Suppression of PI3K/Akt/mTOR Signaling Pathway and Antioxidant System and Reversal of Cancer Cells Resistance to Cisplatin under the Effect of Curcumin.

The effect of curcumin on the resistance of SKOV-3 human ovarian adenocarcinoma cells to cisplatin was studied. It was found that curcumin induced "reversal" of cancer cells resistance, which was associated with suppression of the expression of genes encoding the key antioxidant enzymes (SOD1, SOD2, CAT, GPX1, and HO-1) and transcription factor Nrf2 and a decrease in the expression of genes encoding kinases of the PI3K/Akt/mTOR signaling pathway. The obtained results confirm the role of redox-dependent regulation in the "reversal" of cancer cells resistance to cisplatin.

Role of MicroRNAs in the Regulation of Redox-Dependent Processes.

Cellular redox homeostasis involves a combination of redox processes and corresponding regulatory systems and represents an important factor ensuring cell viability. Redox-dependent regulation of cellular processes is a multi-level system including not only proteins and enzyme complexes, but also non-coding RNAs, among which an important role belongs to microRNAs. The review focuses on the involvement of miRNAs in the redox-dependent regulation of both ROS (reactive oxygen species)-generating enzymes and antioxidant enzymes with special emphasis on the effects of miRNAs on redox-dependent processes in tumor cells. The impact of ROS on the miRNA expression and the role of the ROS/miRNA feedback regulation in the cell redox state are discussed.

Redox-Dependent Expression of Genes Encoding NADPH Oxidase 5 and the Key Antioxidant Enzymes during Formation of Drug Resistance of Tumor Cells to Cisplatin.

Expression of genes that plays a significant role in the control of cellular redox homeostasis was studied during the development of drug resistance of human ovarian adenocarcinoma SKOV-3 cells to cisplatin. It was found that the development of drug resistance was accompanied by enhanced expression of the genes encoding the key antioxidant enzymes (SOD2, CAT, GPX1, and HO-1) and transcription factor Nrf2, as well as reduced expression of the gene encoding NOX5 isoform of NADPH oxidase. The results testify to redox-dependent development of the adaptive antioxidant response as an important process in the mechanism of formation of resistance to cisplatin.

Role of glutathione, glutathione transferase, and glutaredoxin in regulation of redox-dependent processes.

Over the last decade fundamentally new features have been revealed for the participation of glutathione and glutathione-dependent enzymes (glutathione transferase and glutaredoxin) in cell proliferation, apoptosis, protein folding, and cell signaling. Reduced glutathione (GSH) plays an important role in maintaining cellular redox status by participating in thiol-disulfide exchange, which regulates a number of cell functions including gene expression and the activity of individual enzymes and enzyme systems. Maintaining optimum GSH/GSSG ratio is essential to cell viability. Decrease in the ratio can serve as an indicator of damage to the cell redox status and of changes in redox-dependent gene regulation. Disturbance of intracellular GSH balance is observed in a number of pathologies including cancer. Consequences of inappropriate GSH/GSSG ratio include significant changes in the mechanism of cellular redox-dependent signaling controlled both nonenzymatically and enzymatically with the participation of isoforms of glutathione transferase and glutaredoxin. This review summarizes recent data on the role of glutathione, glutathione transferase, and glutaredoxin in the regulation of cellular redox-dependent processes.

Expression of peroxiredoxin 1, 2, 3, and 6 genes in cancer cells during drug resistance formation.

We studied the expression of peroxiredoxin genes (PRDX1, PRDX2, PRDX3, and PRDX6) in human erythroleukemia K652, human breast carcinoma MCF-7, and human ovarian carcinoma SKOV-3 cells during cisplatin resistance development. It was found that drug resistance formation was accompanied by a significant increase in the expression of PRDX1, PRDX2, PRDX3, PRDX6 genes in all cancer cell strains, which confirms the important contribution of redox-dependent mechanisms into the development of cisplatin resistance of cancer cells.

Expression of genes of glutathione transferase isoforms GSTP1-1, GSTA4-4, and GSTK1-1 in tumor cells during the formation of drug resistance to cisplatin.

