Studying Signaling Pathway Activation in TRAIL-Resistant Macrophage-Like Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a malignant neoplasm characterized by extremely low curability and survival. The inflammatory microenvironment and maturation (differentiation) of AML cells induced by it contribute to the evasion of these cells from effectors of antitumor immunity. One of the key molecular effectors of immune surveillance, the cytokine TRAIL, is considered a promising platform for developing selective anticancer drugs. Previously, under in vitro conditions of the inflammatory microenvironment (a three-dimensional high-density culture of THP-1 AML cells), we demonstrated the emergence of differentiated macrophage-like THP-1ad clones resistant to TRAIL-induced death. In the present study, constitutive activation of proinflammatory signaling pathways, associated transcription factors, and increased expression of the anti-apoptotic BIRC3 gene were observed in TRAIL-resistant macrophage-like THP-1ad AML cells. For the first time, a bioinformatic analysis of the transcriptome revealed the main regulator, the IL1B gene, which triggers proinflammatory activation and induces resistance to TRAIL in THP-1ad macrophage-like cells.

[The Redox-Catalytic Properties of Cobalamins].

Vitamin B12, or cobalamin, is essential for normal body function and is used in the therapies of different diseases. Vitamin B12 has anti-inflammatory and antioxidant properties that can play an important role in the prevention of some diseases. On the other hand, it has been reported that vitamin B12 in combination with such reducing agents as ascorbate (vitamin C) and thiols showed prooxidant activity. This review provides information on the roles of vitamin B12 in diseases accompanied by inflammation and oxidative stress and the effects of vitamin B12 administrated alone and in combinations with different reducing agents such as ascorbate and thiols on oxidative stress. In addition, the mechanisms of prooxidant actions of combinations of vitamin B12 with these reducing agents depending on the form of vitamin B12 (hydroxocobalamin and cyanocobalamin) are discussed. Understanding the mechanisms of prooxidant action of vitamin B12 is necessary for developing strategies for therapeutic administration of vitamin B12.

Effect of Morphogenetic Protein BMP-2 on X-Ray Density of Bone Defect in the Experiment.

In experiments on Wistar rats, a simulated defect in the flat bones of the skull was filled with a collagen sponge of animal origin impregnated with BMP-2 or pure sponge; in control rats, the defect was left open. During follow-up, X-ray density of the collagen sponge in the experimental groups differed significantly. The results attest to the absence of spontaneous remodeling of the bone tissue under conditions modeled focal defect. Moreover, stimulation of reparative processes by the collagen matrix did not lead to positive dynamics. Saturation of the collagen sponge with BMP-2 in a concentration of 0.05 mg/ml allowed increasing Xray density of the bone starting from week 4.

Study of Biointegration and Elastic-Strength Properties of a New Xenopericardium-Based Biomaterial for Reconstructive Cardiovascular Surgery.

We analyzed biocompatibility, elastic-strength properties, and biointegration potential of a new biomaterial made of xenopericardium for reconstructive cardiovascular surgery. The biomaterial manufactured by the proposed technology demonstrated high biocompatibility and biointegration potential and its elastic-strength properties 2-4-fold surpassed that of native pericardium. The obtained results attested to good prospects of using the proposed technology for preparing biomaterials for reconstructive cardiovascular surgery.

Hydroxycobalamin catalyzes the oxidation of diethyldithiocarbamate and increases its cytotoxicity independently of copper ions.

It is known that some metals (Cu, Zn, Cd, Au) markedly increase the toxic effect of thiocarbamates. It was shown in the present study that hydroxycobalamin (a form of vitamin B12, HOCbl), which incorporates cobalt, significantly enhances the cytotoxicity of diethyldithiocarbamate (DDC), decreasing its IC50 value in tumor cells three to five times. The addition of HOCbl to aqueous DDC solutions accelerated the reduction of oxygen. No hydrogen peroxide accumulation was observed in DDC + HOCbl solutions; however, catalase slowed down the oxygen reduction rate. Catalase as well as the antioxidants N-acetylcysteine (NAC) and glutathione (GSH) partially inhibited the cytotoxic effect of DDC + HOCbl, whereas ascorbate, pyruvate, and tiron, a scavenger of superoxide anion, had no cytoprotective effect. The administration of HOCbl into DDC solutions (> 1 mM) resulted in the formation of a crystalline precipitate, which was inhibited in the presence of GSH. The data of UV and NMR spectroscopy and HPLC and Mass Spectrometry (LC/MS) indicated that the main products of the reaction of DDC with HOCbl are disulfiram (DSF) and its oxidized forms, sulfones and sulfoxides. The increase in the cytotoxicity of DDC combined with HOCbl occurred both in the presence of Cu2+ in culture medium and in nominally Cu-free solutions, as well as in growth medium containing the copper chelator bathocuproine disulfonate (BCS). The results indicate that HOCbl accelerates the oxidation of DDC with the formation of DSF and its oxidized forms. Presumably, the main cause of the synergistic increase in the toxic effect of DDC + HOCbl is the formation of sulfones and sulfoxides of DSF.

