Under certain conditions, both activators and inhibitors of Nrf2-dependent signaling and autophagy can serve as potential agents for the prevention, treatment, and maintenance therapy of cancer, as well as overcoming chemoresistance. We are developing a panel of structurally related synthetic monophenolic compounds, in addition to a direct antioxidant effect, having the ability to affect intracellular signaling pathways and processes, including the above mentioned. Aim of the study was to investigate the relationship between its structure and the ability to influence the expression of autophagy and Nrf2 pathway genes.Material and methods. Five original hydrophilic phenolic structurally related compounds were synthesized; the prototypical inductor of the Keap1/Nrf2/ARE system, monosubstituted phenol tert-butylhydro-quinone (tBHQ), served as a reference substance. We used cultures of murine Lewis lung carcinoma (LLC) and human breast adenocarcinoma MCF-7 cells, with the test compounds at a concentration of 20 gM cells were incubated for 24 hours. Changes in mRNA expression of Nrf2, Nqo1, LC3b, and Sqstm1 genes encoding Nrf2, NAD(P)H:quinone oxidoreductase-1, LC3B, p62/sequestosome-1, respectively, were determined by TaqMan real-time PCR.Results and discussion. Structurally related synthetic monophenols effectively induced the Keap1/Nrf2/ARE system in LLC and MCF-7 cells, increasing the expression of the Nrf2-driven Nqo1 gene, and ambiguously affected the transcription of genes of proteins mediating autophagy: the content of mRNA of the LC3B protein did not change, while the expression of mRNA of the p62 protein, which affects the operation of both systems (processes), is increased only by the action of ortho-monosubstituted phenol with an «active» sulfur atom in the para-propyl substituent TS-13 and its structural analogue TS-12 with a para-substituent shortened by one methylene. It can be assumed that the observed effect either indicates the ability of TS-13 and TS-12 both to induce the Keap1/Nrf2/ARE system and to stimulate autophagy, or reflects a compound-mediated mutually reinforcing cross-activation of these processes.

The redox‐sensitive signaling system Keap1/Nrf2/ARE is a premier protective mechanism against oxidative stress that plays a key role in the pathogenesis and development of various diseases, including tuberculous granulomatous inflammation. We have previously reported that novel water‐soluble phenolic antioxidant TS‐13 (sodium 3‐(4 ′ ‐methoxyphenyl)propyl thiosulfonate) induces Keap1/Nrf2/ARE and attenuates inflammation. The aim of this study is the examination of the effect of TS‐13 on tuberculous granulomatous inflammation. BALB/c mice were administered TS‐13 (100 mg kg -1 day -1 ) through their drinking water starting immediately after Bacillus Calmette‐Guérin (BCG) intravenous injection. Histological changes, production of reactive oxygen species (ROS) (activity of free‐radical oxidation processes), and mRNA expression of Nrf2‐driven, NF‐ κ B‐, AP‐1‐, and autophagy‐dependent signal pathway genes in the liver and peritoneal exudate were evaluated 30 days later. After the 30th day of infection, the activity of the Keap1/Nrf2/ARE system was decreased and its effector genes entailed increasing ROS production in the liver. Therapeutic intervention with TS‐13 is aimed at activating the Keap1/Nrf2/ARE system that leads to an increase in Nrf2 and Nrf2‐mediated gene expression and a decrease in NF‐ κ B expression. Changes in these pathways resulted in a decline of ROS production and a decrease in the number and the size of granulomas. In total, the results indicate that the Keap1/Nrf2/ARE system can be an effective pharmacological target in host‐adjunctive treatment of tuberculosis.

