The inhibitory effect of dinitrosyl iron complexes (NO donors) on myeloperoxidase activity

The effect of synthetic analogues of dinitrosyl mononuclear iron complexes (DNICs) with functional sulfur-containing ligands (NO donors) on the activity of myeloperoxidase (MPO) was studied, and their efficiency was evaluated. It was shown that the enzyme MPO is the molecular target of DNICs. It was found that six DNICs inhibited the activity of MPO and one compound potentiated it. The evaluation of their efficiency showed that two DNICs effectively inhibited the activity of MPO by 50% at IC₅₀ = 2 × 10^–4 M and IC₅₀ = 5 × 10^–7 M.     

Dinitrosyl iron complexes--structure and biological functions

Formation of dinitrosyl iron complexes (DNICs), which can be described by general formula Fe(NO)2(L)2, where L is carbonyl-, nitrosyl- or imino- complexing ligand, was observed in many kinds of living organisms, in a wide spectrum of physiological conditions associated with inflammation, ischemia/reperfusion and cancer. Accumulation of DNICs coincides with intensified production of nitric oxide in macrophages, neurons, endothelial cells, Langerhans' cells and hepatocytes. Low-molecular thiol-containing DNICs (DNIC-(RS)2) show vasodilatory action and they are proposed to play a role of nitric oxide transducers and stabilizers. DNICs have been shown to modulate redox potential of the cell via inhibition of glutathione-dependent enzymes, such as glutathione reductase, S-transferase and peroxidase. Although there is a convincing experimental evidence for their NO and NO+ donating function, the nature of DNICs formed in biological systems, their stability and biological role is still a matter of discussion.     

High glucose represses the proliferation of tendon fibroblasts by inhibiting autophagy activation in tendon injury

Diabetic foot ulcer (DFU) is a kind of common and disabling complication of Diabetes Mellitus (DM). Emerging studies have demonstrated that tendon fibroblasts play a crucial role in remodeling phase of wound healing. However, little is known about the mechanism underlying high glucose (HG)-induced decrease in tendon fibroblasts viability. In the present study, the rat models of DFU were established, and collagen deposition, autophagy activation and cell apoptosis in tendon tissues were assessed using Hematoxylin–Eosin (HE) staining, immunohistochemistry (IHC), and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, respectively. Tendon fibroblasts were isolated from Achilles tendon of the both limbs, and the effect of HG on autophagy activation in tendon fibroblasts was assessed using Western blot analysis, Cell Counting Kit-8 (CCK-8) assay, and flow cytometry. We found that cell apoptosis was increased significantly and autophagy activation was decreased in foot tendon tissues of DFU rats compared with normal tissues. The role of HG in regulating tendon fibroblasts viability was then investigated in vitro, and data showed that HG repressed cell viability and increased cell apoptosis. Furthermore, HG treatment reduced LC3-II expression and increased p62 expression, indicating that HG repressed autophagy activation of tendon fibroblasts. The autophagy activator rapamycin reversed the effect. More importantly, rapamycin alleviated the suppressive role of HG in tendon fibroblasts viability. Taken together, our data demonstrate that HG represses tendon fibroblasts proliferation by inhibiting autophagy activation in tendon injury.     

Distribution and pharmacokinetics of dinitrosyl iron complexes in rat organs

Dinitrosyl iron complexes (DNICs) have been traced in rat blood and organs after intravenous infusion of Oxacom. It is shown that the active principle (DNIC with glutathione) is rapidly distributed through the organism and deposited in blood and organs as protein-bound DNICs. The specific levels of DNIC in the main body organs are comparable, whereas its apparent lifetimes relate as blood < heart = lung < liver < kidney. Spin trapping assays indicate that protein-bound DNICs are a major but not the only form of NO deposition; the next largest depot is most probably formed by S-nitrosothiols. The gradual release of NO from such pools ensures the smooth and prolonged hypotensive effect of Oxacom.     

