Cytotoxic and antiproliferative effects of thymoquinone on rat C6 glioma cells depend on oxidative stress.

Thymoquinone (TQ) is a highly perspective chemotherapeutic agent against gliomas and glioblastomas because of its ability to cross the blood-brain barrier and its selective cytotoxicity for glioblastoma cells compared to primary astrocytes. Here, we tested the hypothesis that TQ-induced mild oxidative stress provokes C6 glioma cell apoptosis through redox-dependent alteration of MAPK proteins. We showed that low concentrations of TQ (20-50 µM) promoted cell-cycle arrest and induced hydrogen peroxide generation as a result of NADH-quinone oxidoreductase 1-catalyzed two-electron reduction of this quinone. Similarly, low concentrations of TQ efficiently conjugated intracellular GSH disturbing redox state of glioma cells and provoking mitochondrial dysfunction. We demonstrated that high concentrations of TQ (70-100 µM) induced reactive oxygen species generation due to its one-electron reduction. TQ provoked apoptosis in C6 glioma cells through mitochondrial potential dissipation and permeability transition pore opening. The identified TQ modes of action on C6 glioma cells open up the possibility of considering it as a promising agent to enhance the sensitivity of cancer cells to standard chemotherapeutic drugs.

GLIOMA CELL PROLIFERATION MEDIATED BY COENZYME Q10 AT SERUM DEPRIVATION IN VITRO.

Mechanisms of coenzyme Q10 effect on serum-deprived glioma cell proliferation have been studied. Our results have shown that the addition of coenzyme Q10 into serum-free culture medium leads to increase in cell viability, stimulation of cell growth, as well as restoration of mitochondrial potential and increase of quantity of energized mitochondria. It has been found out that coenzyme Q10-induced glioma cell proliferation under serum deficiency is a result of intracellular reduced glutathione concentration decrease with subsequent activation of proteinkinase C, ERK1/2 and phosphoinositol-3-kinase.

Redox regulation of mitochondrial functional activity by quinones.

Quinones are among the rare compounds successfully used as therapeutic agents to correct mitochondrial diseases and as specific regulators of mitochondrial function within cells. The aim of the present study was to elucidate the redox-dependent effects of quinones on mitochondrial function. The functional parameters [respiratory activity, membrane potential, and reactive oxygen species (ROS) generation] of isolated rat liver mitochondria and mitochondria in intact cells were measured in the presence of eight exogenously applied quinones that differ in lipophilicity and one-electron reduction potential. The quinones affected the respiratory parameters of mitochondria, and dissipated the mitochondrial membrane potential as well as influenced (either decreased or enhanced) ROS generation, and restored the electron flow during electron transport chain inhibition. The stimulation of ROS production by juglone and 2,5-di-tert-butyl-1,4-benzoquinone was accompanied by a decrease in the acceptor control and respiration control ratios, dissipation of the mitochondrial membrane potential and induction of the reverse electron flow under succinate oxidation in isolated mitochondria. Menadione and 2,3,5-trimethyl-1,4-benzoquinone, which decreased the mitochondrial ROS generation, did not affect the mitochondrial potential and, vice versa, were capable of restoring electron transport during Complex I inhibition. In intact C6 cells, all the quinones, except for coenzyme Q10, decreased the mitochondrial membrane potential. Juglone, 1,4-benzoquinone, and menadione showed the most pronounced effects. These findings indicate that quinones with the reduction potential values E1/2 in the range from -99 to -260 mV were effective redox regulators of mitochondrial electron transport.

[Pteridine-dependent oxygen activation in neutrophils].