Выдвинутая более 50 лет назад Д. Харманом свободнорадикальная теория старения остается популярной и сегодня. В обзоре проведен анализ возрастных изменений основных эндогенных механизмов продукции активированных кислородных метаболитов (АКМ) и механизмов антиоксидантной защиты. С возрастом генерация АКМ митохондриями, пероксисомами и NAD(P) H -оксидазами усиливается, в то время как транскрипционная активность важной системы поддержания редокс-баланса Keap 1/ Nrf 2/ ARE уменьшается. У старых животных отмечается также низкая активность аутофагии, удаляющей из клеток поврежденные органеллы и агрегированные структуры. Возрастное смещение редокс-баланса в сторону окислительного стресса может являться причиной развития возраст-ассоциированных нейрогеденеративных, аутоиммунных и воспалительных патологий. The free-radical theory of aging, advanced more than 50 years ago by D. Harman, remains popular today. The review analyzes age-related changes in the main endogenous mechanisms of reactive oxygen species (ROS) production and antioxidant defense mechanisms. With age, ROS generation by mitochondria, peroxisomes, and NAD(P)H oxidases is enhanced, while the transcriptional activity of the important system Keap 1/ Nrf 2/ ARE maintaining redox balance decreases. In old animals, autophagy activity is also low, which removes damaged organelles and aggregated structures from cells. The age-related shift of the redox balance towards oxidative stress can cause the development of age-associated neurodegenerative, autoimmune and infl ammatory pathologies.

Pharmacological modification of the redox properties of tumor cells is a promising approach to enhance the efficiency of antitumor therapy. Currently, the transcription factor Nrf2 is considered as a new target for the development of selective chemosensitizers. Nrf2 plays a key role in regulation of cellular redox homeostasis against stress and during adaptation processes. Many natural and synthetic phenolic antioxidants are inducers of Nrf2 transcriptional activity. Due to differences in Nrf2 transcriptional activity between normal and tumor cells, phenolic antioxidants at certain concentrations act as biological regulators the antioxidant activity of which has two different effects: in tumor cells they promote the development of oxidative stress and enhance the effect of antitumor drugs, in normal cells these antioxidants exhibit protective properties. The review discusses the possible molecular mechanisms of action and the prospects for the clinical use of natural and synthetic phenolic antioxidants in antitumor therapy.

Understanding the role of reactive oxygen and nitrogen species in eustress (redox balance) and distress (oxidative stress) development poses new challenges for biomedical scientists and pharmacologists in the search for compounds that can not only have a direct antioxidant (antiradical) effect, but also affect redox-sensitive signaling pathways, primarily Keap1/Nrf2/ARE system. Aim of the study was to investigate the influence of novel water-soluble structurally related monophenols on key elements of Keap1/Nrf2/ARE system induction (activity of Nrf2-driven enzymes, the state of the glutathione system, and intracellular redistribution of transcription factor Nrf2).Material and methods. Five original hydrophilic structurally related monophenols, differing in the number of tert-butyl ortho-substituents, the length of the para-alkyl substituent, and the presence of a divalent sulfur or selenium atom in it were investigated (phenoxane, the potassium salt of phenosan acid, was used as a reference compound). Cell lines U937 and J774 were cultured for 24 h in the presence of tested compounds, and comparative analysis was performed of its ability to induce the synthesis of Nrf2-driven enzymes of phase II xenobiotic detoxification pathway and antioxidant enzymes (NAD(P)H: quinone oxidoreductase 1 (NQO1), glutathione S-transferases (GST), glutathione peroxidases, glutathione reductase (biochemical spectrophotometric methods were used to study their activity), as well as to influence the state of glutathione system (spectrophotometry) and translocation of transcription factor Nrf2 into the nucleus (immunofluorescent staining, confocal microscopy) (key events of Keap1/Nrf2/ARE signaling system activation).Results and discussion. Monophenol TS-13 have found to be the most effective inducer of tested enzymes in U937 cells among the structural analogs, while the structure of the para-alkyl substituent and the degree of OH group hindrance are important for the implementation of this effect; TS-13 also effectively enhanced Nrf2 import into J774 cell nucleus. The NQO1- and GST-inducing abilities of structurally related monophenols are closely interrelated, which indicates the possibility of coordinated induction of these enzymes and the presence of a common regulatory system that ensures their activation in response to cell treatment with phenolic antioxidants.