The effect of the bisphosphonate alendronate on viability of canine osteosarcoma cells in vitro

The objective of this study was to determine the effect of alendronate on the viability of canine osteosarcoma cells and nonneoplastic canine cells. The sample population was composed of canine osteosarcoma tumor cells. Osteosarcoma cells and canine fibroblasts were maintained in culture under standard conditions. The MTT assay for cell viability was performed after 24, 48, and 72 h of incubation with alendronate (0.001 to 1000 microM) or no drug (control). Plates were set up so that each concentration and the control had a sample number of 8. The optical density (OD) of each well was measured at 540 nm using an enzyme-linked immunosorbent assay microplate reader. The percent viability was determined for each concentration and for each incubation time. After 24 h of incubation of POS (parent osteosarcoma) and HMPOS cells with alendronate, there was no significant difference in mean OD at any drug concentration when compared with control samples. A significant concentration- and time-dependent reduction in mean OD of osteosarcoma cells was observed after 48 and 72 h of incubation, with alendronate concentrations ranging from 10 to 1000 microM. The lowest percent cell viability observed in treated cells was 35%. Conversely, alendronate did not significantly affect mean OD in fibroblasts, and the lowest percent cell viability observed was 76%. Our data indicate that alendronate may have the potential to inhibit canine osteosarcoma tumor growth. It will be important to determine the clinical relevance of these in vitro findings. If similar findings are observed in vivo, use of alendronate may also be indicated as an adjuvant to existing chemotherapeutic protocols.     

Dinitrosyl iron complexes with thiol-containing ligands as a base for developing drugs with diverse therapeutic activities: Physicochemical and biological substantiation

Dinitrosyl iron complexes (DNICs) with thiol-containing ligands occur in animal tissues as paramagnetic (EPR-active) mononuclear and diamagnetic (EPR-silent) binuclear species with the presence of nitric oxide. They provide stabilization and storage (within protein-bound DNICs) of nitric oxide, as well as its transport (within low-molecular-weight DNICs) to biological targets to serve as donors of not only nitric oxide itself but also of the nitrosonium ion (NO⁺). The latter function determines the ability of DNICs to S-nitrosylate various thiol-containing proteins. In this way, the complexes participate in a wide range of physiological and biochemical processes. With respect to the high and diverse level of biological activity and dose dependence of DNICs, they mimic the endogenous nitric oxide system. Taken together with the broad occurrence of DNICs (predominantly in the diamagnetic form) in animal tissues, this fact points to their role as an “operating form” of nitric oxide. It is thought that drugs designed on the basis of DNICs can substantially improve the efficiency of modern medical practice.     

Zinc and Propolis Reduces Cytotoxicity and Proliferation in Skin Fibroblast Cell Culture: Total Polyphenol Content and Antioxidant Capacity of Propolis

It has been demonstrated that zinc exerts its beneficial influence on skin fibroblasts. Propolis, a complex mixture of plant-derived and bees’ products, was reported to stimulate cicatrization processes in skin and prevent infections. The aim of this study was to find out how zinc and propolis influence human skin fibroblasts in cell culture and to compare the effect of individual compounds to the effect of a mixture of zinc and propolis. In this study, zinc, as zinc aspartate, at a concentration of 16 μM, increased human fibroblasts proliferation in cell culture, whereas propolis at a concentration of 0.01 % (w/v) revealed antiproliferative and cytotoxic action followed by mild cell necrosis. In culture, zinc was effectively transported into fibroblasts, and propolis inhibited the amount of zinc incorporated into the cells. An addition of propolis to the medium caused a decrease in the Zn(II) amount incorporated into fibroblasts. The obtained results also indicate an appreciable antioxidant property of propolis and revealed its potential as a supplement when applied at doses lower than 0.01 % (w/v). In conclusion, the present study showed that zinc had a protective effect on human cultured fibroblasts’ viability, although propolis revealed its antiproliferative action and caused mild necrosis.     

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor

It is shown that neokyotorphin (the -globin fragment 137–141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.     