We investigated the influence ofneopterin and 7, 8-dihydroneopterin on the activity and secretory degranulation of myeloperoxidase in neutrophils and the ability of pteridines to interact with the main substrate of this enzyme, hydrogen peroxide, and with the intermediate product of halogenation cycle--hypochlorous acid. It was shown that neopterin and 7, 8-dihydroneopterin while being a redox-pair regulated the process of oxygen activation in neutrophils by functioning of myeloperoxidase. Depending on concentration, pteridines can influence the secretion of myeloperoxidase into intracellular medium and decrease the level of hydrogen peroxide and hypochlorous acid that are a substrate and an intermediate product of the enzyme respectively. It was shown that 7, 8-dihydroneopterin in micromolar concentration appeared to be noncompetitive inhibitor of myeloperoxidase. We suppose that myeloperoxidase assists 7, 8-dihydroneopterin oxidation by hypochlorous acid that leads to neopterin concentration increase. These changes modify the concentration of reactive oxygen species in intracellular and extracellular media.

[Vitamin K3-induced activation of molecular oxygen in glioma cells].

It has been shown by the method of fluorescent analysis that the rate of hydrogen peroxide generation in human U251 glioma cells under the effect of lipophilic (menadione) or hydrophilic (vikasol) analogues of vitamin K3 was different. Analyzing experimental data we can conclude that menadione underwent one- and two-electron reduction by intracellular reductases in glioma cells. Reduced forms of menadione interact with molecular oxygen leading to reactive oxygen species (ROS) generation. The theoretical model of ROS generation including two competitive processes of one- and two-electron reduction of menadione has been proposed. Rate constants of ROS generation mediated by one-electron reduction process have been estimated.

[Effect of hydrogen peroxide on ability of neutrophils to generate the reactive oxygen and chlorine species and secrete myeloperoxidase in vitro].

The influence of H2O2 at concentrations of 10(-8)--10(-2) mol/l on neutrophil ability to generate the reactive oxygen and chlorine species (ROCS) and secrete myeloperoxidase (MPO) was studied, and H202 injurious effect on neutrophils was also investigated in this work. It was revealed that H2O2 at concentrations of 2 x 10(-3)--2 x 10(-2) mol/l induced disturbance of the neutrophil membrane barrier properties and lactate dehydrogenase release. The incubation of the neutrophils with the addition of 10(-4)--10(-7) mol/l H2O2 led to an increase in the cell ability to generate ROCS during phagocytosis and decreased neutrophil ability to secrete MPO and ROCS in extracellular medium during adhesion. The mechanisms of H2O2 effect are coupled with arachidonic acid metabolism. Inhibition of metabolic pathways of 5-lipoxygenase or cyclooxygenase increased the destructive effect of H2O2 on the cells. Five-lipoxygenase way prohibition led to cancellation of H2O2 influence on MPO and ROCS secretion and to enhancement of H2O2 effect on neutrophil ability to generate ROCS during phagocytosis. The data obtained testify to the high neutrophil resistance to destructive effect of H2O2 and confirm the regulatory role of H2O2 with respect to the neutrophil functions.

[Formation of reactive oxygen species in monocytes at adhesion to glass].

Processes of oxygen activation in monocytes stimulated with adhesion to glass were studied by methods of luminol-dependent and lucigen-independent chemiluminescence. It was shown that monocyte chemiluminescence was caused by cell adhesion to glass surface. Generation of reactive oxygen species at monocyte adhesion to glass was dependent on calcium ion concentration in the medium. The increase in the level of cytosolic calcium, as the extracellular calcium concentration elevated, was accompanied by the activation of phospholipase A2, 5-lypoxygenase and cycloxygenases. Magnesium ions exerted no influence on oxygen activation by cells. Incubation of cells in glucose-free medium, or the addition of glycolysis blocker (2-deoxy-D-glucose) to cell suspension led to a decrease in chemiluminescence intensity. By means of inhibitory analysis, it has been established that processes of oxygen activation are related to arachidonic acid metabolism, and depend on the activity of phospholipase A2.

[Regulation of morphologicaland functional properties of astroglial cells by hydrogen peroxide].

Effects of hydrogen peroxide on morphological characteristics, proliferation index, menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H2O2 at 1 x 10(-8) - 5 x 10(-7) M concentrations acts as a regulator of morphological and functional properties of astrocytes by inducing their reactivation that is manifested as a cell body hypertrophy and an increase of proliferative activity and of menadione-dependent production of superoxide (O2- ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 microM on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O2- under menadione action. Using lipopolysaccharide B as a functional stimulator it has been shown that H2O2 modifies signaling pathways leading to the increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-enhanced chemiluminescence of astrocytes under menadione action to the level of control values.

