Search

Cobalamin is an essential nutrient required for the normal functioning of cells. Its deficiency can lead to various pathological states. Hydroxocobalamin (HOCbl) and cyanocobalamin (CNCbl) are the forms of vitamin B12 that are most commonly used for supplementation. There is substantial evidence indicating that cobalamins can both suppress and promote oxidative stress; however, the mechanisms underlying these effects are poorly understood. Here, it was shown that the oxidation of thiols catalyzed by HOCbl and CNCbl is accompanied by ROS production and induces, under certain conditions, oxidative stress and cell death. The form of vitamin B12 and the structure of thiol play a decisive role in these processes. It was found that the mechanisms and kinetics of thiol oxidation catalyzed by HOCbl and CNCbl differ substantially. It was discussed how these differences may explain different levels of ROS production and cytotoxicity induced by combinations of thiols with HOCbl and CNCbl. On the whole, the data obtained provide a new insight into the redox processes in which cobalamins are involved and might be helpful in developing new approaches to the treatment of some cobalamin-responsive disorders in which oxidative stress is an important component. In addition, these data may be useful for a better understanding of mechanisms underlying induction of different types of death of cancer cells and in a search for new targets for anticancer therapy.

Cobalamin is an essential nutrient required for the normal functioning of cells. Its deficiency can lead to various pathological states. Hydroxocobalamin (HOCbl) and cyanocobalamin (CNCbl) are the forms of vitamin B12 that are most commonly used for supplementation. There is substantial evidence indicating that cobalamins can both suppress and promote oxidative stress; however, the mechanisms underlying these effects are poorly understood. Here, it was shown that the oxidation of thiols catalyzed by HOCbl and CNCbl is accompanied by reactive oxygen species (ROS) production and induces, under certain conditions, oxidative stress and cell death. The form of vitamin B12 and the structure of thiol play a decisive role in these processes. It was found that the mechanisms and kinetics of thiol oxidation catalyzed by HOCbl and CNCbl differ substantially. HOCbl increased the rate of oxidation of thiols to a greater extent than CNCbl, but quenched ROS in combination with certain thiols. Oxidation catalyzed by CNCbl was generally slower. Yet, the absence of ROS quenching resulted in their higher accumulation. The aforementioned results might explain a more pronounced cytotoxicity induced by combinations of thiols with CNCbl. On the whole, the data obtained provide a new insight into the redox processes in which cobalamins are involved. Our results might also be helpful in developing new approaches to the treatment of some cobalamin-responsive disorders in which oxidative stress is an important component.

Previously, we found a significant increase in TRAIL (TNFα-related apoptosis-inducing ligand) resistance of acute myeloid leukemia (AML) cells in multicellular aggregates in vitro that simulate the location of leukemia cells in the bone marrow. In this study, we tried to assess whether the increased resistance of the AML cells to the cytotoxic effect of TRAIL in multicellular aggregates in vitro can be associated with the launch of mechanisms mediated by cell differentiation or the acquisition of a pro-inflammatory phenotype. The phenotype of THP-1 cells in aggregated and disaggregated cultures was studied in comparison with the phenotype of a disaggregated THP-1 cell culture exposed to factors that induce monocyte-like differentiation or pro-inflammatory phenotype. It was found that an increase in the TRAIL resistance of the THP-1 cells in the aggregated culture was accompanied by the appearance of signs characteristic of monocyte-like differentiation and a pro-inflammatory phenotype. The results are of interest for developing approaches for suppressing TRAIL resistance of AML cells in human bone marrow.