We studied the expression of genes encoding glutathione-S-transferase isoforms GSTP1-1, GSTA4-4, and GSTK1-1 during the development of the resistance of human erythroleukemia (K562), mammary adenocarcinoma (MCF-7) and ovary adenocarcinoma (SKOV-3) cells to cisplatin (CDDP). It was found that drug resistance development in all three strains of tumor cells is associated with significant increase in hGSTP1 and hGSTA4 gene expression, whereas increased hGSTK1 gene expression was detected only in resistant K562/CDDP and MCF-7/CDDP cells.

[Current views on antioxidative activity of glutathione and glutathione-depending enzymes].

Reduced glutathione (GSH), gamma-glutamyl-L-cysteineglycine, is a tripeptide widespread in plants, animals, and man as a low-molecular weight SH-containing compound. This review presents results of published and original studies concerned with the synthesis and the role of glutathione and glutathione-dependent enzymes in antioxidative processes, such as maintenance and regulation of cell status, glutathionilation and deglutathionilation, redox-dependent signaling, and apoptosis.

Involvement of thio-, peroxi-, and glutaredoxins in cellular redox-dependent processes.

Among the key antioxidant enzymes, thioredoxin and glutaredoxin systems play an important role in cell defense against oxidative stress and maintenance of redox homeostasis owing to the regulation of thiol-disulfide exchange. The thioredoxin isoforms Trx1 (cytoplasmic form) and Trx2 (mitochondrial form) can reduce inter- and intramolecular disulfide bonds in proteins, in particular, in oxidized peroxiredoxins, which disrupt organic hydroperoxides, H2O2, and peroxynitrite. NADPH-dependent thioredoxin reductase, which reduces a broad range of substrates including oxidized form of thioredoxin, can also directly reduce lipid hydroperoxides, H2O2, and dehydroascorbic and lipoic acids. Glutaredoxin, whose major isoforms in mammals are Grx1, Grx2, and Grx5, as well as thioredoxin, catalyzes S-glutathionylation and deglutathionylation of proteins to protect SH-groups from oxidation and restore functionally active thiols. However, in contrast to thioredoxin, glutaredoxin reduces GSH-mixed disulfides and catalyzes the reaction not only via a dithiol mechanism but also via monothiol reduction. In addition to the role in cellular antioxidant defense, all of the reviewed redox proteins (thioredoxin, thioredoxin reductase, peroxiredoxin, and glutaredoxin) have a number of significant functions required for cell viability: they regulate transcription factor activities, play the role of growth factors, serve as enzyme cofactors, take part in regulation of cell cycle, and are involved in antiapoptotic mechanisms.

Expression of genes for thioredoxin 1 and thioredoxin 2 in multidrug resistance ovarian carcinoma cells SKVLB.

The expression of genes for thioredoxin isoforms Trx1 and Trx2 was studied in sensitive SKOV-3 and resistant SKVLB human ovarian carcinoma cells. The development of doxorubicin resistance was accompanied by a significant increase in the expression of TRX1 gene and less pronounced increase in TRX2 gene expression.

Expression of genes for redox-dependent glutathione S-transferase isoforms GSTP1-1 and GSTA4-4 in tumor cell during the development doxorubicin resistance.

Expression of genes for redox-dependent glutathione S-transferase isoforms GSTP1-1 and GSTA4-4 in tumor cells K562, MCF-7, and SKOV-3 was studied during the development of resistance to doxorubicin. It was found that the development of resistance was accompanied by predominant increase in the expression of hGSTP1 gene in MCF-7 cells, and hGSTA4 gene in resistant K562/DOX and SKVLB cells.

Changes in expression of genes encoding antioxidant enzymes, heme oxygenase-1, Bcl-2, and Bcl-xl and in level of reactive oxygen species in tumor cells resistant to doxorubicin.

