N. K. Zenkov

Mechanism of the Nrf2/Keap1/ARE signaling system.

Nrf2 regulates expression of genes containing antioxidant-respons(iv)e element (ARE) in their promoters and plays a pivotal role among all redox-sensitive transcription factors. Nrf2 is constitutively controlled by repressor protein Keap1, which acts as a molecular sensor of disturbances in cellular homeostasis. These molecular patterns are in close inter-connection and function as parts of the integrated redox-sensitive signaling system Nrf2/Keap1/ARE. Depending on cellular redox balance, activity of this signaling system changes at the levels of transcription, translation, posttranslational modification, nuclear translocation of transcription factor, and its binding to ARE-driven gene promoters. This review summarizes current conceptions of Nrf2/Keap1/ARE induction and inactivation.

Keap1/Nrf2/ARE redox-sensitive signaling system as a pharmacological target

The redox-sensitive signaling system Keap1/Nrf2/ARE plays a key role in maintenance of cellular homeostasis under stress, inflammatory, carcinogenic, and proapoptotic conditions, which allows us to consider it as a pharmacological target. Here we review the basic regulatory mechanisms of the Keap1/Nrf2/ARE system, key targets for pharmacological intervention, and interconnection of this system with other redox-sensitive transcriptional factors. We also discuss the range of currently available pharmaceuticals. Finally, we promote “indirect” antioxidants as a promising strategy for prevention and treatment of wide range of diseases associated with oxidative stress.

Plant Phenols and Autophagy.

Many plant phenols (stilbenes, curcumins, catechins, flavonoids, etc.) are effective antioxidants and protect cells during oxidative stress. Extensive clinical studies on the potential of phenolic compounds for treatment of cardiovascular, neurodegenerative, oncological, and inflammatory diseases are now being conducted. In addition to direct antioxidant effect, plant phenols may provide a protective effect via activation of the Keap1/Nrf2/ARE redox-sensitive signaling system and regulation of autophagy. In this review, mechanisms of effects of the most common plant phenols on autophagy are presented.

Mazes of Nrf2 regulation

Nrf2 transcription factor plays a key role in maintaining cellular redox balance under stress and is a perspective target for oxidative stress-associated diseases. Under normal conditions, Nrf2 transcriptional activity is low due to its rapid ubiquitination and degradation in the 26S proteasome, as well as through various modifications of amino acid residues of this transcription factor that regulate its transport to the nucleus and binding to DNA. Continuous activation of Nrf2 is possible due to autophagy and epigenetic regulation that may underlie the increased resistance of tumor cells to radiotherapy and chemotherapy. This review deals with the mechanisms of regulation of Nrf2 transcriptional activity and its main elements, and pharmacological approaches to activation of the Keap1/Nrf2/ARE system.

Phenolic antioxidant TS-13 regulating ARE-driven genes induces tumor cell death by a mitochondria-dependent pathway

Effects of water-soluble phenolic antioxidant sodium 3-(3′-tert-butyl-4′-hydroxyphenyl)-propyl thiosulfonate (TS-13), potassium 3,5-dimethyl-4-hydroxybenzyl thioethanoate (BEP-11-K), and potassium 3-(3′,5′-di-tert-butyl-4′-hydroxyphenyl)-propionate (potassium phenosan) on the proliferative activity of tumor cells and the role of redox-dependent and calcium-dependent signaling mechanisms in realization of tumor cell response to antioxidants were studied. Potassium phenosan and BEP-11-K were found to stimulate proliferation, whereas the ARE-inducing phenolic antioxidant TS-13 inhibited tumor cell growth in culture. The rate of tumor cell growth depended on the rate of intracellular reactive oxygen species production and was suppressed by apocynin (a NADPH oxidase inhibitor) and antimycin A (an ubiquinol-cytochrome c oxidoreductase inhibitor). The action of TS-13 on tumor cells was accompanied by a transient increase in the intracellular production of reactive oxygen species and the intracellular calcium concentration, whereas cell incubation with potassium phenosan and BEP-11-K did not influence the level of reactive oxygen species and intracellular calcium ions. Cyclosporin A blocked the inhibitory effect of TS-13. Thus, it can be reasonably speculated that phenolic antioxidant TS-13 triggers mitochondria-dependent apoptosis in tumor cells by opening mitochondrial permeability transition pores.