The combined effect of melatonin and cytarabin at low concentration on the cells of acute promyelocytic leukemia HL-60 was studied. It has been shown that the combined effect of these substances led to a decrease in the number of cells by 70% and the mitotic activity index by 75%. It was found that the combined effect of melatonin and cytarabin also contributed to a decrease in the expression of mitochondrial proteins. It has been demonstrated that the combined use of melatonin with low concentration of cytarabin (2 nM) reduced the expression of the voltage-dependent anion channel (VDAC1). Melatonin in combination with cytarabin similarly affected the expression of translocator protein (TSPO) and antiapoptotic protein Bcl-2. The synergistic effect of melatonin in the combined action with cytarabin has been revealed, which provides an increase in the effectiveness of its antitumor action.

—The resistance of leukemic cells to the action of chemotherapeutic drugs is the main reason for insufficient efficacy in the treatment of acute myeloid leukemia. Microenvironmental factors play one of the key roles in the formation of the phenotype of drug resistance of leukemic cells. The elucidation of the nature of drug resistance mediated by the conditions of the microenvironment is important for the identification of new pharmacological targets for conservative directed therapy. We conducted a study of the role of differentiation in the macrophage direction in increasing the drug resistance of acute myeloid leukemia cells in multicellular aggregates. It was shown that maturation does not occur in the macrophage direction in multicellular aggregates. These data indicate that other mechanisms contribute to an increased drug resistance of acute myeloid leukemia cells in multicellular aggregates.

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.

—Different synthetic calcium phosphate compounds are used as osteoplastic materials for filling of bone defects during surgical reconstruction of bone. Synthetic nanosized hydroxyapatite is of particular interest because of its similarity to the chemical composition of bone and its osteoinductive potential. However, it is also known that nanosized hydroxyapatite can inhibit the processes of biointegration of biomaterials, although we have no clear understanding of the mechanisms that are responsible for this effect. In our in vitro studies suppression of cell adhesion on nanosized hydroxyapatite that may lead to anoikis was shown. Studies in which rat subcutaneous heterotopic implantation methods were used showed that nanosized hydroxyapatite in vivo formed a slightly resorbable agglomerate that blocks cell migration into the material, as well as neocollagenesis processes in it, including the processes of encapsulation of the material. In this case, the processes of neocollagenesis along the periphery of the sample were observed only over a dense layer of dead cells (cellular debris) surrounding the agglomerated nanosized hydroxyapatite. Addition of demineralized bone collagen matrix to nanosized hydroxyapatite as a conductive component contributed to colonization of the material by the cells, resorption of nanosized hydroxyapatite, and active construction of the neocollagen matrix. These results show that modification of nanosized hydroxyapatite with a conductive component is required in order to prevent its agglomeration and thus increase its biointegration and osteoinductive potential in the body.

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., Graphical abstract fx1, Highlights • HOCbl synergistically enhances the cytotoxicity of DDC against tumor cells. • HOCbl catalyzes the oxidation of DDC and the formation of DSF and its oxidized forms. • HOCbl enhances DDC cytotoxicity by catalyzing extracellular DSF oxidation. • HOCbl catalyzes DSF oxidation in DDC solutions in a radical-mediated manner. • HOCbl enhances DDC cytotoxicity independently of extracellular Cu ions.

Сalcium phosphate materials have been applied in clinical medicine since 1920. Among calcium phosphate materials, hydroxyapatite (HAp) is traditionally of the greatest interest, because HAp is the main inorganic component of bone tissues. However, synthetic HAр ceramics subjected to high-temperature processing, as it turned out, have a rather limited use as an osteoplastic material. Since 1990, due to advances in chemical technology, new materials of pasty nanocrystalline HAр have been developed, which are promising for the directed influence on the process of bone tissue regeneration. This review briefly summarizes the experimental and clinical data related to the application of micro- and nano-sized hydroxyapatite, and evaluated the potential of pasty nanocrystalline HAp as a material for guided bone regeneration.

