Heat Shock Protein HSP27 and the Status of the Glutathione System in Dexamethasone-Induced Apoptosis of Jurkat Tumor Cells.

We studied the effect of the HSP27 inhibitor, 5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)-isoxazole, at a final concentration of 0.1 µM and/or the apoptosis inducer dexamethasone at a final concentration of 10 µM on the content of hydroxyl radical, reduced and oxidized glutathione, HSP27, activity of glutathione reductase, glutathione peroxidase, caspase-3, and the number of Annexin+ Jurkat tumor cells. The involvement of HSP27 in apoptosis of Jurkat tumor cells was demonstrated. Simultaneous exposure to the HSP27 inhibitor and dexamethasone resulted in an increase in the level of HSP27 against the background of developing oxidative stress (increase in the concentration of hydroxyl radicals and changes in the state of the glutathione system).

The Role of Redox Status Changes in Dexamethasone-Induced Apoptosis in Jurkat Tumor Cells.

The apoptotic death and its regulation was studied in intact Jurkat tumor cells and under the influence of buthionine-sulfoximine (de novo glutathione synthesis inhibitor; 1 mM) and/or apoptosis inducer dexamethasone (10 µM). The role of glutathione system components in dexamethasone-induced apoptosis in Jurkat tumor cells (both receptor-mediated and mitochondrial pathways) was analyzed. Under conditions of dexamethasone-induced apoptosis, glutathione system blockage mostly affects presentation of TNF RI- and Fas-receptors in Jurkat tumor cells, as well as change in content of transcription factors Apaf-1 and NF-κB, thereby promoting cell death. The decrease in the content of oxidized glutathione produced a potentiating effect on dexamethasone-induced apoptotic death of Jurkat tumor cells.

Oxidative Modification of Redox Proteins: Role in the Regulation of HBL-100 Cell Proliferation.

HBL-100 breast epithelial cells were cultured with a blocker (N-ethylmaleimide) and protector (1,4-dithioerythritol) of SH groups. The study assessed changes in redox potential of glutathione and thioredoxin systems, intensity of oxidative modification of proteins, ROS production, and cell proliferation. The roles of thioredoxin system and protein oxidative modification in HBL-100 cell proliferation under redox status modulation were established. The role of carbonylated thioredoxin in arrest of the cell cycle in S-phase was demonstrated, which could be used for targeted therapy of the diseases accompanied by oxidative stress and disturbed redox status.

The Role of the Glutathione System in Oxidative Modification of Proteins and Dysregulation of Apoptosis in Jurkat Tumor Cells.

We compared changes in the redox status and intensity of oxidative modification of proteins in intact Jurkat tumor cells and cells cultured with buthionine sulfoximine, an inhibitor of the key enzyme of glutathione synthesis γ-glutamylcysteine synthetase. The glutathione system components play a role in modulation of the content of protein-bound glutathione, protein carbonyl derivatives, bityrosine, and oxidized tryptophan, and in dysregulation of apoptosis in Jurkat tumor cells. Inhibition of de novo synthesis of glutathione in Jurkat tumor cells was followed by accumulation of hydroxyl radical, a reduction in the level of protein-bound glutathione and oxidized tryptophan, and a rise in the concentration of protein carbonyl derivatives. These changes were accompanied by activation of programmed cell death.

[Redox-dependent mechanisms of regulation of breast epithelial cell proliferation]. / Redoks-zavisimye mekhanizmy reguliatsii proliferatsii kletok épiteliia molochnoi zhelezy.

Activation of free radical oxidation in different cell types, including breast epithelial cells, may result in damage to macromolecules, in particular, proteins taking part in regulation of cell proliferation and apoptosis. The glutathione, glutaredoxin and thioredoxin systems play an essential role in maintaining intracellular redox homeostasis. Due to this fact, modulation of cellular redox status under the effect of an SH group inhibitor and an SH group protector may be used as a model for studying the role of redox proteins and glutathione in regulating cell proliferation in different pathological processes. In this study we have evaluated the state of the thioredoxin, glutaredoxin and glutathione systems as well as their role in regulating proliferation of HBL-100 breast epithelial cells under redox status modulation with N-ethylmaleimide (NEM) and 1,4-dithioerythriol (DTE). Modulating the redox status of breast epithelial cells under the effect of NEM and DTE influences the functional activity of glutathione-dependent enzymes, glutaredoxin, thioredoxin, and thioredoxin reductase through changes in the GSH and GSSG concentrations. In HBL-100 cells under redox-status modulation, we have found an increase in the number of cells in the S-phase of the cell cycle and a decrease in the number of cells in the G0/G1 and G2/М phases, as opposed to the values in the intact culture. The proposed model of proliferative activity of cells under redox status modulation may be used for development of new therapeutic approaches for treatment of diseases accompanied by oxidative stress generation.

[The role of heat shock proteins 27 and 70 in redox-dependent regulation of apoptosis in Jurkat tumor cells]. / Uchastie belkov teplovogo shoka 27 i 70 v redoks-zavisimoi reguliatsii apoptoza opukholevykh kletok linii Jurkat.

