Sulfurtransferases and Cystathionine Beta-Synthase Expression in Different Human Leukemia Cell Lines.

The studies concerned the expression of sulfurtransferases and cystathionine beta-synthase in six human leukemia cell lines: B cell acute lymphoblastic leukemia-B-ALL (REH cells), T cell acute lymphoblastic leukemia-T-ALL (DND-41 and MOLT-4 cells), acute myeloid leukemia-AML (MV4-11 and MOLM-14 cells), and chronic myeloid leukemia-CML (K562 cells). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were performed to determine the expression of thiosulfate sulfurtransferase, 3-mercaptopyruvate sulfurtransferase, gamma-cystathionase, and cystathionine beta-synthase on the mRNA and protein level. Interestingly, we found significant differences in the mRNA and protein levels of sulfurtransferases and cystathionine beta-synthase in the studied leukemia cells. The obtained results may contribute to elucidating the significance of the differences between the studied cells in the field of sulfur compound metabolism and finding new promising ways to inhibit the proliferation of various types of leukemic cells by modulating the activity of sulfurtransferases, cystathionine beta-synthase, and, consequently, the change of intracellular level of sulfane sulfur as well as H2S and reactive oxygen species production.

Circadian Changes of Dendritic Spine Geometry in Mouse Barrel Cortex.

The circadian rhythmicity changes the density and shape of dendritic spines in mouse somatosensory barrel cortex, influencing their stability and maturation. In this study, we analyzed the main geometric parameters of dendritic spines reflecting the strength of synapses located on these spines under light/dark (12:12) and constant darkness conditions, in order to distinguish between endogenously regulated and light-driven parameters. Using morphological analysis of serial electron micrographs, as well as three-dimensional reconstructions, we found that the light induces elongation of single-synapse spine necks and increases in the diameter of double-synapse spine necks, increasing and decreasing the isolation of synapses from the parent dendrite, respectively. During the subjective night of constant darkness, we observed an enlargement of postsynaptic density area in inhibitory synapses and an increase in the number of polyribosomes inside double-synapse spines. The results show that both endogenous effect (circadian clock/locomotor activity) and light affect the morphological parameters of single- and double-synapse spines in the somatosensory cortex: light reduces the efficiency of excitatory synapses on single-synapse spines, increases the effect of synaptic transmission in double-synapse spines, and additionally masks the endogenous clock-driven enlargement of inhibitory synapses located on double-synapse spines. This indicates a special role of double-synapse spines and their inhibitory synapses in the regulation of synaptic transmission during both circadian and diurnal cycles in the mouse somatosensory cortex.

Identification of CD34+/PGDFRα+ Valve Interstitial Cells (VICs) in Human Aortic Valves: Association of Their Abundance, Morphology and Spatial Organization with Early Calcific Remodeling.

Aortic valve interstitial cells (VICs) constitute a heterogeneous population involved in the maintenance of unique valvular architecture, ensuring proper hemodynamic function but also engaged in valve degeneration. Recently, cells similar to telocytes/interstitial Cajal-like cells described in various organs were found in heart valves. The aim of this study was to examine the density, distribution, and spatial organization of a VIC subset co-expressing CD34 and PDGFRα in normal aortic valves and to investigate if these cells are associated with the occurrence of early signs of valve calcific remodeling. We examined 28 human aortic valves obtained upon autopsy. General valve morphology and the early signs of degeneration were assessed histochemically. The studied VICs were identified by immunofluorescence (CD34, PDGFRα, vimentin), and their number in standardized parts and layers of the valves was evaluated. In order to show the complex three-dimensional structure of CD34+/PDGFRα+ VICs, whole-mount specimens were imaged by confocal microscopy, and subsequently rendered using the Imaris (Bitplane AG, Zürich, Switzerland) software. CD34+/PDGFRα+ VICs were found in all examined valves, showing significant differences in the number, distribution within valve tissue, spatial organization, and morphology (spherical/oval without projections; numerous short projections; long, branching, occasionally moniliform projections). Such a complex morphology was associated with the younger age of the subjects, and these VICs were more frequent in the spongiosa layer of the valve. Both the number and percentage of CD34+/PDGFRα+ VICs were inversely correlated with the age of the subjects. Valves with histochemical signs of early calcification contained a lower number of CD34+/PDGFRα+ cells. They were less numerous in proximal parts of the cusps, i.e., areas prone to calcification. The results suggest that normal aortic valves contain a subpopulation of CD34+/PDGFRα+ VICs, which might be involved in the maintenance of local microenvironment resisting to pathologic remodeling. Their reduced number in older age could limit the self-regenerative properties of the valve stroma.