The composite material based on reinforcement of polyamide filaments enclosed by a nonwoven matrix of nanoscaled bioresorbable poly(3-hydroxybutyrate) fibers was developed for application as an artificial ligament implant. The aim of this study was to investigate biodegradability and biocompatibility of the developed implant, as well as its stress-strain properties. The study results show the polyamide core of the implant has stress-strain properties comparable with a natural ligament. Simultaneously, the polyhydroxybutyrate external layer provides high biocompatibility and bioresorbability of the developed implant. The material has proven to be effective under in vivo tests with experimental rats as a ligament replacement for damaged Achilles tendons. Due to cell attachment and growth on the fibrous matrix during 5 weeks postsurgery, regenerated connective tissue was formed substituting for the polymeric implant, which confirmed its efficiency in contrast to the polyamide filament implant with a much longer resorption time. The results obtained indicate application prospects of polyamide-polyhydroxybutyrate implants for reconstructive surgery. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2708-2713, 2018.

—A comparative study of the expression of pro- and anti-apoptotic receptors to cytokine TRAIL (TNF-related apoptosis-inducing ligand) on immature blast cells of acute myeloid leukemia, on the cells of acute myeloid leukemia differentiated into monocyte-like and macrophage-like cells, and on normal monocytes and macrophages has been performed. It has been shown that a decrease in the expression of proapoptotic receptors to TRAIL and the acquisition of resistance to TRAIL-induced apoptosis occurs not only during the differentiation of acute myeloid leukemia cells into monocyte-like and macrophage-like cells, but also during the maturation of normal monocytes into macrophages. Thus, the differentiation of both acute myeloid leukemia cells and normal myeloid cells in the direction of monocyte-macrophages mediates a decrease in the expression of proapoptotic receptors to TRAIL, which in turn can determine the resistance of normal myeloid cells to TRAIL-induced apoptosis.

Melatonin (N-acetyl-5-methoxytryptamine), a neuroendocrine hormone of the pineal gland, participates in the modulation of the mitochondrial nonspecific pore (mitochondrial permeability transition pore, mPTP). According to the results we obtained, protoporphyrin IX (PPIX, a ligand of the TSPO translocator protein) induces the opening of the mPTP in brain mitochondria; melatonin slows the induction of the mPTP in rat brain mitochondria incubated with PPIX. Induction of the mPTP activates protein kinases/protein phosphatases that are involved in the regulation of protein phosphorylation. The TSPO modulates protein phosphorylation; in the presence of PPIX, the phosphorylation of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) increases. Here, we showed an increase in the degree of phosphorylation of CNPase in PPIX-treated rat brain mitochondria, a melatonin-induced decrease in level of CNPase phosphorylation in mitochondria incubated with PPIX; hence, we propose that melatonin participates in TSPOmodulated protein phosphorylation.

The mechanisms underlying cancer cell resistance to TRAIL-induced apoptosis are highly diverse and are of scientific and practical interest. We have demonstrated earlier that the human skin cancer A431 cell line sensitive to TRAIL-induced apoptosis in low-density culture (3 × 104 cells/cm2) acquired transient TRAIL resistance in dense, confluent cultures (3 × 105 cells/cm2). We have derived two cell lines, TRAILresistant (A431-R) and TRAIL-sensitive (A431-S), regardless of cell culture density, from parental A431 cell line using selection and clonal technique. We have obtained the genome-wide transcriptome data for these cell lines in low and high (confluent) density cultures and analyzed signaling pathway changes in these cells with respect to confluence-dependent and confluence-independent resistance to TRAIL-induced apoptosis. Signaling pathways were assayed using GSEA, which provides an entire cell transcriptome analysis and takes into account even small significant changes in the genes belonging to different signaling pathways that may be more important than a significant increase in a single gene. It is found that the confluence-dependent TRAIL resistance of cells A431 is associated with activation of a vast number of signaling pathways linked with the protective effects of RAS and interferon signaling, a change in the cytokine microenvironment, and suppression of the antitumor immune response. In contrast, the confluence-independent resistance of the A431- R cells is associated with activation of Nuclear factor erythroid 2-related factor 2 (NFE2L2) and Peroxisome proliferator-activated receptor alpha (PPARA) transcription factors. The TRAIL sensitivity of A431-S cells in confluent culture correlates with a decrease of the RAS and PPARA signaling pathways activity.