Pharmacological modification of the redox properties of tumor cells is a promising approach to enhance the efficiency of antitumor therapy. The Nrf2 transcription factor is considered as a new target for the development of selective chemosensitizers. Nrf2 plays a key role in the regulation of cellular redox homeostasis against stress and during adaptation processes. Many natural and synthetic phenolic antioxidants are inducers of Nrf2 transcriptional activity. Because of differences in Nrf2 transcriptional activity between normal and tumor cells, phenolic antioxidants at certain concentrations act as biological regulators of the antioxidant activity, which has two different effects. They promote the development of oxidative stress and enhance the effect of antitumor drugs in tumor cells and exhibit protective properties in the normal cells. This review discusses possible molecular mechanisms of the action and the prospects for clinical use of natural and synthetic phenolic antioxidants in antitumor therapy.

The aim of the study was to study investigate the relationship between the antioxidant activity of new synthetic structurally related water-soluble monophenols and their effect on the tumor cell viability in vitro. Material and methods. Five original hydrophilic sulfur- and selenium-containing monophenols with varying length of the hydrocarbon chain of the para -alkylthiosulfonate substituent, the amount of tert -butyl ortho -substituents and the «S-S» fragment structure are synthesized: sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)ethyl thiosulfonate (TS-12), sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)propyl sulfonate (S-13), sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)propyl seleniumsulfonate (SeS-13), sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)propyl thiosulfonate (TS-13), sodium 3-(3',5'-di- tert -butyl-4’-hydroxyphenyl)propyl thiosulfonate (TS-17). The antioxidant activity of the compounds was determined in a cell-free model test system by the ability to inhibit the luminol-dependent chemiluminescence of free radical-generating azo compound AAPH. Antitumor cytotoxicity was evaluated by their effect on the viability of human histiocytic lymphoma cell line U937 (monocyte/macrophage-like cells) and human breast adenocarcinoma cell line MCF-7 in the MTT test. Results and discussion. All tested phenolic compounds exerted antioxidant activity in the cell-free model system (inhibition of azo initiator radical AAPH • ) and cytotoxicity against U937 and MCF-7 cells, the effect depended on the dose and structure of the molecule. There was a direct relationship between the structure of monophenols and their ability to inhibit the viability of tumor cells of different lines, regardless of the origin of the latter, myeloid (U937) or epithelial (MCF-7), and growth type (respectively anchorage-independent and attached), as well as the concentration range of compounds (respectively from 100 to 500 μM and from 2 to 150 μM). At the same time, the relationship between the antioxidant activity of monophenols and their cytotoxicity was inverse (when SeS-13 was excluded from analysis), which may be related both to the ability of the tumor to self-defense against reactivate oxygen metabolites and to the indirect pro-oxidant effect of phenolic compounds.

Autophagy is the main catabolic process required for the removal of damaged organelles, aggregated proteins and intracellular pathogens from cells. Oxidative stress is accompanied by an increase in autophagy, which has a protective effect by maintaining the qualitative composition of mitochondria (mitophagy) and peroxisomes (pexophagy) followed by lysosomal degradation of organelles with high production of reactive oxygen species. Aggrephagy also removes toxic products formed during oxidative and carbonyl stress. Furthermore, autophagy can activate the antioxidant response element system and increase the expression of antioxidant enzyme genes. The protective role of autophagy can be useful in many pathological processes accompanied by the development of oxidative stress while at the same time it may cause chemoresistance, reducing the effectiveness of anti-tumor therapy.

Endogenous mechanisms of reactive oxygen (ROS) and nitrogen species production and of antioxidant defense systems in tumor cells are analyzed. Increased ROS production is an important regulator of metabolic changes in these cells: enhanced proliferation, apoptosis inhibition, resistance to hypoxia and to cytostatics (doxorubicin, carboplatin, cisplatin, etc.). The most active ROS sources in tumor cells are mitochondria, NAD(P)H oxidases and peroxisomes, which synthesize O2 • – and H2O2. In mitochondria, the superoxide anion radical is generated mainly by complexes I and III; membrane NAD(P)H oxidases Nox1, Nox2, Nox3, and Nox5 produce O2 • –, Nox4, and dual oxidases DUOX-1, DUOX-2 – mainly H2O2. Increasing ROS stationary concentration activates endogenous antioxidant defense mechanisms, such as redox-dependent antioxidant respons(iv)e element system Keap1/Nrf2/ARE and autophagy, which allows tumor cells to survive under oxidative stress and may underlie resistance to radio- and chemotherapy. The possibilities of tumor cell redox balance regulation by antioxidants with targeted action and by specific inhibitors of ROS enzymatic production are discussed.