Combined effect of G3139 and TSPO ligands on Ca(2+)-induced permeability transition in rat brain mitochondria.

Permeability of the mitochondrial outer membrane is determined by the activity of voltage-dependent anion channels (VDAC) which are regulated by many factors and proteins. One of the main partner-regulator of VDAC is the 18 kDa translocator protein (TSPO), whose role in the regulation of membrane permeability is not completely understood. We show that TSPO ligands, 1 µM PPIX and PK11195 at concentrations of 50 µM, accelerate opening of permeability transition pores (mPTP) in Ca(2+)-overloaded rat brain mitochondria (RBM). By contrast, PK11195 at 100 nM and anti-TSPO antibodies suppressed pore opening. Participation of VDAC in these processes was demonstrated by blocking VDAC with G3139, an 18-mer phosphorothioate oligonucleotides, which sensitized mitochondria to Ca(2+)-induced mPTP opening. Despite the inhibitory effect of 100 nM PK11195 and anti-TSPO antibodies alone, their combination with G3139 considerably stimulated the mPTP opening. Thus, 100 nM PK11195 and anti-TSPO antibody can modify permeability of the VDAC channel and mPTP. When VDAC channels are closed and TSPO is blocked, permeability of the VDAC for calcium seems to be the highest, which leads to accelerated pore opening.

[Inhibition of NF-kB Activation Decreases Resistance in Acute Myeloid Leukemia Cells to TRAIL-induced Apoptosis in Multicellular Aggregates].

Suppression of resistance in acute myeloid leukemia cells to TRAIL-induced apoptosis in multicellular aggregates, was studied using small molecule inhibitors of the activation of the transcription factor NF-kB - NF-k9 Activation Inhibitor IV and JSH-23 at non-toxic concentrations. NF-kB Activation Inhibitor IV and JSH-23 reduced resistance in the acute myeloid leukemia cells in multicellular aggregates to cytotoxic action of recombinant protein izTRAIL. It is shown that the use of these inhibitors decreased the phosphorylation of the RelA (p65) as a main marker activation of the transcription factor NF-kB. We discuss a possible reason for increasing resistance in acute myeloid leukemia cells to TRAIL-induced apoptosis in multicellular aggregates.

[Density compensation effect in arboreal communities of the West Caucasus].

Density compensation effect (DCE) is qualified as increase of some species abundance after dropping of other species out of a community, as a consequence of unutilized resources appearance and interspecies competition abatement. DCE was first noticed about 50 years ago but till now the questions on causes of its origin, its scale and intensity remain open due to the lack of field data as well as methodological problems. In the article, these questions have been tested using arboreous tier of the West Caucasus forest phytocenoses as a case study. An analysis of actual data is conducted by means of numerical experiments. Three scenarios have been modeled: in the first one there has been assumed no manifestations of compensatory processes; in the second, after dropping some species out of community, all the other species rose in abundance; in the third, DCE was manifested through abundance increase of dominant species only. Model predictions on the analyzed parameters relationships have been checked against actual measurements. The results obtained suggest that for the DCE to occur, the absolute number of species in a community is not so much important as the ratio between their specific capacities, which is determined by environmental conditions, and the actual number of species, which may not match the capacity due to interference of regional processes (e.g., the history of cenoses formation, isolation, etc.). Thus, DCE does not appear to be an invariable consequence of environmental extremity and may occur in cenoses consisted of both many and few species.

Thiol antioxidants in combination with vitamin B12 induce apoptotic death of human lymphocytic leukemia cells by destabilization of lysosomes with the involvement of iron ions.