Gaseous nitric oxide exhibits minimal effect on skin fibroblast extracellular matrix gene expression and immune cell viability

NO (nitric oxide) molecule is produced by various mammalian cell types and plays a significant role in inflammation, infection and wound healing processes. Recently, gNO (gaseous nitric oxide) therapy has been utilized for its potential clinical application as an antimicrobial agent, with special focus on skin infection. In a previous study, we demonstrated that 200 ppm gNO, 8 h/day for three consecutive days significantly reduced the number of bacteria in dermal wounds without compromising the viability and function of skin cells. To increase the feasibility and ease of its clinical use, we propose that different doses of gNO (5 to 10 K ppm) for 8 h and as short as 10 min be used, respectively. To achieve this, we set up in vitro experiments and asked whether (i) different doses of gNO have any toxic effect on immune cells and (ii) gNO has any modulating effect on key ECM (extracellular matrix) components in fibroblasts. To further investigate the effect of gNO, expression of more than 100 key ECM genes have been examined using gene array in human fibroblasts. As immune cells play an important role in wound healing, the effect of gNO on proliferation and viability of human and mouse lymphocytes was also examined. The findings showed that, the 5, 25, 75 and 200 ppm of gNO for 8 h slightly increased the expression of Col 5A3 (collagen type V alpha 3), and gNO at 5 ppm decreased the expression of MMP-1 (matrix metalloproteinase 1), while exposure of fibroblast to 10 K ppm of gNO for 10 min does not show any significant changes in ECM genes. Exposure to gNO resulted in inhibition of lymphocyte proliferation without affecting the cell viability. Taken together, our findings show that skin could be treated with gNO without compromising the role of ECM and immune cells in low concentrations with long time exposure or high concentrations for a shorter exposure time.     

Differential effects of trichostatin A on gelatinase A expression in 3T3 fibroblasts and HT-1080 fibrosarcoma cells: implications for use of TSA in cancer therapy

Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor with potential in cancer therapeutics. In a recent communication, we demonstrated that TSA is a selective, potent inhibitor of gelatinase A in 3T3 fibroblasts. In the present study, we extend these observations and examine the effects of TSA in 3T3 fibroblasts compared to HT-1080 fibrosarcoma cells with respect to gelatinase A expression, cell viability, and apoptosis. We find that while expression of gelatinase A in 3T3 fibroblasts is exquisitely sensitive to inhibition by TSA, expression of this enzyme in HT-1080 cells is minimally affected by this compound. Moreover, we show that TSA is pro-apoptotic in HT-1080 cells, but is anti-apoptotic in 3T3 cells. We propose a two-pronged model for the therapeutic action of TSA. On the one hand TSA selectively decreases cancer cell viability, while enhancing the viability of stromal cells. On the other hand, by selectively decreasing gelatinase A expression in stromal but not cancer cells, TSA acts to control metastatic potential by reducing the ability of metastatic cells to recruit stromal cells to secrete gelatinase A.     

Coenzyme Q10 reduces ethanol-induced apoptosis in corneal fibroblasts

Dilute ethanol (EtOH) is a widely used agent to remove the corneal epithelium during the modern refractive surgery. The application of EtOH may cause the underlying corneal fibroblasts to undergo apoptosis. This study was designed to investigate the protective effect and potential mechanism of the respiratory chain coenzyme Q(10) (CoQ(10)), an electron transporter of the mitochondrial respiratory chain and a ubiquitous free radical scavenger, against EtOH-induced apoptosis of corneal fibroblasts. Corneal fibroblasts were pretreated with CoQ(10) (10 μM) for 2 h, followed by exposure to different concentrations of EtOH (0.4, 2, 4, and 20%) for 20 s. After indicated incubation period (2-12 h), MTT assay was used to examine cell viability. Treated cells were further assessed by flow cytometry to identify apoptosis. Reactive oxygen species (ROS) and the change in mitochondrial membrane potential were assessed using dichlorodihydrofluorescein diacetate/2',7'-dichlorofluorescein (DCFH-DA/DCF) assays and flow-cytometric analysis of JC-1 staining, respectively. The activity and expression of caspases 2, 3, 8, and 9 were evaluated with a colorimetric assay and western blot analysis. We found that EtOH treatment significantly decreased the viability of corneal fibroblasts characterized by a higher percentage of apoptotic cells. CoQ(10) could antagonize the apoptosis inducing effect of EtOH. The inhibition of cell apoptosis by CoQ(10) was significant at 8 and 12 h after EtOH exposure. In EtOH-exposed corneal fibroblasts, CoQ(10) pretreatment significantly reduced mitochondrial depolarization and ROS production at 30, 60, 90, and 120 min and inhibited the activation and expression of caspases 2 and 3 at 2 h after EtOH exposure. In summary, pretreatment with CoQ(10) can inhibit mitochondrial depolarization, caspase activation, and cell apoptosis. These findings support the proposition that CoQ(10) plays an antiapoptotic role in corneal fibroblasts after ethanol exposure.     