[Relationship between the oxidation-reduction system of astrocytes with production of active forms of oxygen]. / Sviaz' okislitel'no-vosstanovitel'noi sistemy astrotsitov s produtsirovaniem aktivnykh form kisloroda.

Cells of neuroglia--the astrocytes are of interest from the point of view of their participation in phagocytosis. Phagocyte ability to generate active oxygen forms (AOF) as used as the basic criterion of the estimation of their functional activity. For the purpose to clear up molecular and cellular mechanisms of phagocytosis a study of astrocyte redox-systems, participating in production of AOF, was undertaken. Registration of AOF in astrocytes was carried out using a method of luminol-dependent chemiluminescence. Primary culture of guinea pig astrocytes was used. Spontaneous chemiluminescence of low intensity was found for the astrocytes at the presence of luminol. The destruction of the cells was accompanied by a significant growth of the intensity of spontaneous chemiluminescence. Suspension of endocutosis inductors, particle of latex and phytohemagglutinin, added to astrocytes did not result in formation of AOF, characteristic for other cells, possessing phagocytosis. It was established, that addition of hydrogen peroxide destroys astrocytes at the presence of luminol and gives rise to the emission. Chemiluminescence was not observed in similar experiments with intact cells. A conclusion was made that inside astrocytes there are structures, which show peroxidase-like activity.

[Aggregation of neutrophils and their generation of active forms of oxygen as affected by lectins]. / Agregatsiia neitrofilov i generatsiia imi aktivnykh form kisloroda pri deistvii lektinov.

It was shown that neutrophil aggregation is caused by all lectins with different specificity for carbohydrates but generation of active oxygen forms is induced only by some lectins. Polyspecific lectins-erythroagglutinin and phytohemagglutinins have the greatest activity in relation to both processes.

[The effect of recombinant interleukin-1 beta on concanavalin A-induced lymphocyte reactions]. / Vliianie rekombinantnogo interleikina-1 beta na indutsirovannye konkanavalinom A reaktsii limfotsitov.

As revealed by investigations made by the use of the method of luminol-dependent chemiluminescence and light dispersion, the addition of concanavalin A (ConA) to the suspension of human peripheral blood lymphocytes and subsequent incubation with recombinant interleukin-1 beta leads to a sharp increase in the yield of chemiluminescence and the rate of cell aggregation in comparison with similar parameters obtained for cells, not treated with interleukin-1 beta. The study revealed the potentiating action of recombinant interleukin-1 beta on ConA-induced proliferative response of thymocytes in the culture. The mechanisms of the priming action of recombinant interleukin-1 beta on ConA-induced reaction of lymphocytes are discussed.

[A comparative study of the reactions of the peripheral blood neutrophils from donors and from lymphogranulomatosis patients to arachidonate stimulation of the cells]. / Sravnitel'noe issledovanie reaktsii neitrofilov perifericheskoi krovi donorov i bol'nykh limfogranulematozom pri stimuliatsii kletok arakhidonatom.

Arachidonate-induced aggregation and generalization of active oxygen forms (OAF) by peripheral blood neutrophils in donors were studied in donors and Hodgkin's disease patients. Leukocytes of the latter had incomplete ability to produce AOF in response to cell stimulation with arachidonic acid. The study of arachidonate-induced aggregation of neutrophils indicated no differences in the speed of the process in the patients and donors. AOF catchers did not act on the rate of leukocyte aggregation in the patients though accelerated the process in the donors. It is inferred that Hodgkin's disease is associated with dysfunction of oxygen activation by neutrophils. The findings suggest that defects in leukocytes ability to activate oxygen in Hodgkin's disease may entail deranged regulation of other processes essential for functional activity of polymorphonuclear leukocytes.