The aim of the study was to determine the dose of a virulent strain of Mycobacterium tuberculosis H37Rv that is optimal for modeling experimental tuberculosis granulomatosis in mice and to investigate the effect of the original inductor of the Keap1/Nrf2/ARE system TS-13 (sodium 3- (3'- tert -butyl-4'-hydroxyphenyl) propylthiosulfonate) on animal survival and the dynamics of granuloma formation. Material and methods. Generalized tuberculosis granulomatosis was modeled by a single injection into the tail vein of male BALB/c mice of the 2-month-old M. tuberculosis strain H37Rv at doses of 106, 107 and 108 microbial bodies. Another group of animals on the day of infection with M. tuberculosis (107 microbial bodies) began to receive TS-13 with drinking water (100 mg/kg body weight). Survival was fixed daily; after 5 weeks, mice were euthanized and liver samples were taken for histological examination. Results and discussion. The dose of 107 microbial bodies was found to be the most adequate when modeling in BALB/c mice the tuberculosis granulomatosis caused by the intravenous injection of virulent M. tuberculosis strain H37Rv. At the 36th day after the injection of 107 microbial bodies, mortality was significantly lower in the group of mice receiving the inducer of the signal system Keap1/Nrf2/ARE monophenol TS-13 with drinking water (44 and 15% mice survived, respectively). At the same time, these two groups did not differ in the number and diameter of liver granulomas. The results show a high prospect of studying the role of oxidative stress and the redox-sensitive signal system Keap1/Nrf2/ARE in tuberculosis granulomatosis.

Endogenous mechanisms of reactive oxygen (ROS) and nitrogen species production and of antioxidant defense systems in tumor cells are analyzed. Increased ROS production is an important regulator of metabolic changes in these cells: enhanced proliferation, apoptosis inhibition, resistance to hypoxia and to cytostatics (doxorubicin, carboplatin, cisplatin, etc.). The most active ROS sources in tumor cells are mitochondria, NAD(P)H oxidases and peroxisomes, which synthesize O2 • – and H2O2. In mitochondria, the superoxide anion radical is generated mainly by complexes I and III; membrane NAD(P)H oxidases Nox1, Nox2, Nox3, and Nox5 produce O2 • –, Nox4, and dual oxidases DUOX-1, DUOX-2 – mainly H2O2. Increasing ROS stationary concentration activates endogenous antioxidant defense mechanisms, such as redox-dependent antioxidant respons(iv)e element system Keap1/Nrf2/ARE and autophagy, which allows tumor cells to survive under oxidative stress and may underlie resistance to radio- and chemotherapy. The possibilities of tumor cell redox balance regulation by antioxidants with targeted action and by specific inhibitors of ROS enzymatic production are discussed.

The effect of novel water-soluble structurally related monophenolic compounds on the activity of two most important mechanisms of maintaining intracellular homeostasis, autophagy and the redox-sensitive signal system Keap1/Nrf2/ARE, has been studied in human breast adenocarcinoma cell line MCF-7 using confocal microscopy. Autophagy processes were analyzed on the basis of the amount of intracellular vesicles that were positive for the autophagy marker (LC3B). The activation of the Keap1/Nrf2/ARE system was determined by the translocation of the transcription factor Nrf2 into the nucleus. It was found that the effect of the tested compounds depended on their structure and concentration. When the inhibitor of autophagosome–lysosome fusion chloroquine was added to the culture medium (20 μM), the asymmetrically hindered by the tert-butyl group phenols with thiosulfonate (TS-13) and sulfonate group in the para-propyl substituent increased the rate of autophagosome elimination in MCF-7 cells. Shortening of the para-alkyl substituent by one methylene unit abolished the effect. The addition of the second ortho-tert-butyl substituent had the reverse result. Both tested compounds enhanced the translocation of the transcription factor Nrf2 into the nucleus of MCF-7 cells (which is a critical step in Keap1/Nrf2/ARE activation). It was observed after incubation with asymmetrically hindered by the tert-butyl group phenol with selenosulfonate group in para-propyl substituent (5−100 μM) for 4 h and with TS-13 (5−100 μM) for 24 h. Taking into account our previous findings on the toxicity of this group of compounds for MCF-7 cells we can conclude that these compounds exert different effect on autophagy and activation of the antioxidant response element signaling system Keap1/Nrf2/ARE.

