Migration of cancer cells from the primary tumor site to nearby tissues is the starting point of the metastatic process. The invasive properties of cells are especially important for carcinomas, since tumor cells need to overcome the basement membrane and go beyond its boundaries to the underlying tissues. Substances that reduce the invasive ability of malignant cells are promising as antimetastatic agents. In the present work, the possibility of inhibiting the ability of different cancer cell lines to migrate under the influence of the Bacillus pumilus ribonuclease (binase) was analyzed using the scratch-wound assay. It was established that binase at non-toxic concentrations (10 μg/mL) reliably suppressed the migratory ability of HuTu 80 human duodenum adenocarcinoma cells incubated with RNase for 48-72 h. The antimetastatic potential of binase is confirmed by molecular modeling data demonstrating the ability of binase to inhibit cellular metalloproteinases that determine the migration of tumor cells.

Lipopolysaccharides (LPS), components of the cell wall of gram-negative bacteria, activate neutrophils that trigger pathological processes, including gram-negative sepsis. LPS inhibit spontaneous apoptosis of neutrophils that leads to inflammation. In this work we tested the action of H2S donor (GYY4137) on the activation of human neutrophils by E. coli LPS. We estimated the changes in redox status (ROS level, intracellularglutathione, NO), apoptosis and mitochondrial potential of neutrophils under the LPS action in the presence and absence of GYY4137. GYY4137 reduces the ROS level, slightly reduces GSH, does not influence the NO level and has no apoptogenic effect. LPS induce the increasing of ROS level and inhibit spontaneous apoptosis of neutrophils. We found that GYY4137 prevents the growth of ROS caused by LPS and leads to a reduction of LPS-induced inhibition of neutrophil apoptosis. Thus the mechanism of GYY4137 protection against inflammation, triggered by bacterial infection, is concerned with the neutralization of LPS effect on neutrophils.

Na,K-ATPase is a transmembrane enzyme that creates a gradient of sodium and potassium, which is necessary for the viability of animal cells. The activity of Na,K-ATPase depends on the redox status of the cell, decreasing with oxidative stress and hypoxia. Previously, we have shown that the key role in the redox sensitivity of Na,K-ATPase is played by the regulatory glutathionylation of cysteine residues of the catalytic alpha subunit, which leads to the inhibition of the enzyme. In this study, the effect of reducing agents (DTT, ME, TCEP) on the level of glutathionylation of the alpha subunit of Na,K-ATPase from rabbit kidneys and the enzyme activity has been evaluated. We have found that the reducing agents partially deglutathionylate the protein, which leads to its activation. It was impossible to completely remove glutathionylation from the native rabbit kidney protein. The treatment of a partially denatured protein on the PVDF membrane with reducing agents (TCEP, NaBH4) also does not lead to the complete deglutathionylation of the protein. The obtained data indicate that Na,K-ATPase isolated from rabbit kidneys has both regulatory and basal glutathionylation, which appears to play an important role in the redox regulation of the function of Na, K-ATPase in mammalian tissues.

Na,K-ATPase maintains sodium and potassium homeostasis. It is the only known receptor for cardiotonic steroids such as ouabain. Binding of ouabain to Na,K-ATPase leads to the activation of Src kinase and the subsequent initiation of intracellular signaling pathways, including the induction of apoptosis. Changes in Na,K-ATPase activity is one of the earliest responses to hypoxia and is most critical for cell survival. However, it is not known how the hypoxia affects the functioning of Na,K-ATPase as a receptor. We have shown that, under the conditions of hypoxia and ischemia, ouabain is less toxic for murine fibroblast cells (SC-1 cell line) and ouabain does not cause an increase in the level of reactive oxygen species, which is typically observed at 20% pO2. Under hypoxia, the treatment of cells with ouabain also does not lead to the activation of Na,K-ATPase-associated Src kinase. Thus, at low oxygen content, the receptor function of Na,K-ATPase is altered, and cells become less sensitive to cardiotonic steroids. The decrease in sensitivity to cardiotonic steroids, which is evident at hypoxic conditions, should be taken into account in clinical practice. At the same time, in the presence of ouabain the cells are less sensitive to hypoxia, which indicates that cardiotonic steroids can be protective in acute ischemia.