The relationship between expression of genes encoding key antioxidant enzymes, heme oxygenase-1, Bcl-2, and Bcl-xl and change in production of reactive oxygen species (ROS) resulting from development of resistance of cancer cells K562, MCF-7, and SKOV-3 to the prooxidant chemotherapeutic agent doxorubicin (DOX) has been studied. Significant increase in mRNA level and activity of Mn-superoxide dismutase (Mn-SOD), catalase, and selenium-dependent glutathione peroxidase-1 (GPx-1) and reduced ROS level was found in resistant K562/DOX and SKVLB cells. In contrast, no change in ROS level was observed in MCF-7/DOX cells in parallel with decrease in Mn-SOD and catalase mRNAs and corresponding activities concurrently with high increase in GPx-1 mRNA and activity. As a result of the development of resistance, a similarity was found between the change in ROS level and the change in ho-1 and bcl-2 gene expression, whereas elevation of bcl-xl gene expression was observed in all three types of resistant cells. Particular features of development of adaptive antioxidant response as well as redox-dependent change in bcl-2 gene expression under formation of DOX resistance of cancer cells of different genesis are discussed.

[NP-detecting DNA technologies: solving problems of applied biochemistry].

Heterozygosity of CANP3, ACTN3, and GHR genes in specialized collections was studied using state-of-the-art DNA technologies for DNA analysis. A new dinucleotide deletion (AC) at the beginning of exon 21 was identified in five individuals with heterozygous CANP3 gene. Analysis of polymorphism (SNP1747 C-->T) of ACTN3 gene demonstrated a positive association of allele C with a high muscular performance. Real-time PCR assay of SNP1630 (A-->C) in GHR gene suggested a putative negative association of allele C of this SNP with a high muscular performance.

Subunit separation in reversed micelle system reveals the existence of active centers both on light and heavy gamma-glutamyltransferase subunits.

Regulation of supra-macromolecular composition and catalytic activity of a heterodimeric enzyme, gamma-glutamyltransferase, in the system of Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) reversed micelles in octane were studied. Variation of the surfactant hydration degree (parameter, determining dimensions of the polar inner cavity of the micelle) causes a reversible dissociation of the enzyme to light and heavy subunits. Both enzyme subunits possess catalytic activity. The light and heavy subunits of the enzyme were separated on a preparative scale in a reversed micelle system using ultracentrifugation. The active centers of gamma-glutamyltransferase were studied using its irreversible inhibitor--AT-125 (L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). Separation of the gamma-glutamyltransferase subunits results in the 'opening' of a new active center located at the heavy subunit. In the dimer form of the enzyme this center is masked and it is not accessible to both substrate and inhibitor molecules.

[Comparative study of the regulation of catalytic activity of soluble and membrane forms of enzymes in reverse micellar systems. Gamma-glutamyltransferase and aminopeptidase]. / Sravnitel'noe izuchenie reguliatsii kataliticheskoi aktivnosti rastvorimykh i membrannykh form fermentov v sistemakh obrashchennykh mitsell. Gamma-glutamiltransferaza i aminopeptidaza.

The regulations of functioning of water soluble and membrane forms of enzymes in the systems of reversed micelles of surfactants in organic solvents are compared. By an examples of gamma-glutamyltransferase (in AOT reversed micelles in octane) and amino-peptidase (in Brij 96 reversed micelles in cyclohexane) the principal difference in the catalytic activity regulation of water soluble and membrane forms is demonstrated. The catalytic activity of the membrane form depends largely on the surfactant concentration at the constant hydration degree, whereas the activity of the water soluble form is constant under these conditions. The catalytic activity dependence on the surfactant concentration is regarded as a "test for the enzyme's membrane activity".

A new strategy for the study of oligomeric enzymes: gamma-glutamyltransferase in reversed micelles of surfactants in organic solvents.