The extensively used thiol antioxidants (dithiothreitol, glutathione, and N-acetylcysteine) in combination with hydroxycobalamine (vitamin B12) gain toxic activity in relation to human lymphocytic leukemia cell line HL60. Combined treatment with thiol and vitamin B12 was followed by early destabilization of lysosomes and apoptotic death of cells. The cytotoxic effect was abolished by caspase inhibitors. An iron-chelating agent deferoxamine partly prevented cell death, while lysosomal protease inhibitor pepstatin produced no protective effect.

[Multikinase inhibitor sorafenib and HDAC inhibitor suberoylanilide hydroxamic acid suppress confluent resistance of cancer cells to recombinant protein izTRAIL].

Suppression of human tumor cell resistance to TRAIL-induced apoptosis in confluent cultures, using molecular target drugs (sorafenib and SAHA) at non-toxic concentrations was studied. Sorafenib, a multikinase inhibitor, and SAHA, an inhibitor of histone deacetylase, effectively suppressed resistance of confluent human cells derived from the skin carcinoma (A431 cell line) and fibrosarcoma (HT-1080 cell line). The effectiveness of suppression of confluent resistance with these inhibitors for human carcinoma A431 cells was significantly higher than that for the human ovarian carcinoma OVCAR-3 cells. For all cell lines studied, suppression of confluent resistance with SAHA was more effective than when sorafenib was used. The possible reason for increasing tumor cell resistance in confluent cultures and the importance of this phenomenon for understanding drug resistance of cells in the tumor tissue are discussed.

[Increase in resistance of A431 cancer cells to TRAIL-induced apoptosis in confluent cultures].

It was shown that cancer cells acquired resistance to TRAIL-induced apoptosis in confluent cultures. Recombinant protein izTRAIL induced apoptosis of human carcinoma A431 cells in the first hours after cell plating at a concentration of 3-10 ng/ml, while in confluent cultures these cells acquire resistance to protein izTRAIL even at the concentration of 2 mkg/ml. Detachment and suspending of the cells of confluent cultures immediately suppressed the resistance to izTRAIL. The cells of confluent cultures, being resistant to TRAIL-induced apoptosis continue progression through the cell cycle, as evidenced by the DNA cytograms and the Ki67p-GFP reporter system. Thus, the results showed that tumor A431 cells can acquire resistance to TRAIL-induced apoptosis in confluent cultures, while continue progression through the cell cycle, keeping the proliferative potential.

[60 years of the equilibrium theory of insular biogeography: problems of testing, results of the field studies, applied importance].

A review is presented of publications dealing with analysis of species richness of island biological communities and habitat islands based on the equilibrium theory of insular biogeography by MacArthur and Wilson (1963). Principal points of the theory are considered along with its shortcomings, problems and results of its testing. Also, possibilities are appraised for using recommendations elaborated on the base of the theory in nature conservation practice. The results of island and habitat island biota studies indicate that in many cases data corroborate the equilibrium theory while in many other cases they do not. In particular, for cenoses fragmented 50-250 years ago, especially for the ones formed by long living species, there have been no conspicuous effects of species relaxation detected. At that, the theory prediction of substantial reduction in species richness of fragmented communities in the long run is hardly disputed. The results of studies conducted in the field of insular biogeography are taken as a basis for recommendations on the long-term conservation of isolated communities integrity, although mostly they are of qualitative nature.

[Relationship between dominance and richness of local species: an analysis of the underlying reasons with arboreal and avian communities of the West Caucasus as an example].