Anti-Infl ammatory Activity of TS-13, ARE-Inducing Phenol Antioxidant

The protective effect of water-soluble TS-13 monophenol inducing the antioxidant-responsive element (ARE) system was studied on the models of acute inflammation. Intragastric administration of TS-13 to rats significantly reduced the severity of acute aseptic inflammation induced by intravenous injection of zymosan particles: granulocyte blood count and volume density of infiltrates in the liver decreased on day 3, spontaneous production of activated oxygen metabolites and respiratory burst in blood granulocytes decreased on days 2 and 3. A single dose of TS-13 improved survival of mice with endotoxin shock induced by intraperitoneal injection of E. coli LPS. These results confirmed high anti-inflammatory activity of TS-13.

Protective Effect of ARE-Inducing Phenol Antioxidant TS-13 in Chronic Inflammation

The protective effect of partially substituted monophenol TS-13 inducing the Nrf2/Keap1/ARE signaling system was studied on the model of chronic inflammation in vivo. It was found that during simulation of inflammation in an air pouch lined with synovial-like membrane, ТS-13 did not affect the exudate volume, protein content, and cell count, but significantly reduced the intensity of oxidative metabolism in leukocytes of the exudate. In rheumatoid polyarthritis induced by heterologous collagen, TS-13 reduced the severity of clinical signs of inflammation only at the early stages, but inhibited H2O2 generation by monocytes and, partially, by blood neutrophils. These results suggest that the phlogolytic effect of the redox sensitive Nrf2/Keap1/ARE signaling system is less pronounced in chronic immune-mediated inflammatory processes than in acute inflammation.

Effect of phenol inducing antioxidant responsive element on D. melanogaster lifespan

The effect of hydrophilic synthetic antioxidant TS-13 (sodium 3-(3′-tert-butyl-4′-hydroxyphenyl)propyl thiosulfonate) on the lifespan of different lines of Drosophila melanogaster under normal conditions and the survival in the oxidative stress induced by hydrogen peroxide and paraquat were studied. The addition of 1% TS-13 to diets extended the lifespan of males and females of the long-living Canton S line of D. melanogaster, had no effect on the lifespan of short-living Oregon R line and shortened the average lifespan of males of the lgl558OR/Cy line of D. melanogaster containing a heterozygous recessive lethal mutation of tumor suppressor. Under the conditions of oxidative stress induced by hydrogen peroxide, TS-13 increased the survival of Canton S males and Oregon R females; under the action of paraquat, the protective effect of antioxidant was manifested toward Canton S females and Oregon R flies of both sexes. Despite the fact that anti-aging and protective properties of synthetic phenolic antioxidant TS-13 strongly depend on the genotype and gender, under extreme conditions of oxidative stress. its positive effect is pronounced.

Effect of Phenol Inducing the Antioxidant Responsive Element on Drosophila Melanogaster Lifespan

The effect of hydrophilic synthetic antioxidant TC-13 (3-(3’-tert-butyl-4’-hydroxyphenyl) propylthiosulfonate sodium) inducing the antioxidant-responsive element on the lifespan of Drosophila melanogaster was studied. Addition of 1% TC-13 to diets prolonged the lifespan of long-lived D. melanogaster Canton S strain females and males, but not of short-lived Oregon R insects and reduced the mean lifespan of D. melanogaster males of the lgl558OR/Cy strain containing a recessive lethal mutation of tumor suppressor in the heterozygotic state. The geroprotective effects of TC-13 synthetic phenol antioxidant depended on D. melanogaster genotype and gender.

Synthetic water-soluble phenolic antioxidant regulates L-arginine metabolism in macrophages: A possible role of Nrf2/ARE

Synthetic water-soluble phenolic antioxidant TS-13 exhibits pronounced anti-inflammatory properties in vivo and induces intracellular signal system Nrf2/ARE. At concentrations 150–1000 μM it inhibits nitric oxide (NO) production in mouse peritoneal macrophages. However, this compound at low concentrations (1–100 μM) paradoxically increases NO production and decreases activity of arginase. These results are indicative of an ambiguous role of NO and its metabolites in the mechanism of development of inflammatory reaction.