Nonwoven microfibrous materials based on poly(3-hydroxybutyrate) (PHB) were produced by electrospinning. The fiber supramolecular structure was investigated using DSC and EPR. It was shown that the PHB fiber consisted of perfect and less perfect crystallites with an amorphous phase that combined regions of different density. A model of an artificial implant for reconstructive surgery was developed based on a nonwoven fibrous PHB matrix with the required physicomechanical characteristics, high biocompatibility, and the optimal morphology for growth of cell cultures and increased implant regenerative capability.

We studied the effect of isoquinoline carboxamide (PK11195) applied alone or in combination with melatonin on the differentiation of N1E-115 mouse neuroblastoma cells (clone C-1300). PK11195 is a synthetic ligand of the mitochondrial translocator protein (TSPO), which is one of the mitochondrial proteins that are responsible for the opening of the mitochondrial pore (mPTP); expression of this protein is enhanced in different types of cancer cells. PK11195 is considered as a potential anticancer drug. It has been shown that PK11195 at a nontoxic/subtoxic concentration induces the differentiation of the N1Е-115 mouse neuroblastoma cells and suppresses cell proliferation; the magnitude of this effect coincides with the effect induced by melatonin at a nontoxic concentration. The combination of PK11195 with melatonin did not intensify its effect as a differentiation inducer. Based on the results of Western blot analysis, it has been hypothesized that the differentiation of N1Е-115 neuroblastoma cells is associated with the expression of mitochondrial 2’,3’-cyclonucleotide-3’-phosphodiesterase but not with the expression of the mitochondrial translocator protein TSPO.

Creation of implants for the restoration of ligaments and tendons in their complete/massive injury is an urgent task. The efficacy of the restoration of completely resected Achilles tendons after implantation of a new material consisting of absorbable pledged threads Dar-Vin USP 5/0 and conductive nonofibrous scaffolds of polyhydroxybutyrate placed around the threads into the zone of defect was studied in Wistar rats. Either nanofibrous implant (experiment) or pledged threads (control 1) were placed into the zone of completely resected Achilles tendon (control 1), or the postresection wound was sutured without placement of any material (control 2). It was stated that from 2nd to 13th week after implantation of nanofibrous implant the animals from the experimental group used their hind paws in full volume. In control groups 1 and 2 the animals moved with difficulties. In the experimental group of animals in 6 and 13 weeks after implantation a soft tissue regenerate connecting the calcanean tuber and gastrocnemius muscle was present with the absence of gastrocnemius muscle contracture as compared with the control groups. Histologic examination showed the similarity of the regenerate on nanofibrous implant with the native Achilles tendon. Thus, the suggested implant ensured functional and morphologic restoration of totally resected Achilles tendon in rats and can be considered as a perspective prototype of new materials initiating effective regeneration of ligaments and tendons in their massive injuries.

Calcium as a secondary messenger regulates the phosphorylation of several membrane-bound proteins in brain and liver mitochondria. Regulation of the activity of different protein kinases and phosphatases by Ca2+ occurs through its binding with calmodulin. The protein phosphorylation is strongly dependent on the Ca2+-induced mitochondrial permeability transition pore (mPTP) opening. 2&prime, 3&prime, Cyclic nucleotide-3&prime, phosphodiesterase (CNPase) was phosphorylated by protein kinases A and C. CNPase and melatonin (MEL) might interact with calmodulin. The effects of the calmodulin antagonist calmidazolium and the inhibitor of protein kinase A H89 on mPTP opening in rat brain mitochondria of male Wistar rats were investigated. In addition, the role of CNPase, serine/threonine kinases, and MEL in the mPTP opening was examined. The anti-CNPase antibody added to rat brain mitochondria (RBM) reduced the content of CNPase in mitochondria. The threshold [Ca2+] decreased, and mitochondrial swelling was accelerated in the presence of the anti-CNPase antibody. H89 enhanced the effect of anti-CNPase antibody and accelerated the swelling of mitochondria, while CmZ abolished the effect of anti-CNPase antibody under mPTP opening. The levels of phospho-Akt and phospho-GSK3&beta, increased, while the MEL content did not change. It can be assumed that CNPase may be involved in the regulation of these kinases, which in turn plays an important role in mPTP functioning.