Heat shock proteins Hsp) act as molecular chaperones, protecting enzymes and other proteins against reactive oxygen species. The objective of the study was to investigate the role of Hsp27 in maintaining the balance of the glutathione system and Hsp70 concentrations as well as in implementing Jurkat tumor cell apoptosis. Addition of the Hsp27 inhibitor KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)-isoxazol) to Jurkat cells resulted in glutathione redox imbalance (increased GSSG and increased glutathione reductase activity), a decrease in Hsp70 concentrations, and also increased cell apoptosis as compared with to the intact cell culture. The proposed selective regulation of chaperone activity is a promising direction in regulating apoptosis at the cellular level.

[The role of oxidative protein modification and the gluthatione system in modulation of the redox status of breast epithelial cells].

The effects of the SH-group blocker N-ethylmaleimide (NEM) and thiol group protector 1,4-dithioerythritol (DTE) on the redox status of cells HBL-100 cells, oxidative modification of their proteins and the state of glutathione and thioredoxin systems have been investigated. Breast epithelial cells cultivated in the presence of NEM were characterized by decreased redox status, increased glutathione reductase activity, and increased concentrations of products of irreversible oxidative modification of protein and amino acids. Cultivation of HBL-100 cells in the presence of DTE resulted in a shift of the redox status towards reduction processes and increased reversible protein modification by glutathionylation. The proposed model of intracellular redox modulation may be used in the development of new therapeutic approaches to treat diseases accompanied by impaired redox homeostasis (e.g. oncologic, inflammatory, cardiovascular and neurodegenerative disease).

Role of Glutathione System Redox Potential in Apoptosis Dysregulation in MCF-7 Breast Adenocarcinoma.

MCF-7 breast cancer cells and HBL-100 breast epithelial cells were cultured with N-ethylmaleimide, a blocker of SH groups. Changes in redox potential of the glutathione system, activities of glutathione reductase, glutathione peroxidase, and intensity of apoptotic cell death were evaluated. The results indicate that incubation with N-ethylmaleimide led to glutathione system imbalance, reduced tumor cell redox potential, and induced their programmed death, which seemed useful for prospective target therapy of tumor diseases.

The Role of Protein Oxidative Modification and the Cellular Redox Status in Realization of Apoptosis of MCF-7 Breast Adenocarcmoma Cells.

The aim of this study was to establish the role of redox modification of proteins and redox status in the realization of apoptosis of MCF-7 breast adenocarcinoma cells du-ing cultivation with the SH-group blocker N-ethylmaleimide (NEM) and the SH-group protector 1,4-dithioerythritol (DTE). The activation of apoptosis in MCF-7 breast adenocarcinoma cells was shown to be due to the irreversible modification of redox sensitive protein molecules. The presence of DTE in the culture medium of cancer.cells caused reversible glutathionylation of protein molecules and did not change the: number of apoptotic MCF-7 cells.

Effect of insulin, the glutathione system, and superoxide anion radical in modulation of lipolysis in adipocytes of rats with experimental diabetes.

Spontaneous lipolysis was found to be increased in adipocytes of rats with alloxan-induced diabetes. In addition, isoproterenol-stimulated hydrolysis of triacylglycerols was inhibited against the background of oxidative stress and decreased redox-status of cells. A decrease in the ability of insulin to inhibit isoproterenol-stimulated lipolysis in adipocytes that were isolated from adipose tissue of rats with experimental diabetes was found, which shows a disorder in regulation of lipolysis in adipocytes by the hormone in alloxan-induced diabetes. Based on these findings, we concluded that there is an influence of reactive oxygen species, superoxide anion radical in particular, and redox potential of the glutathione system on molecular mechanisms of change in lipolysis intensity in rat adipocytes in alloxan-induced oxidative stress. Activation of spontaneous lipolysis under conditions of oxidative stress might be a reason for the high concentration of free fatty acids in blood plasma in experimental diabetes, and this may play a significant role in development of insulin resistance and appearance of complications of diabetes.

Role of heat shock protein 27 in regulation of glutathione system and apoptosis of Jurkat tumor cells and blood lymphocytes.

Reaction of the glutathione system of Jurkat tumor cells and blood lymphocytes was evaluated under conditions of culturing with 5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole (KRIBB3), a selective inhibitor of heat shock protein Hsp27. The results indicated the regulatory role of Hsp27 in the maintenance of the functional activities of glutathione reductase, glutathione peroxidase, and realization of apoptotic death of Jurkat cells and blood lymphocytes. Inhibition of Hsp27 in Jurkat tumor cells led to imbalance of the glutathione system and increase of the share of annexin-positive cells.

[Molecular mechanisms of modulation of lipolysis in adipose tissue and development of insulinresistance in diabetes].

Analysis of modern literature data as well as the results of personal research on development of oxidative stress in adipose tissue in diabetes is presented. Mechanisms of modulation of spontaneous and induced lipolysis in adipocytes in conditions of oxidative stress are discussed. Participation of adipose tissue in forming insulin resistance in types 1 and 2 diabetes is considered.