Combination of ERK2 and STAT3 Inhibitors Promotes Anticancer Effects on Acute Lymphoblastic Leukemia Cells.

BACKGROUND/AIM: Deregulated activation of signaling through the RAS/RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/RAF/MEK/ERK) and signal transducer and activator of transcription (STAT) pathways is involved in numerous hematological malignancies, making it an attractive therapeutic target. This study aimed to assess the effect of the combination of ERK2 inhibitor VX-11e and STAT3 inhibitor STA-21 on acute lymphoblastic leukemia cell lines REH and MOLT-4. MATERIALS AND METHODS: REH and MOLT-4 cell lines were cultured with each drug alone and in combination. Cell viability, ERK activity, cell cycle distribution, apoptosis and oxidative stress induction were assessed by flow cytometry. Protein levels of STAT3, phospho-STAT3, protein tyrosine phosphatase 4A3 (PTP4A3), survivin, p53 and p21 were determined by western blotting. RESULTS: VX-11e in combination with STA-21 significantly inhibited cell viability, induced G0/G1 cell-cycle arrest, enhanced production of reactive oxygen species, and induced apoptosis. These effects were associated with an increased level of p21 protein in REH cells and with reduced levels of phopho-STAT3, survivin and PTP4A3 proteins in MOLT-4 cells. CONCLUSION: Our findings provide a rationale for combined inhibition of RAS/RAF/MEK/ERK and STAT3 pathways in order to enhance anticancer effects against acute lymphoblastic leukemia cells.

Targeting the MAPK/ERK and PI3K/AKT Signaling Pathways Affects NRF2, Trx and GSH Antioxidant Systems in Leukemia Cells.

The mitogen-activated protein kinase (MAPK)/extracellular signal kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signal transduction pathways have been implicated in the pathogenesis of leukemia. The aim of this study was to investigate the effect of the combination of ERK1/2 inhibitor AZD0364 and PI3K inhibitor ZSTK474 on acute lymphoblastic leukemia (ALL) REH, MOLT-4, acute myeloid leukemia (AML) MOLM-14, and chronic myeloid leukemia (CML) K562 cell lines. To evaluate the interactions of the drugs, cells were treated for 48 h with AZD0364 or ZSTK474 alone and in combination at fixed ratios. The combinatorial effects of both inhibitors were synergistic over a wide range of concentrations in REH, MOLT-4, and MOLM-14 cell lines. However, in K562 cells, the effects were found to be antagonistic. Furthermore, AZD0364 and ZSTK474 significantly decreased both ERK1/2 and AKT activation in REH, MOLT-4, and MOLM-14 cells. The results showed that incubation with both AZD0364 and ZSTK474 inhibited cell viability, increased reactive oxygen species (ROS) production, and induced apoptosis in leukemia cells. We observed that combined treatment with AZD0364 and ZSTK474 affected nuclear factor-κB (NF-κB) and antioxidant protein levels: NF-E2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), thioredoxin (Trx), thioredoxin reductase (TrxR), and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio. These effects were accompanied with decreased antiapoptotic survivin protein level. However, distinct cell line dependent effects were observed. In conclusion, the combination of AZD0364 and ZSTK474 can exert a synergistic anticancer effect in ALL and AML cells, which is associated with the induction of oxidative stress and the involvement of cellular antioxidant defense mechanisms.