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.

Melatonin is produced by the pineal gland. It can be regarded as an anticancer agent and used for combined therapy, owing to its oncostatic, antioxidant, and immunoregulatory activities. Retinoic acid is widely used for the treatment of acute promyelocytic leukemia, however, it has adverse effects on the human organism. We investigated the effect of melatonin and reduced concentrations of retinoic acid on the activation of proliferation in acute promyelocytic leukemiaon a cell model HL-60. The combined effect of these compounds leads to a reduction in the number of cells by 70% and the index of mitotic activity by 64%. Combined treatment with melatonin and retinoic acid decreased the expression of the Bcl-2. The mitochondrial isoform VDAC1 can be a target in the treatment of different tumors. The combined effect of and retinoic acid at a low concentration (10 nM) decreased VDAC1 expression. Melatonin in combination with retinoic acid produced a similar effect on the expression of the translocator protein. The coprecipitation of VDAC with 2&prime, 3&prime, cyclonucleotide-3&prime, phosphodiesterase implies a possible role of its in cancer development. The combined effect of retinoic acid and melatonin decreased the activity of the electron transport chain complexes. The changes in the activation of proliferation in HL-60 cells, the mitotic index, and Bcl-2 expression under combined effect of retinoic acid (10 nM) with melatonin (1 mM) are similar to changes that are induced by 1 &mu, M retinoic acid. Our results suggest that MEL is able to improve the action the other chemotherapeutic agent.

Excessive generation of reactive oxygen species (ROS) in mitochondria and the opening of the nonselective mitochondrial permeability transition pore are important factors that promote cardiac pathologies and dysfunction. The hormone melatonin (MEL) is known to improve the functional state of mitochondria via an antioxidant effect. Here, the effect of MEL administration on heart mitochondria from aged rats with acute cardiac failure caused by isoprenaline hydrochloride (ISO) was studied. A histological analysis revealed that chronic intake of MEL diminished the age-dependent changes in the structure of muscle fibers of the left ventricle, muscle fiber swelling, and injury zones characteristic of acute cardiac failure caused by ISO. In acute heart failure, the respiratory control index (RCI) and the Ca2+ retention capacity in isolated rat heart mitochondria (RHM) were reduced by 30% and 40%, respectively, and mitochondrial swelling increased by 34%. MEL administration abolished the effect of ISO. MEL partially prevented ISO-induced changes at the subunit level of respiratory complexes III and V and drastically decreased the expression of complex I subunit NDUFB8 both in control RHM and in RHM treated with ISO, which led to the inhibition of ROS production. MEL prevents the mitochondrial dysfunction associated with heart failure caused by ISO. It was shown that the level of 2′,3′-cyclicnucleotide-3′-phosphodiasterase (CNPase), which is capable of protecting cells in aging, increased in acute heart failure. MEL also retained the CNPase content in RHM both in control experiments and after ISO-induced heart damage. We concluded that an increase in the CNPase level promotes cardioprotection.

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.