Показали, что причиной ингибирования Na,K-АТФазы при её длительной инкубации с бета-амилоидом (А 42), белком болезни Альцгеймера, является изменение тиолового редокс-статуса клеток, приводящее к индукции глутатионилирования -субъединицы Na,K-АТФазы. Для восстановления активности Na,K-АТФазы предложено использовать восстановители, способствующие нормализации редокс-статуса клеток и деглутатионилированию белка.

Experimental evidences indicate that heat-shock protein 70 (HSP70) can serve as a prospective therapeutic agent to treat Alzheimer's disease (AD). It has demonstrated a neuroprotective effect in vivo on mice models of AD. Moreover, HSP70 decreases oxidative stress in neurons induced by amyloid-β (Aβ42) and its more toxic form with isomerized Asp7 (isoAβ42). The dysfunction of Ubiquitin-proteasome system (UPS) is observed in AD. UPS is responsible for the degradation of the majority of cellular proteins and plays an important role in protecting cells from oxidative stress. Here, we have shown that the incubation of human neuroblastoma cells SK-N-SH with isoAβ42 increases the activity of intracellular proteasomes, which are the principal elements of the UPS. On the contrary, the proteasomal activity was decreased in isoAβ42-treated cells in the presence of exogenous HSP70. These results highlight the existence of an interplay between Aβ peptides, proteasomes, and HSP70.

The progress of neurodegeneration in Alzheimer's disease is closely associated with inflammatory processes in the brain tissues induced by beta-amyloid peptides (Aβ). In this paper, we showed that Aβ(1-42) and isoAβ(1-42) in human neuroblastoma cells SK-N-SH and promonocyte THP-1 activated the production of tumor necrosis factor (TNFα). Notably, isoAβ(1-42) had the strongest effect on the increase in the level of TNFα. The addition of recombinant heat-shock protein HSP70 reduces TNFα production induced by Aβ, which leads to a decrease in neuronal cell damage at the organism level.

Zinc ions form complexes with β-amyloid peptides and play an important role in Alzheimer's disease pathogenesis. It has been demonstrated by turbidimetry and correlation spectroscopy that synthetic peptide Aβ16 representing the metal-binding domain of β-amyloid is able to interact with nucleic acids, chondroitin polysulfate, and dextran sulfates in the presence of zinc ions. The amino acid D7H substitution enhanced the peptide binding to polyanions, whereas the H6R and H6A-H13A substitutions abolished this interaction. It is suggested that the metal-binding domain may serve as a zinc-dependent site of β-amyloid interaction with biological polyanions including DNA, RNA, and glycosaminoglycans.

The progress of neurodegeneration in Alzheimer's disease is closely associated with inflammatory processes in the brain tissues induced by beta-amyloid peptides (Aβ). In this paper, we showed that Aβ(1-42) and isoAβ(1-42) in human neuroblastoma cells SK-N-SH and promonocyte THP-1 activated the production of tumor necrosis factor (TNFα). Notably, isoAβ(1-42) had the strongest effect on the increase in the level of TNFα. The addition of recombinant heat-shock protein HSP70 reduces TNFα production induced by Aβ, which leads to a decrease in neuronal cell damage at the organism level.

Bacterial ribonuclease binase is a potential anticancer agent. In the present study, we have determined the toxic effect of binase towards cell lines of T-cell acute lymphoblastic leukemia Jurkat and CEMss. We have shown that binase induces apoptosis in these cells. At the same time, binase does not cause toxic effects in leukocytes of healthy donors, which suggests that binase activity towards leukemic cells is selective. We have found that the treatment of cancer cells with binase leads to a reduction in reactive oxygen species and transcription factor NFκB levels, and demonstrated that these effects are a common feature of the action of RNases on cancer cells.