Effect of alloxan on spontaneous lipolysis and glutathione system in isolated rat adipocytes.

In vitro experiments on cultured adipocytes from epididymal adipose tissue showed that addition of alloxan (0.5-10.0 mmol/liter) to the incubation medium induced the development of oxidative stress accompanied by an increase in the concentration of reactive oxygen species, TBA-reactive substances, and lipid hydroperoxides in cells. The redox state of adipocytes changed significantly under these conditions, which was associated with a decrease in the amount of reduced tripeptide, an increase in the content of glutathione disulfide, and a decrease in the reduced/oxidized glutathione ratio. The development of oxidative stress in adipocytes was accompanied by activation of spontaneous lipolysis, which probably plays an important role in the mechanisms of insulin resistance.

Role of thiol-disulfide system in mechanisms of functional changes in neutrophils under conditions of oxidative stress.

We studied the state of the thiol-disulfide system (contents of reduced and oxidized glutathione, their ratio, and concentrations of protein SH-groups and protein-bound glutathione) and functional properties of neutrophils (production of hydroxyl radicals, IL-8, and TNF-α and myeloperoxidase activity) from healthy donors under conditions of oxidative stress in vitro induced by H(2)O(2)in a final concentration of 200 µM and from patients with community-acquired pneumonia. We evaluated the role of reduced and protein-bound glutathione in the regulation of functional state of blood neutrophils from patients with community-acquired pneumonia and during oxidative stress in vitro under conditions cell incubation with N-ethylmaleimide or 1,4-dithioerythritolsulfhydryl, the blocker and protector of sulfhydryl groups, respectively.

[Erythrocyte membranes in metabolic syndrome].

The authors discuss results of investigations into the structure and functional status of erythrocyte membranes in patients with metabolic syndrome. It is shown that the pathological process involves not only well known changes in serum lipid metabolism but also highly specialized cells, such as erythrocytes. Specifically, erythrocytes undergo marked disorganization of membranous lipid phase in conjunction with a relative increase of cholesterol fraction and a decrease in phospholipids levels. Analysis of fractional lipid composition in erythrocyte membranes reveals reduced content of phospholipids, sphingomyelin, and phosphatidylcholine coupled to increased content of cholesterol, lysophosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. It is concluded that disturbances of carbohydrate metabolism in patients with metabolic syndrome aggravate manifestations of the underlying disease.

Pathomorphosis of erythrocytes in patients with acquired valvular heart diseases and under conditions of their correction.

Scanning electron microscopic study of the morphology and surface architectonics of erythrocytes in patients with acquired aortic and mitral valve diseases showed signs of morphological restructuring of the erythrocyte population. Reversibly transformed transitional red blood cells and irreversibly changed prehemolytical and degenerative erythrocytes were much more incident in these patients than in donors. The number of functionally intact biconcave discocytes notably decreased in comparison with donors. Morphological heterogeneity of the erythrocyte pool increased during the immediate period after replacement of heart valves with mechanical disc prostheses. Disorganization of the surface relief of red blood cells persisted 12-24 months after surgery.

Typical changes of erythrocytes in chronic inflammation.

Structural and metabolic status and function of erythrocytes were studied in patients with chronic bronchitis, chronic inflammatory diseases of the upper airways, and chronic colitis. Complex study of the peripheral component of the erythron showed that disorders in erythrocyte morphology and function (decreased erythrocyte dry weight, low concentration of sulfhydryl groups and lipoproteins, decreased content of high molecular weight polypeptides in parallel with increased content of low molecular weight proteins, increased number of transformed cells, and enhanced reversible aggregation) were nonspecific and did not depend on the location of the inflammatory process.

Typical changes of reversible erythrocyte aggregation in various pathological processes.

Examinations of patients with malignant tumors of different location (lung cancer, stomach, colorectal cancer, and head and neck tumors), patients with paranoid schizophrenia and neurotic disorders revealed enhanced reversible erythrocyte aggregation. It was found that enhancement of erythrocyte aggregation is associated with structural modification of their membrane and appearance of transformed cells (echinocytes, stomatocytes, etc.). The phenomenon of enhanced reversible erythrocyte aggregation in somatic and mental diseases can be regarded as a typical pathological process.

Structure and properties of lipid bilayer of erythrocyte membranes in patients with malignant tumors.

The study of erythrocyte membranes in untreated patients with malignant tumors of different location (lung cancer, tumors of the head and neck, stomach and colorectal cancer) revealed changes in the fatty-acid spectrum of phospholipid fraction paralleled by an increase in the viscosity of the lipid bilayer, including the area of protein-lipid contacts. The degree of changes depended on tumor location.

Ultrastructural changes in erythrocytes in patients with mental disorders.

Transmission electron microscopy of peripheral blood erythrocytes from patients with paranoid and residual schizophrenia, mental retardation, and neurotic disturbances revealed nonspecific ultrastructural changes in the membrane and matrix of red blood cells.