One of the major causes of low efficiency of the therapy for acute myeloid leukemia is drug resistance of leukemic cells. Microenvironment plays a key role in formation of the phenotype of leukemic cell drug resistance. Investigation of the mechanisms of microenvironment-mediated drug resistance is important to identify novel pharmacological targets for the acute myeloid leukemia therapy. We studied the role of cell aggregation in the drug resistance of leukemic cells. We showed the increased resistance of acute myeloid leukemia cells THP-1 to bortezomib, doxorubicin and fludarabine in multicellular aggregates. In the multicellular aggregates of THP-1 with the higher drug resistance, cell proliferation activity did not change, while the intracellular level of anti-apoptotic protein Bcl-2 increased. Inhibition of the aggregation of THP-1 cells prevented drug resistance. This work demonstrates the involvement of cell aggregation in the formation of drug resistance phenotype in leukemic cells.

TRAIL (TNF-alpha Related Apoptosis-Inducing Ligand) is an attractive candidate for anticancer therapy. TRAIL selectively kills tumor cells, without damaging normal cells. It is known that cancer cells can acquire resistance to chemotherapeutic drugs, oxidative stress in high density culture. This phenomenon appears as a multi-cell resistance, cell adhesion-dependent resistance, or density-dependent resistance of tumor cells. However, it is unclear whether such resistance appears in TRAIL-induced apoptosis. We observed that the resistance to izTRAIL of all tumor cell lines used was considerably increased in confluent cultures. The increase in tumor cell resistance in dense populations is not related to their proliferative status. It was shown that the dissociation of calcium-dependent cell-cell contacts with EGTA did not suppress tumor cell resistance to izTRAIL in confluent cultures. This phenomenon of cancer cells resistance to TRAIL-induced apoptosis should be considered in the development of methods of anticancer therapy.

The process of aging is considered to be tightly related to mitochondrial dysfunction. One of the causes of aging is an increased sensitivity to the induction of mitochondrial permeability transition pore (mPTP) opening in the inner membrane of mitochondria. Melatonin, a natural antioxidant, is a hormone produced by the pineal gland. The role of melatonin whose level decreases with aging is well understood. In the present study, we demonstrated that long-term treatment of aged rats with melatonin improved the functional state of mitochondria; thus, the Ca2+ capacity was enhanced and mitochondrial swelling was deaccelerated in mitochondria. Melatonin prevented mPTP and impaired the release of cytochrome c and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) from mitochondria of both young and aged rats. Our data suggest that melatonin retains СNPase inside mitochondria, thereby providing the protection of the protein against deleterious effects of 2',3'-cAMP in aging.

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 μg/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.

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.

Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of α-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting α-LA – OA samples. In this study we present a novel method for preparation of α-LA – OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLA–OA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLA–OA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLA–OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca2+ transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active α-LA – OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties.

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.

A specific state of the human milk Ca(2+) binding protein alpha-lactalbumin (hLA) complexed with oleic acid (OA) prepared using an OA-pretreated ion-exchange column (HAMLET) triggers several cell death pathways in various tumor cells. The possibility of preparing a hLA-OA complex with structural and cytotoxic properties similar to those of the HAMLET but under solution conditions has been explored. The complex was formed by titration of hLA by OA at pH 8.3 up to OA critical micelle concentration. We have shown that complex formation strongly depends on calcium, ionic strength, and temperature; the optimal conditions were established. The spectrofluorimetrically estimated number of OA molecules irreversibly bound per hLA molecule (after dialysis of the OA-loaded preparation against water followed by lyophilization) depends upon temperature: 2.9 at 17 degrees C (native apo-hLA; resulting complex referred to as LA-OA-17 state) and 9 at 45 degrees C (thermally unfolded apo-hLA; LA-OA-45). Intrinsic tryptophan fluorescence measurements revealed substantially decreased thermal stability of Ca(2+)-free forms of HAMLET, LA-OA-45, and OA-saturated protein. The irreversibly bound OA does not affect the Ca(2+) association constant of the protein. Phase plot analysis of fluorimetric and CD data indicates that the OA binding process involves several hLA intermediates. The effective pseudoequilibrium OA association constants for Ca(2+)-free hLA were estimated. The far-UV CD spectra of Ca(2+)-free hLA show that all OA-bound forms of the protein are characterized by elevated content of alpha-helical structure. The various hLA-OA complexes possess similar cytotoxic activities against human epidermoid larynx carcinoma cells. Overall, the LA-OA-45 complex possesses physicochemical, structural, and cytotoxic properties closely resembling those of HAMLET. The fact that the HAMLET-like complex can be formed in aqueous solution makes the process of its preparation more transparent and controllable, opening up opportunities for formation of active complexes with specific properties.

