This review provides an overview of molecular mechanisms of intracellular signal transduction involving free radicals. The structure and functions of enzymes that can produce superoxide anion-radical and hydrogen peroxide are considered in detail. The mechanisms of regulation of cell properties with the participation of specialized redox chains formed by a group of proteins interacting through electron transport processes are discussed. Genetically mediated mechanisms of regulation of cellular redox homeostasis are analyzed. Particular attention is paid to the issue concerning quantitative characterization of the network of interactions of oxidizing and reducing agents, which determines the species-specific and individual characteristics of redox homeostasis and the stress response of cells.

Fluorescence assay and Raman spectroscopy were used to study the mechanisms of action of 2-isopropyl-5-methyl- 1,4-benzoquinone (thymoquinone) on HEp-2 human larynx carcinoma cells. Thymoquinone was found to have a more pronounced toxic effect than 1,4-benzoquinone and 2,3,5-trimethyl-1,4-benzoquinone. The action of thymoquinone leads to a decrease in the mitochondrial membrane potential and release of cytochrome c from the mitochondria, indicating activation of apoptosis of tumor cells through the mitochondrial-mediated pathway. These results indicate the possibility of using Raman spectroscopy in the study of programmed cell death.

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.

The mechanisms of 2-isopropyl-5-methyl-1,4-benzoquinone (thymoquinone) action on human larynx carcinoma cells of the HEp-2 line were studied using methods of fluorescence assay and Raman spectroscopy. It was found that thymoquinone has a more pronounced toxic effect compared to 1,4-benzoquinone and 2,3,5-trimethyl-1,4-benzoquinone. A decrease of the mitochondrial potential and the release of cytochrome c from the mitochondria are observed under the action of thymoquinone , which indicates the activation of apoptosis of the tumor cells along the mitochondrial-mediated pathway. The obtained results demonstrate the prospects of using Raman spectroscopy in the study of programmed cell death.

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 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.

Effects of water-soluble sulfur-containing phenolic antioxidants sodium 3-(3′-tert-butyl-4′- hydroxyphenyl)propyl thiosulfonate and potassium 3,5-dimethyl-4-hydroxybenzyl thioethanoate on chemoresistance in tumor cells have been studied. The studied phenolic antioxidants cause oppositely directed changes in the redox properties and chemoresistance in tumor cells. Potassium 3,5-dimethyl-4-hydroxybenzyl thioethanoate increases redox buffering capacity and doxorubicin resistance in tumor cells. Sodium 3-(3′- tert-butyl-4′-hydroxyphenyl)propyl thiosulfonate reduces the redox buffering capacity, which leads to a decrease in the chemoresistance of tumor cells. These observations suggest that one of the key mechanisms responsible for the formation of tumor cell resistance to antitumor compounds is the attenuation of apoptosis through increase of redox buffering capacity. The dependence of protein sensor redox state on oxidant concentrations and on redox buffering capacity in cells has been determined based on the proposed biophysical model of redox-dependent mechanism of apoptosis activation.

Mechanisms of tumor-cell responses to 2-isopropyl-5-methyl-1,4-benzoquinone (thymoquinone) and 1,4-benzoquinone were studied using fluorescence and the inhibition assay. It was shown that quinones enhanced the intracellular production of reactive oxygen species, reduced the mitochondrial membrane potential, and induced tumor-cell death through different pathways. It was found that thymoquinone, which induced lower production of reactive oxygen species than 1,4-benzoquinone, was more toxic to tumor cells. It was established that reactive oxygen species produced due to exposure to thymoquinone are involved in redox signaling processes that lead to the formation of mitochondrial permeability transition pores and activation of programmed cell death. These results suggest that the functioning of the established redox signaling mechanism is enabled by the colocalization of mitochondrial oxidoreductases involved in the production of reactive oxygen species and of protein targets of reactive oxygen species involved in the activation of apoptosis.

It is established that thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) and 1,4-benzoquinone regulate the intracellular reactive oxygen species (ROS) production and induce the death of tumor cells by different mechanisms. It is shown that the toxic action of 1,4-benzoquinone is associated with the inhibition of electron transfer in the mitochondrial respiratory chain and with the development of cellular oxidative stress. Thymoquinone initiating a lower level of ROS production in comparison with 1,4-benzoquinone is more toxic to tumor cells. It is established that thymoquinone-induced ROS are involved in the redox signaling processes that lead to the opening of mitochondrial transition pores of high permeability and the activation of the programmed death of cells.