ROS are important intracellular messengers; their ambiguous role in malignant processes was demonstrated in many studies. The effects of a synthetic phenolic antioxidant sodium 3-(3’-tert-butyl-4’-hydroxyphenyl)propyl thiosulfonate sodium (TS-13) on the tumor growth and oncolytic properties of doxorubicin were studied in the experimental model of Lewis lung carcinoma in mice. In mice receiving TS-13 with drinking water (100 mg/kg), suppression of tumor growth by 32.3% was observed on day 21 after inoculation of Lewis lung carcinoma cells. Two-fold intraperitoneal injections of doxorubicin in a cumulative dose of 8 mg/kg were followed by inhibition of tumor growth by 49.5%. Combined treatment with TS-13 and doxorubicin suppressed the tumor growth by 55.4%. In contrast to doxorubicin, TS-13 inhibited NO generation by peritoneal macrophages. The results show the prospect of studying TS-13 in the context of overcoming drug-resistance of tumors.

The effect of ascorbate in physiological concentrations on the proliferative activity and chemoresistance in human larynx carcinoma HEp-2 cells was studied. Ascorbate in a concentration of 60 mu M was found to increase the cancer cells proliferation rate 1.5 times. Ascorbate changes the functional state of the cancer cells, thereby increasing their resistance to doxorubicin and thymoquinone. It was shown that apocynin (NADPH oxidase inhibitor) blocks the stimulating effect of the antioxidant. The results obtained suggest that reactive oxygen species produced by NADPH oxidase participate in the mechanism of cell adaptive response induced by ascorbate.

Reactive oxygen and nitrogen species are important intracellular messengers that are strongly implicated in tumor processes, yet their precise role in cancerogenesis is still largely unclear and often debatable. Plant and synthetic phenols and polyphenols are known to have antitumor effect through multiple mechanisms most importantly the one that activates Keap1/Nrf2/ARE pathway. We have previously demonstrated that water-soluble synthetic phenolic antioxidant sodium 3-(3′-tert-butyl-4′-hydroxyphenyl) propyl thiosulfonate (TS-13) increased cell susceptibility to doxorubicin in cell culture. Antitumor action of TS-13 was attributed to its effect on mitochondria followed by Keap1/Nrf2/ARE activation and subsequent autophagy and apoptosis. The effect of TS-13 on tumor growth and the oncolytic effect of doxorubicin was studied in experimental model of transplantable tumor (Lewis lung carcinoma, LLC) in mice. On day 21 after inoculation of LLC cells, tumor growth was inhibited by 32.3% in mice that received TS-13 with drinking water (100 mg/kg). Two intraperitoneal injections of doxorubicin in a cumulative dose of 8 mg/kg body weight reduced tumor growth by 49.5%, while co-administration of TS-13 and doxorubicin resulted in tumor growth inhibition by 55.4%. Animals with inoculated tumor (+ or - doxorubicin) had significantly higher rate (by 27 and 39%, respectively, p < 0.05) of nitric oxide production by their peritoneal macrophages compared to intact controls. However, the ability of mouse macrophages in LLC + TS-13 group to produce nitric oxide was reduced to the control levels. Animals that received TS-13 demonstrated significant oncostatic effect as histologically revealed by a smaller zones of necrosis, edema and hemorrhage in the tumor nodes as well as less cellular polymorphism and the lack of inflammatory infiltration in underlying muscle tissue compared to the LCC and LCC+ doxorubicin animals. These data provide a basis for exploring TS-13 as a potential pharmaceutical for overcoming tumor chemoresistance.

The present research is devoted to the study of the relationship between the structure of the original synthetic monophenolic antioxidants and their ability to influence the activity of autophagy in tumor cells.

Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson’s disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucleus, probably due to its capacity to induce EPO synthesis.