Prooxidant and cytotoxic effects of thiols N-acetylcysteine (NAC) and glutathione (GSH) were studied in combinations with vitamin B12b. Both GSH and NAC at physiological doses when combined with B12b were shown to cause initiation of apoptosis. It was established that the prooxidant action of NAC (or GSH) combined with B12b, i.e., generation and accumulation of hydrogen peroxide in culture medium, led to intractellular oxidative stress and cell redox imbalance. These effects are completely prevented by nonthiol antioxidants catalase and pyruvate. The chelators of iron phenanthroline and deferoxamine do not suppress the H2O2 accumulation in culture medium, but inhibit cell death induced by NAC combined with B12b or by GSH combined with B12b. Therefore, the thiols GSH or NAC in combination with vitamin B12b reveal prooxidant properties and induce, with participation of intracellular iron, apoptotic HEp-2 cell death.

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.

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.

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF–EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor–PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83) 1 (PA63) 6 or (PA83) 2 (PA63) 5 heteroheptamer with LF–EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.

Incubation of human laryngeal epidermoid carcinoma HEp-2 cells with hydroxocobalamin (vitamin B12b) and ascorbic acid (vitamin C) for 1 h initiated oxidative stress accompanied by damage to mitochondria and increase in intracellular oxidative activity. Studies of the kinetics of these processes showed that the increase in intracellular H2O2 activity and mitochondrial damage are more likely a result, but not the cause of cell apoptosis during the first hour of their incubation with vitamins B12b and C.

The paper reviews the structure formation of fibrous materials based on poly-3-hydroxybutyrate depending on parameters of electrospinning and characteristics of polymer solution. Fiber structure was studied by DSC, ESR and SEM. The molecular weight affects the diameter and uniformity of the fiber. An electromechanical impact leads to an orientation of crystalline structure in the fiber. The design of an artificial bioresorbable implant based on nano- and microfibers of poly-3-hydroxybutyrate is created. Dynamics of growth of mesenchymal stem cells on poly-3-hydroxybutyrate scaffolds is studied. Successful field tests of implants of the Achilles tendon in Wistar rats are conducted.

The combination of hydroxocobalamin (vitamin B12b) and ascorbicacid (vitamin C) can cause the death of tumor cells at the concentrationsof the components at which they are nontoxic when administeredseparately. This cytotoxic action on epidermoid human larynx carcinomacells HEp-2 in vitro is shown to be due to the hydrogen peroxidegenerated by the combination of vitamins B12b and C. The drop inthe glutathione level preceding cell death was found to be the result ofcombined action of the vitamins. It is supposed that the induction of celldeath by combined action of vitamins B12b and C is connected to the damageof the cell redox system.

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.