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.

Исследовано влияние водорастворимых фенольных антиоксидантов 3-(3'-трет-бутил-4'-гидро ксифенил)-пропилтиосульфоната натрия (Т C-13), 3,5-диметил-4-гидроксибензилтиоэтаноата калия (БЭК-11-К) и 3-(3 /,5 /-ди-трет-бутил-4 /-гидроксифенил)-пропионата калия (фенозан-калий) на пролиферативную активность опухолевых клеток и роль редокс-зависимых и кальций-зависимых сигнальных механизмов в реализации отклика опухолевых клеток на действие антиоксидантов. Обнаружено, что фенозан-калий и БЭК-11-К стимулировали пролиферацию, а индуктор сигнальной системы Keap1/Nrf2/ARE фенольный антиоксидант ТС-13 ингибировал рост опухолевых клеток в культуре. Скорость роста опухолевых клеток зависела от скорости внутриклеточной продукции активных форм кислорода и снижалась при действии ингибитора НАДФН-оксидазы апоцинина и ингибитора убихинол-цитохром с оксидоредуктазы антимицина А. Действие ТС-13 на опухолевые клетки сопровождалось кратковременным повышением внутриклеточной продукции активных форм кислорода и ростом внутриклеточной концентрации ионов Са 2+, тогда как инкубация клеток с фенозан-калием и БЭК-11-К не приводила к повышению уровня активных форм кислорода и внутриклеточной концентрации ионов Са 2+. Циклоспорин А блокировал ингибирующий эффект ТС-13. Это позволило предположить, что при действии фенольного антиоксиданта ТС-13 в опухолевых клетках через открытие пор высокой проводимости запускается митохондриально-опосредованный апоптоз.

A model for the redox regulation of the functional state of the cell has been constructed on the basis of representation of electron transfer processes by equivalent electric circuits. The mechanism of action of redox-active molecules on biosystems has been discussed in terms of circuit theory. A method for determining the parameters of cellular redox sensors has been proposed. It has been established that the concentration and redox potential of compounds entering the cell are the main regulatory parameters of redox signals for the cell. It has been experimentally shown that the calcium response to hydrogen peroxide in rat C6 glioma cells and human FL amnion cells depends on the redox-buffer capacity of cells.

The parameters of the acid-base state and redox state of erythrocytes have been studied with the use of the fluorescent probes 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and 2',7'-dichlorodihydrofluorescein. It has been found that the value of redox state parameters in erythrocytes depends both on the extracellular and intracellular concentrations of hydrogen ions. It has been shown that hydrogen peroxide induces a decrease in the value of the intracellular pH. The interrelation of cellular homeostasis parameters characterizing the acid-base and redox states of erythrocytes has been theoretically and experimentally substantiated.

Effects of ascorbic acid on calcium homeostasis of human laryngeal carcinoma cells were studied. Intracellular concentration of free calcium and intracellular pH were measured by fluorescent analysis. Ascorbic acid in concentrations of 3–10 mM caused pH drop and sharply increased concentrations of free Ca ions in HEp-2 cells. Intracellular concentration of free Ca ions resulted from Ca ion release from the thapsigargin-sensitive Ca depots.

Mechanisms of regulation of redox homeostasis of biological systems are analyzed in the present work. Methods of quantitative description of redox phenomena in biological systems are discussed. It has been shown that the redox homeostasis parameters are to be considered as the regulatory factors of signal transduction in cells.

The introduction of the parameters characterizing the redox state of the cell, such as the effective redox potential and the redox buffer capacity has been theoretically substantiated. A comparative study of the parameters of the redox state of erythrocytes from healthy donors and patients with diabetes and acute coronary syndrome has been performed. It was found that the redox buffer capacity in erythrocytes from patients with diabetes and acute coronary syndrome was reduced by 30-40% in comparison with the redox buffer capacity of erythrocytes from healthy donors. The largest change in the effective redox potential was observed for erythrocytes from patients with diabetes, which indicates a more expressed oxidative stress in this pathology.