A heterodimeric enzyme (gamma-glutamyltransferase) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. As was shown earlier, the size (radius) of inner cavity of the AOT-reversed micelles is determined by their hydration degree, i.e., [H2O]/[AOT] molar ratio, in the system. Owing to this, the dependence of hydrolytic, transpeptidation and autotranspeptidation activities of the enzyme on the hydration degree was investigated using L- and D-isomers of gamma-glutamyl(3-carboxy-4-nitro)anilide and glycylglycine as substrates. For all of the reaction types, the observed dependences are curves with three optima. The optima are found at the hydration degrees, [H2O]/[AOT] = 11, 17 and 26 when the inner cavity radii of reversed micelles are equal to the size of light (Mr 21,000) and heavy (Mr 54,000) subunits of gamma-glutamyltransferase, and to their dimer (Mr 75,000), respectively. Ultracentrifugation experiments showed that a change of the hydration degree resulted in a reversible dissociation of the enzyme to light and heavy subunits. The separation of light and heavy subunits of gamma-glutamyltransferase formed in reversed micelles was carried out and their catalytic properties were studied. The two subunits catalyze hydrolysis and transpeptidation reactions; autotranspeptidation reaction is detected only in the case of the heavy subunit. These findings imply that the reversed micelles of surfactants in organic solvents function as the matrices with adjustable size permitting to regulate the supramolecular structure and the catalytic activity of oligomeric enzymes.

[Regulation of the supramolecular structure and the catalytic activity of gamma-glutamyltransferase in the reversed micelle system]. / Reguliatsiia nadmolekuliarnoi struktury i kataliticheskoi aktivnosti gamma-glutamiltransferazy v sistemakh obrashchennykh mitsell.

Regulation mechanisms of the supramolecular structure and the catalytic activity of a heterodimeric enzyme, gamma-glutamyltransferase, in the system of Aerosol OT (AOT) reversed micelles in octane have been studied. gamma-(3-carboxy-4-nitro)-glutamic acid anilide (L- and D-isomers) and glycylglycine were used as substrates to explore the enzyme-catalyzed hydrolase, autotransferase, and transferase reactions. For all types of reactions, the catalytic activity of gamma-glutamyltransferase as a function of the hydration degree has a shape of curves with three optima. The optima of the catalytic activity were detected at hydration degrees [( H2O]/[AOT] = 11, 17, and 26) when radii of the micelle's inner cavity are commensurate with the light and heavy subunits (Mr 21,000 and 54,000, respectively) of gamma-glutamyltransferase as well as with the dimer (Mr 75,000). As ultracentrifugation the change in hydration degree caused a reversible dissociation of the enzyme to the light and heavy subunits. Both subunits catalyze the hydrolase and transferase reactions, whereas the autotransferase activity was detected only for the heavy subunit. Dependencies of catalytic activities of the subunits on the hydration degree have one optimum each (at [H2O]/[AOT] = 11 and 17 for the light and heavy subunits, respectively). When mixing micellar solutions containing both subunits, a third optimum was detected corresponding to the dimer [( H2O]/[AOT] = 26).

[Latent form of glutathione reductase in the rat liver]. / Latentnaia forma glutationreduktazy v pecheni krys.

The existence of membrane-bound forms of glutathione reductase in rat liver and transplantable hepatoma G-27 was demonstrated, using differential centrifugation techniques. The activity of the sedimentable form of the liver enzyme was detected only in the presence of detergents. Conditions for the manifestation of the latent glutathione reductase activity in whole liver homogenates and in the 105000 g pellet were determined. Solubilization of the latent form of the enzyme in the presence of sodium deoxycholate increases 2-fold the glutathione reductase activity in liver homogenates (but not in hepatoma). Simultaneous determination of the disulfidereductase, nonspecific NADPH-oxidase and gamma-glutamyltransferase (membrane-bound enzyme of glutathione metabolism) activities was performed.

[Phosphorylation and inhibition of gamma-glutamyl transferase activity by cAMP-dependent protein kinase]. / Fosforilirovanie i tormozhenie aktivnosti gamma-glutamiltransferazy cAMP-zavisimoi proteinkinazoi.

It was shown that preparations of bovine kidney gamma-glutamyl transferase with different degree of purity are phosphorylated by cAMP-dependent protein kinase. Phosphorylation is accompanied by a simultaneous decrease of both transferase and hydrolase activities of the enzyme. Hence, gamma-glutamyltransferase may serve as a substrate and target of regulation by cAMP-dependent protein kinase.