The translocator protein (TSPO; 18 kDa) is a high-affinity cholesterol-binding protein located in the outer membrane of mitochondria. A domain in the C-terminus of TSPO was characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide may participate in the regulation of mitochondrial membrane permeability. Herein, we report the effect of the synthetic CRAC peptide, VLNYYVW, on mitochondrial permeability transition pore (mPTP) opening. It was found that the CRAC peptide alone prevents the mPTP from opening, as well as the release of apoptotic factors (cytochrome c, AIF, and EndoG) in rat brain mitochondria (RBM). Co-incubation of CRAC, together with the TSPO drug ligand, PK 11195, resulted in the acceleration of mPTP opening and in the increase of apoptotic factor release. VLNYYVW did not induce swelling in rat liver mitochondria (RLM). 3,17,19-androsten-5-triol (19-Atriol; an inhibitor of the cholesterol-binding activity of the CRAC peptide) alone and in combination with the peptide was able to stimulate RLM swelling, which was Ca2+- and CsA-sensitive. Additionally, a combination of 19-Atriol with 100 nM PK 11195 or with 100 µM PK 11195 displayed the opposite effect: namely, the addition of 19-Atriol with 100 µM PK 11195 in a suspension of RLM suppressed the Ca2+-induced swelling of RLM by 40%, while the presence of 100 nM PK 11195 with 19-Atriol enhanced the swelling of RLM by 60%. Taken together, these data suggest the participation of the TSPO’s CRAC domain in the regulation of permeability transition.

Incubation of Ehrlich ascites carcinoma and HEp-2 human epidermoid laryngeal carcinoma cells with hydroxycobalamin (vitamin B12b) and ascorbic acid induced generation and accumulation of double-stranded DNA fragments (23,000 b.p. and longer) in cells. The same vitamins alone in the same concentrations produced no such effects. DNA degradation in HEp-2 cells caused by long-term (4 h) incubation with 5-25 microM hydroxycobalamin and ascorbic acid (1:10-1:40 molar ratio) at 37 degrees C was comparable with that induced by gamma-irradiation in a dose of 150 Gy at 4 degrees C.

The probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) is widely used for studying the superoxide anion production and the efficiency of antioxidants in biological systems. Here we report that a number of sulfur-containing compounds applied in biochemical and cytological studies are able to suppress MCLA-derived chemiluminescence (MDCL) independent of their capability to scavenge superoxide anion. The most effective MDCL quenchers appeared to be the substances with thiocarbamoyl and thiocarbonyl groups coupled to cyclic molecules and several thiol- and disulfide-containing compounds. The analysis of MDCL kinetics in a xanthine oxidase system allows one to rapidly discriminate between true antioxidants and the quenchers of chemiluminescence.

OBJECTIVES Rejection is thought to contribute to the degeneration of valved allografts. Most proposed methods of decellularisation allow usage of treated valves in pulmonic position. We developed a new protocol of devitalization, which provides cell death and suppression of calcification using digitonin and ethylenediaminetetraacetic acid. The aim of the study was to evaluate new allografts in a chronic canine model. METHODS Two groups of adult mongrel dogs (5 in each) were used for allograft implantation. The cryopreserved viable (group 1) and devitalized (group 2) heart valve aorta allografts were tested. Allografts were implanted as valved patches into the thoracic aorta and explanted after four months. Histologic examination and fluorescence microscopy were used to test tissue matrix and cells in allografts. Mineralized calcium in the samples was detected using absorption spectroscopy. RESULTS The fluorescence microscopy proved that a significant number of cells were viable in the allografts after their cryopreservation (group 1) and all the cells were dead after anticalcinosis devitalisation (group 2). No damage of tissue matrix was observed in group 2 after devitalisation. After explantation, the cusps in both groups were either stuck to aorta wall of the allografts, or there were thrombus clots between the cusps and the wall. Internal surface was covered with neointima. Media of aortic wall was acellular. Repopulation of the viable and devitalized tissues with recipient cells during a 4-month follow-up period was not observed. In non-treated allografts, aortic walls had areas of dissection and infiltration of lymphoid cells. Devitalized patches were homogenous without dissection areas. There was no immune-cell infiltration in devitalized matrix as opposed to cryopreserved vital tissue. CONCLUSIONS The new devitalizing technology seems effective in decreasing immune response to homologous tissue. It does not affect elasto-mechanic properties and collagenous structure of allografts. The presented data stimulate interest to the anticalcinosis devitalisation technology as an affective tool for improving biocompatibility of allografts.