Polyelectrolyte Multilayer Films Modification with Ag and rGO Influences Platelets Activation and Aggregate Formation under In Vitro Blood Flow.

Surface functionalization of materials to improve their hemocompatibility is a challenging problem in the field of blood-contacting devices and implants. Polyelectrolyte multilayer films (PEMs), which can mimic functions and structure of an extracellular matrix (ECM), are a promising solution to the urgent need for functional blood-contacting coatings. The properties of PEMs can be easily tuned in order to provide a scaffold with desired physico-chemical parameters. In this study chitosan/chondroitin sulfate (Chi/CS) polyelectrolyte multilayers were deposited on medical polyurethane. Afterwards PEMs were modified by chemical cross-linking and nanoparticles introduction. Coatings with variable properties were tested for their hemocompatibility in the cone-plate tester under dynamic conditions. The obtained results enable the understanding of how substrate properties modulate PEMs interaction with blood plasma proteins and the morphotic elements.

Rolling or Two-Stage Aggregation of Platelets on the Surface of Thin Ceramic Coatings under in Vitro Simulated Blood Flow Conditions.

The process of modern cardiovascular device fabrication should always be associated with an investigation of how surface properties modulate its hemocompatibility through plasma protein adsorption as well as blood morphotic element activation and adhesion. In this work, a package of novel assays was used to correlate the physicochemical properties of thin ceramic coatings with hemocompatibility under dynamic conditions. Different variants of carbon-based films were prepared on polymer substrates using the magnetron sputtering method. The microstructural, mechanical, and surface physicochemical tests were performed to characterize the coatings, followed by investigation of whole human blood quality changes under blood flow conditions using the "Impact R" test, tubes' tester, and radial flow chamber assay. The applied methodology allowed us to determine that aggregate formation on hydrophobic and hydrophilic carbon-based coatings may follow one of the two different mechanisms dependent on the type and conformational changes of adsorbed blood plasma proteins.

Circadian clock regulates the shape and content of dendritic spines in mouse barrel cortex.

Circadian rhythmicity affects neuronal activity induced changes in the density of synaptic contacts and dendritic spines, the most common location of synapses, in mouse somatosensory cortex. In the present study we analyzed morphology of single- and double-synapse spines under light/dark (12:12) and constant darkness conditions. Using serial electron micrographs we examined the shape of spines (stubby, thin, mushroom) and their content (smooth endoplasmic reticulum, spine apparatus), because these features are related to the maturation and stabilization of spines. We observed significant diurnal and circadian changes in the shape of spines that are differentially regulated: single-synapse spines remain under circadian clock regulation, while changes of double-synapse spines are driven by light. The thin and mushroom single-synapse spines, regardless of their content, are more stable comparing with the stubby single-synapse spines that show the greatest diversity. All types of double-synapse spines demonstrate a similar level of stability. In light/dark regime, formation of new mushroom single-synapse spines occurs, while under constant darkness new stubby single-synapse spines are formed. There are no shape preferences for new double-synapse spines. Diurnal and circadian alterations also concern spine content: both light exposure and the clock influence translocation of smooth endoplasmic reticulum from dendritic shaft to the spine. The increasing number of mushroom single-synapse spines and the presence of only those mushroom double-synapse spines that contain spine apparatus in the light phase indicates that the exposure to light, a stress factor for nocturnal animals, promotes enlargement and maturation of spines to increase synaptic strength and to enhance the effectiveness of neurotransmission.

Combination of ERK2 inhibitor VX-11e and voreloxin synergistically enhances anti-proliferative and pro-apoptotic effects in leukemia cells.