Dominance level is traditionally expressed as a ratio between the number of individuals belonging to the most abundant species and the total number of individuals in a biological community. It is known that local species richness is usually higher in biological communities with high dominance level than in communities with low one. Taking into account a complex nature of the dominance phenomenon, the underlying reasons (or mechanisms) may be diverse: 1. Dominance level may be determined by bioecological traits of the most abundant species as well as stochastic impacts. The more abundant is dominant species, the fewer amount of resources goes to concomitant species and, therefore, the lower is community species richness. 2. The part of community resources used by the dominant species may be not a special case but can be a reflection of general pattern of resources distribution among species under specific environmental conditions. Correspondingly, in communities with higher dominance level there might be more "strict" distribution of resources among concomitant species, which, in turn, might influence community species richness. 3. The relationship between dominance level and community species richness may be caused by their dependence on the third variable, namely regional species pool. In the present paper we tackle the problem using arboreal and insectivorous bird communities of the West Caucasus as a case study. The data were collected in different altitudinal belts on both macroslopes of the western part of the Main Caucasian Ridge. The number of tree species and individual trees was counted within homogenous patches of arboreal phytocenoses 300 m2 in area. Species richness and numbers of insectivorous birds were estimated in course of route surveys with a route length being about 5 km. An analysis of empirical data was carried out using univariate and multiple correlation-regression techniques. The results indicate that the relationship between dominance and local species richness is determined to a large extent (by 50-60%) by a dominant taking over greater or lesser amount of the resources (mechanism 1). The role of two other mechanisms (2 and 3) is not so prominent--together, they are responsible for 25-40% of the relationship power. Relative contribution of different mechanisms to the relationship under consideration depends on conformity of species abundance rank structure with the geometric series model. At those sites where this conformity is manifested, the relationship between dominance level and species richness is due mainly to mechanisms 1 and 2, i.e., is determined by local processes. At other sites, where the conformity of species abundance rank structure with the geometric series model is not so good, a certain role belongs to the size of regional species pool (mechanism 3).

[The role of recipient cells in the mechanism of pathological calcification of heart valve and vascular transplants].

It was found using the model of subcutaneous implantation in rats that the calcification of the aorta wall occurs by two mechanisms of which one is dependent on, and the other independent of the migration of recipient cells to the transplant.

[Species saturation and invasibility of the plant communities: a hypothesis of species pools correlation].

Within a set of plant communities of Western Caucasus (open phytogroups of mountain near-riverbed shoals, phytocoenoses of glades and stepped meadows, tree tiers of riverbed forests), analysis is given to the correlation between levels of their species completeness, and number and total abandons of the adventive species. An assessment of the species completeness is based on an assumption that its increase is directed toward maximal species diversity with minimal abundance of any particular species in the species pool. The results indicate that both the open phytogroups and closed communities with relatively high species completeness are characterized by a less number of the adventive species, as well as by their lower average and total abundance, as compared to the species-incomplete ones. A hypothesis is suggested to explain correlation between species completeness and invasiveness of communities. It is based on assumption of an approximate average equivalence of aboriginal and adventive species and of stochastic processes of their immigrations and extinctions in particular communities. In accordance to this hypothesis, a number of the adventive species in particular parts of the communities is determined by their ratio to the aboriginal ones in these communities, with their average abundance being determined by the ratio of the species number to the total individuals number in those parts of the communities. The both ratios are relatively lower in more species-complete communities, which is caused by their lower saturation by adventive species. However, the impact of the latter factor on the stability of plant communities is not especially significant, as it is decreases with increase of intensiveity of adventive species invasions and respective increase of their portion in the species pools.

[Prooxidant and cytotoxic action of N-acetylcysteine and glutathione combined with vitamin Bl2b].

We studied the prooxidant and cytotoxic action of thiols N-acetylcystein (NAC) and glutathione (GSH) combined with vitamin Bl2b. The synergism of action of the thiols and Bl2b resulted in human carcinoma cell damage was found. It was shown that GSH and NAC in physiological doses combined with Bl2b caused the initiation of apoptosis. It was established that prooxidant action of the thiols combined with vitamin Bl2b, i. e. generation and accumulation of hydrogen peroxide in culture medium, led to intracellular oxidative stress and injury of cell redox system. These effects were completely abolished by nonthiol antioxidants catalase and pyruvate. The chelators of iron phenanthroline and deferoxamine did not suppress the H2O2 accumulation in culture medium but significantly inhibited the cell death induced by the thiols combined with Bl2b. Therefore, the thiols GSH and NAC widely used as antioxidants, in combination with vitamin Bl2b show prooxidant characteristics and induce, with the participation of intracellular iron, apoptotic HEp-2 cell death.

Improving biocompatibility of heart valve and vascular transplants by their devitalization and repopulation by recipient cells.

Inoculation of cells derived from the aorta of Wistar rats on devitalized porcine aortic walls 2-4-fold reduced their calcinosis after subcutaneous implantation to Wistar rats. Inoculation of Wistar rat bone marrow mesenchymal cells selected by adhesion activity did not reduce tissue calcinosis. The results indicate good prospects of repopulation of devitalized heart valve and vessel transplants by recipient vascular cells for reducing transplant calcinosis and improvement of their biocompatibility.