Снижение содержания кислорода в тканях, наблюдаемое при целом ряде патологических процессов, неизбежно приводит к их повреждению. Одна из главных причин повреждения и гибели клеток в условиях гипоксии – нарушение работы систем поддержания ионного баланса. Ингибирование активности Na,K-АТРазы, основного ион-транспортирующего белка плазматической мембраны животных клеток, происходящее при снижении концентрации кислорода, считается одним из самых ранних и критичных для жизнеспособности клеток событий. В настоящее время проводится активный поиск модуляторов активности Na,K-АТРазы. Традиционно с этой целью используют сердечные гликозиды, однако вызываемые ими тяжелые побочные эффекты вынуждают искать альтернативные ингибиторы Na,K-АТРазы. Ранее нами было обнаружено, что глутатионилирование каталитической субъединицы Na,K-АТРазы приводит к полному ингибированию фермента. В представленной работе показано, что вещества, увеличивающие уровень глутатионилирования Na,K-АТРазы: этилглутатион (et-GSH), окисленный глутатион (GSSG) и N-ацетилцистеин (NAC), повышают выживаемость клеток в условиях недостатка кислорода, предотвращают падение уровня АТР и нормализуют редокс-статус клеток. Установлен диапазон концентраций, в котором проявляется максимальный защитный эффект этих веществ и отсутствуют выраженные цитотоксические свойства: 0.2–0.5 мМ – у et-GSH, 0.2–1 мМ – у GSSG, 10–15 мМ – у NAC. Полученные результаты показывают перспективность разработки методов защиты тканей от повреждения в условиях кислородного голодания путем изменения степени глутатионилирования Na,K-АТРазы.

Bacterial ribonucleases (RNases) are considered to be potential anticancer agents. One of most important determinants of RNase cytotoxicity is the net charge of the molecule. In this work a set of mutants of the RNase from Streptomyces aureofaciens (RNase Sa), differing in the net charge of the protein molecules (from -7 to +6) and localization of additional positive charge at the N- or C-terminus of the molecule is used to study inhibition of cell growth. It has been found that the mutants of RNase with increased cationicity most effectively inhibit the growth of HEKhSK4 cells. Additional positive charge at the C-terminus of the molecule also increases the cytotoxic properties of RNases. It has been shown that RNase cytotoxicity correlated with the level of inhibition of the K+-current in cells.

Decreasing the amount of oxygen in the tissues under hypoxic and ischemic conditions, observed at a number of pathologic processes, inevitably leads to their damage. One of the main causes of cell damage and death is a violation of the systems maintaining ionic balance. Na,K-ATPaseis a basic ion-transporting protein of animal cell plasma membrane and inhibition of the Na,K-ATPase activity at lower concentrations of oxygen is one of the earliest and most critical events for cell viability. Currently there is an active search for modulators of Na,K-ATPase activity. For this purpose traditionally used cardiac glycosides but the existence of serious adverse effects forced to look for alternative inhibitors of Na,K-ATPase. Previously we have found that the glutathionylation of Na,K-ATPase catalytic subunit leads to a complete-inhibition of the enzyme. In this paper it is shown that the agents which increase the level of Na,K-ATPase glutathionylation: ethyl glutathione (et-GSH), oxidized glutathione (GSSG) and N-acetyl cysteine (NAC), increase cell survival under oxygen deficiency conditions, prevent decline of ATP in the cells and normalize their redox status. Concentration range in which these substances have a maximum protective effect, and does not exhibit cytotoxic properties was defined: for et-GSH 0.2-0.5 mM, for GSSG 0.2-1 mM, for NAC 10 to 15 mM. The results show prospects for development of methods for tissues protection from damage caused by oxygen starvation by varying the degree of Na,K-ATPase glutathionylation.

Neuronal cell death in Alzheimer's disease is associated with the development of oxidative stress caused by the reactive oxygen species (ROS), which can be generated as a result of the effect of beta-amyloid peptides. One of the sources of ROS is hydrogen peroxide, inducing the apoptosis and necrosis of neural tissue cells. The mechanism of hydrogen peroxide apoptotic action includes launching signaling pathways that involve protein kinases PI3K, p38MAPK, JNK and ERK. Oxidative stress leads to increased synthesis of heat-shock proteins in the cells including HSP70. It was shown that the exogenous HSP70 could reduce generation of ROS in cells. In this study, we determined how HSP70 affected apoptosis and necrosis in human neuroblastoma cells SK-N-SH, induced by hydrogen peroxide and β-amyloid peptide Aβ(1-42). It was shown that HSP70 reduces the cytotoxic effects of hydrogen peroxide and beta-amyloid, and protein kinases PI3K and JNK play an important role in the mechanism of HSP70 protective effect on the peroxide induced apoptosis in SK-N-SH cells.