Iron-sulfur proteins are the major source of protein-bound dinitrosyl iron complexes formed in Escherichia coli cells under nitric oxide stress

Protein-bound dinitrosyl iron complexes (DNICs) have been observed in prokaryotic and eukaryotic cells under nitric oxide (NO) stress. The identity of proteins that bind DNICs, however, still remains elusive. Here we demonstrate that iron-sulfur proteins are the major source of protein-bound DNICs formed in Escherichia coli cells under NO stress. Expression of recombinant iron-sulfur proteins, but not proteins without iron-sulfur clusters, almost doubles the amount of protein-bound DNICs formed in E. coli cells after NO exposure. Purification of recombinant proteins from the NO-exposed E. coli cells further confirms that iron-sulfur proteins, but not proteins without iron-sulfur clusters, are modified, forming protein-bound DNICs. Deletion of the iron-sulfur cluster assembly proteins IscA and SufA to block the [4Fe-4S] cluster biogenesis in E. coli cells largely eliminates the NO-mediated formation of protein-bound DNICs, suggesting that iron-sulfur clusters are mainly responsible for the NO-mediated formation of protein-bound DNICs in cells. Furthermore, depletion of the "chelatable iron pool" in wild-type E. coli cells effectively removes iron-sulfur clusters from proteins and concomitantly diminishes the NO-mediated formation of protein-bound DNICs, indicating that iron-sulfur clusters in proteins constitute at least part of the chelatable iron pool in cells.     

Effect of lactoferrin on the viability of Arn8 and HaCaT cell lines

The results on the effect of LF from different sources on the viability of two cell lines (Arn8 and HaCaT) are presented in the work. It is shown that both hLF and bLF have an effect on the viability of these two cell lines, and that bLF decreases the cell viability in much more extend than hLF. Possible action mechanisms of LF are discussed.     

Reversible inhibition of cAMP-induced axon formation in neuroblastoma cells by concanavalin a and vinblastine : Relationship to microtubules and cytotoxicity

The formation of axons induced by dibutyryl-adenosine 3′,5′-cyclic monophosphate (db-cAMP) in neuroblastoma cells was inhibited by concanavalin A (ConA) and vinblastine. These compounds also caused the retraction of existing axons. After removal of ConA or vinblastine, addition of db-cAMP again resulted in axon formation. The cytotoxicity of ConA and vinblastine for neuroblastoma cells was reduced when cell multiplication was inhibited by db-cAMP. Linearly growing normal fibroblasts were also more sensitive to the cytotoxic effect of ConA than confluent non-multiplying fibroblasts. The effects of ConA and vinblastine were additive both in their effects on axon formation and cytotoxicity. Wheat germ agglutinin (WGA) and lumicolchicine did not affect axon formation or reduce cell viability. It is suggested that ConA bound to the cell surface can interfere with the assembly of cytoplasmic microtubules involved in axon formation and cell division.     