ERK1/2 inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination of ERK2 inhibitor VX-11e and voreloxin on MOLM-14, K562, REH and MOLT-4 leukemia cell lines. We found that VX-11e alone and in combination with voreloxin significantly decreased ERK activation in all cell lines tested. To evaluate the interactions of the drugs, cells were treated for 24 h with VX-11e or voreloxin alone and in combination at fixed ratios based on IC50 values. The combinatorial effects of both drugs were synergistic over a wide range of concentrations in MOLM-14, REH and MOLT-4 cell lines. In K562 cells, three effects were found to be additive, one antagonistic and only one synergistic. The results showed that incubation with both VX-11e and voreloxin inhibited the growth of leukemia cells, affected cell cycle and induced apoptosis. Furthermore, the molecular mechanism of these effects might be attributed to an increased expression of p21 and a decreased expression of survivin and NF-κB in all cell lines tested except from K562 cells. In conclusion, combination of VX-11e and voreloxin can exert a synergistic anticancer effect in leukemia cells.

TAK-733, a Selective MEK Inhibitor, Enhances Voreloxin-induced Apoptosis in Myeloid Leukemia Cells.

BACKGROUND/AIM: MEK inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination treatment of voreloxin with the MEK inhibitor TAK-733 on HL60 myeloid leukemia cells. MATERIALS AND METHODS: MAPK activity, cell viability, apoptosis, oxidative stress induction and AIF (apoptosis-inducing factor) distribution were assessed in HL60 cells cultured with each drug alone or with both drugs. RESULTS: TAK-733 alone at 5 µM significantly reduced MAPK activity and did not influence viability and apoptosis in HL60 cells. Voreloxin at concentration of 0.03-0.48 µM reduced cell viability and increased apoptosis rate. Incubation with both drugs caused further inhibition of cell viability and increased apoptosis associated with generation of reactive oxygen species (ROS) and nuclear translocation of AIF. CONCLUSION: Combination of TAK-733 and voreloxin can exert a synergistic anticancer effect in myeloid leukemia cells.

New aspects of antiproliferative activity of 4-hydroxybenzyl isothiocyanate, a natural H2S-donor.

The effect of 4-hydroxybenzyl isothiocyanate (HBITC), a natural H2S-donor from white mustard seeds (Sinapis alba), on the proliferation of human neuroblastoma (SH-SY5Y) and glioblastoma (U87MG) cells was studied and some aspects of the mechanism of its activity were suggested. The inhibition of both SH-SY5Y and U87MG cell proliferation was associated with an increase in the thiosulfate level, the number of cells with the inactive form of Bcl-2 protein, and with a decrease of mitochondrial membrane potential. Interestingly, HBITC results in downregulation of p53 protein and upregulation of p21 protein levels in SH-SY5Y cells. In the presence of elevated levels of H2S and thiosulfate, the sulfhydryl groups of p53 protein as well as Bcl-2 protein could be modified via HBITC-induced S-sulfuration or by oxidative stress. It seems that the induction of p21 protein level is mediated in SH-SY5Y cells by p53-independent mechanisms. In addition, HBITC-treatment caused downregulation of the level of mitochondrial rhodanese and 3-mercaptopyruvate sulfurtransferase, and consequently increased the level of the reactive oxygen species in SH-SY5Y cells.

A possible mechanism of inhibition of U87MG and SH-SY5Y cancer cell proliferation by diallyl trisulfide and other aspects of its activity.

The study was conducted to elucidate the mechanism of antiproliferative and antioxidative action of diallyl trisulfide (DATS), a garlic-derived organosulfur compound. Changes in the L-cysteine desulfuration, and the levels of cystathionine and non-protein thiols in DATS-treated human glioblastoma (U87MG) and neuroblastoma (SH-SY5Y) cells were investigated. The inhibition of proliferation of the investigated cells by DATS was correlated with an increase in the inactivated form of Bcl-2. In U87MG cells, an increased level of sulfane sulfur and an increased activity of 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese, the enzymes involved in sulfane sulfur generation and transfer, suggest that DATS can function as a donor of sulfane sulfur atom, transferred by sulfurtransferases, to sulfhydryl groups of cysteine residues of Bcl-2 and in this way lower the level of active form of Bcl-2 by S-sulfuration. Diallyl trisulfide antioxidative effects result from an increased level of cystathionine, a precursor of cysteine, and an increased glutathione level. MPST and rhodanese, the level of which is increased in the presence of DATS, can serve as antioxidant proteins.