[Chemiluminescence in the peroxidase oxidation of luminol with hydrogen peroxide in various media]. / Khemiliuminestsentsiia pri peroksidaznoi reaktsii okisleniia liuminola perekis'iu vodoroda v razlichnykh sredakh.

The effects of culture media of various compositions on chemiluminescence developing in peroxidase oxidation of luminol with hydrogen peroxide were under study. The findings evidence that the presence of carbonate and bicarbonate ions in the medium results in a two-staged chemiluminescence kinetics and in more intensive chemiluminescence in the peroxidase-luminol-hydrogen peroxide system. This fact has brought the authors to a conclusion that carbonate and bicarbonate-containing media are more effective for the detection of low peroxidase concentrations by the chemiluminescence technique.

[Oxygen activation by lymphocytes during interaction of HLA antibodies with HLA antigens]. / Aktivatsiia kisloroda limfotsitami pri vzaimodeistvii HLA-antitel s HLA-antigenami.

The method of luminol-dependent chemiluminescence was used to detect oxygen activation during interaction of HLA antibodies with HLA antigens expressed on lymphocyte surface. Introduction of specific anti-HLA serum into the lymphocyte suspension leads to a rapid decrease of intensity in chemiluminescence intensity follows. The maximum yield of induced chemiluminescence in case of using a specific antiserum is significantly lower than in the control. The method could be used for detection of HLA antigens on lymphocyte surface.

[Generation of active forms of oxygen by human peripheral blood neutrophils and lymphocytes during adhesion to glass]. / Generatsiia aktivnykh form kisloroda neitrofilami i limfotsitami perifericheskoi krovi cheloveka pri adgezii na steklo.

Oxygen activation by neutrophils and human blood lymphocytes at adhesion to glass have been studied by luminol--dependent chemiluminescence. It has been established that the cell interaction with glass leads to the formation of O2-., O2', .OH and H2O2. Chemiluminescence kinetics and the excited--oxygen forms ratio at adhesion were different for neutrophils and lymphocytes. The desorption of cells resulted in a decrease of the chemiluminescent response to neutrophils and lymphocytes when they again adhered to glass and in practically complete inhibition of chemiluminescence induced by adding concanavalin A. It has been determined that at adhesion of neutrophils and lymphocytes to glass different mechanisms of oxygen activation take place.

[Effect of medium pH on oxygen activation by human blood neutrophils and lymphocytes during adhesion to glass and treatment with concanavalin A]. / Vliianie pH sredy na aktivatsiiu kisloroda neitrofilami i limfotsitami krovi cheloveka pri adgezii na steklo i deistvii konkanavalina A.

It has been established that oxygen activation by neutrophils and human blood lymphocytes at adhesion to glass and under the action of concanavalin A differently depends on pH. It has been suggested that oxygen activation by neutrophils and lymphocytes occurs through different mechanisms.

Chemiluminescence of luminol caused by interaction of cytochrome P-450 and cytochrome C with cumene hydroperoxide: comparative studies.

A comparative study of the interaction of cumene hydroperoxide with cytochrome P-450LM2 and with cytochrome C has been undertaken using the chemiluminescence method in the presence of luminol. Considerable differences in the mechanisms of action of both hemoproteins have been revealed with various inhibitors of radical stages, i.e. superoxide dismutase, mannitol, sodium azide, and alpha-tocopherol. It is shown that molecular oxygen participates in the process of hemoprotein-catalyzed hydroperoxide oxidation of luminol.

[Effect of ozone on the permeability of flat bilayer lipid membranes]. / Vliianie ozona na pronitsaemost' ploskikh bisloinykh lipidnykh membran.

The influence of the liposome ozonolysis products on ionic permeability and stability of the planar bilayer lipid membrane ( PBLM ) was studied by electroconductivity . Addition of ozonized liposomes and PBLM preparation from the products of the ozonized lipids results in a decrease of the PBLM resistance. It is suggested that the enhancement of PBLM stability from ozonized lipids may be caused by production during lipid peroxidation of the intermolecular bonds between lipid molecules and by an increase of the lipid bilayer rigidity.