkin

Ryazan State Medical University named after Academician I.P. Pavlov

Email: abalenihina88@mail.ru
Russian Federation, Ryazan, 390026

A. F. Topunov

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Email: abalenihina88@mail.ru
Russian Federation, Moscow, 119071

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Supplementary files

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2. Fig. 1. Fluorescence intensity (U. fl.) in HepG2 cell lysate after exposure to GSNO for 24 h (a) and 72 h (b) when stained with DAF-FM.

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3. Fig. 2. Changes in NO levels in HepG2 cells exposed to GSNO for 24 h (a) and 72 h (b). Cells were stained with DAF-FM, nuclei were stained with DAPI. Cell visualization was performed using an Olympus CKX-53 inverted microscope (“Olympus”, Japan), magnification ×400.

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4. Fig. 3. Concentration of non-protein SH-groups (μmol/mg protein) inside HepG2 cells after exposure to GSNO for 24 (a) and 72 h (b).

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5. Fig. 4. The effect of GSNO for 24 h (a, c) and 72 h (b, d) on the relative amount of 3-nitrotyrosine in protein molecules of HepG2 cells: a, b — blotting results; c, d — densitometric analysis results.

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6. Fig. 5. Dityrosine concentration (nmol/mg protein) in HepG2 cells exposed to GSNO for 24 h (a) and 72 h (b).

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7. Fig. 6. Change in viability (%) of HepG2 cells exposed to GSNO for 24 h (a) and 72 h (b).

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8. Fig. 7. The effect of GSNO for 24 h and 72 h on the relative amount of Nrf2 in the nucleus (a, c) and cytoplasm (b, d) in HepG2 cells: a, b — blotting results; c, d — densitometric analysis results.

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9. Fig. 8. Glutathione transferase activity (μmol*CDNB/mg protein) in HepG2 cells exposed to GSNO and in combination with inhibitors of sGC (ODQ) and antioxidant response element (AEM1) for 24 h (a) and 72 h (b).

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10. Fig. 9. Viability of HepG2 cells after exposure to GSNO together with antioxidant response element inhibitor (AEM1) for 24 h (a) and 72 h (b).

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11. Fig. 10. The mechanism of activation of Nrf2 and glutathione transferase in HepG2 cells under the influence of GSNO.

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