Combined orcein and martius scarlet blue (OMSB) staining for qualitative and quantitative analyses of atherosclerotic plaques in brachiocephalic arteries in apoE/LDLR-/- mice.

Numerous cellular and extracellular components should be analyzed in sections of atherosclerotic plaques to assess atherosclerosis progression and vulnerability. Here, we combined orcein (O) staining for elastic fibers and martius scarlet blue (MSB) polychrome to visualize various morphological contents of plaque in brachiocephalic arteries (BCA) of apoE/LDLR-/- mice. Elastic fibers (including broken elastic laminae and 'buried' fibrous caps) were stained purple and they could be easily distinguished from collagen fibers (blue). Orcein allowed clear identification of even the finest elastic fibers. Erythrocytes were stained yellow and they could easily be discerned from mature fibrin (red). Old fibrin tends to acquire blue color. The method of OMSB staining is simple, takes less than 1 h to perform and can be adapted to automatic stainers. Most importantly, the color separation is good enough to allow digital automatic segmentation of specific components in tissue section and quantitative analysis of the plaque constituents. OMSB was used to compare atherosclerotic plaques in proximal and distal regions of BCA in apoE/LDLR-/- mice. In conclusion, OMSB staining represents a novel staining that could be routinely used for qualitative and quantitative microscopic assessments of formaldehyde-fixed and paraffin-embedded sections of arteries with atherosclerotic lesions.

Influence of osteoclasts and osteoprotegerin on the mode of calcific degeneration of aortic valves.

INTRODUCTION: Calcific aortic valve disease is associated with inflammation and calcification, thus the osteoprotegerin (OPG), receptor activator of nuclear factor κB (RANK) and its ligand (RANKL) system involved in osteoclastogenesis and inflammation may play a significant role in valve degeneration. OBJECTIVES: The aim of this study was to assess whether circulating OPG, sRANKL, and other bone metabolism markers can predict the presence of osteoclasts in stenotic valves and to evaluate their impact on the mode of degeneration. PATIENTS AND METHODS: The study involved 60 patients with aortic stenosis who underwent valve replacement surgery and subsequently were divided into 2 groups: osteoclastic (n = 12) and nonosteoclastic (n = 48), according to the presence or absence of intravalvular osteoclasts. Before the surgery, we measured serum levels of OPG, sRANKL, osteocalcin, osteopontin, tumor necrosis factor α (TNF-α), interleukin (IL) 1ß, and IL-6. Immunohistochemistry and morphometry were used to determine the extent of valve calcification, lipid accumulation, neovascularization, and the number and phenotype of macrophages. RESULTS: Compared with the nonosteoclastic group, patients with intravalvular osteoclasts had lower levels of OPG (P = 0.0006) and TNF-α (P = 0.02) and less frequently had diabetes (P = 0.04). Their valves showed higher incidence of ossification (P = 0.002), higher total (P = 0.008) and M2 macrophage counts (P = 0.0002), increased neovascularization (P = 0.003), and lower accumulation of lipids (P = 0.04). They also showed a negative correlation between valve calcification and age (r = -0.79, P = 0.002), which was not observed in patients without osteoclasts. In a multivariate analysis, low circulating OPG levels and the absence of diabetes were predictors of intravalvular osteoclastic differentiation. CONCLUSIONS: The presence of osteoclasts in stenotic valves associated with low circulating OPG levels and an enhanced proportion of M2 macrophages can represent a variant of calcific aortic valve disease with a specifically regulated calcification process.