Evaluation of gene targeting by homologous recombination in ovine somatic cells

Mouse models for some human genetic diseases are limited in their applications since they do not accurately reproduce the phenotype of the human disease. It has been suggested that larger animals, for example sheep, might produce more useful models, as some aspects of sheep physiology and anatomy are more similar to those of humans. The development of methods to clone animals from somatic cells provides a potential novel route to generate such large animal models following gene targeting. Here, we assess targeting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in ovine somatic cells using homologous recombination (HR) of targeting constructs with extensive (>11 kb) homology. Electroporation of these constructs into ovine fetal and post-natal fibroblasts generated G418-resistant clones, but none analyzed had undergone HR, suggesting that at least for this locus, it is an extremely inefficient process. Karyotyping of targeted ovine fetal fibroblasts showed them to be less chromosomally stable than post-natal fibroblasts, and, moreover, extended culture periods caused them to senesce, adversely affecting their viability for use as nuclear transfer donor cells. These data stress the importance of donor cell choice in somatic cell cloning and suggest that culture time be kept to a minimum prior to nuclear transfer in order to maximize cell viability.     

Cryopreservation of cell-containing poly(ethylene) glycol hydrogel microarrays

Here we describe the fabrication and preservation of mammalian cell-containing hydrogel microarrays that have potential applications in drug screening and pathogen detection. Hydrogel microstructures containing murine fibroblasts were fabricated on silicon substrates and subjected to a "stage-down" freezing process. The percent viability of both immortal and primary embryonic murine fibroblast cells within the gels was determined at various stages in the freezing process, showing that cells entrapped in hydrogel microstructures remained viable throughout the process. When compared to immortalized adherent cultures subjected to the same freezing process, cells within hydrogel structures had higher cell viabilities at all stages during preservation. Finally, the necessity of using a cryoprotectant, dimethyl sulfoxide (DMSO), was investigated. Cells in hydrogels were cryopreserved with and without DMSO. The addition of DMSO altered cell viability after the freeze-thaw process, enhancing viability in an immortalized cell line and decreasing viability in a primary cell line.     

Failure of hydrocortisone or growth factors to influence the senescence of fibroblasts in a new culture system for assessing replicative lifespan

It has been reported that the replicative lifespan of human fibroblasts can be substantially extended by supplementing the growth medium with hydrocortisone or increased levels of serum proteins. These observations have been made only on cell populations transferred many times at high cell density, and cumulative population doublings have been recorded, rather than a more direct measure of cell division potential. We have measured the replicative potential of human fibroblasts cultured so as to avoid conditions of high cell density, medium depletion, and departure from exponential growth. Two fetal lung and two newborn foreskin fibroblast strains were serially passaged in the presence or absence of hydrocortisone (HC), epidermal growth factor (EGF), and fibroblast growth factor (FGF) until they senesced. At each passage cells were plated at densities sufficiently low that colony-forming efficiency could be calculated. We determined cumulative population doublings and also estimated the number of cell generations attained under each condition. FGF caused small but possibly significant changes, while HC and EGF failed to substantially alter replicative lifespan. The reported effect of HC on the doubling potential of fetal lung fibroblasts is therefore not an inevitable action of this hormone on the senescence mechanism, but may instead depend for its apparent activity on the passage regimen used. The fibroblast's insensitivity to EGF as a modulator of replicative potential, as compared with the keratinocyte, whose lifespan can be tripled by EGF, implies that the mechanisms limiting the replicative potential of these two cell types are not identical.     

Influence of oxidized low-density lipoproteins (LDL) on the viability of osteoblastic cells

Cardiovascular diseases have recently been noted as potential risk factors for osteoporosis development. Although it is poorly understood how these two pathologies are related, it is a known fact that oxidized low-density lipoproteins (OxLDL) constitute potential determinants for both of them. The current study investigated the metabolism of OxLDL by osteoblasts and its effect on osteoblastic viability. The results obtained show that OxLDL are internalized but not degraded by osteoblasts while they can selectively transfer their CE to these cells. It is also demonstrated that OxLDL induce proliferation at low concentrations but cell death at high concentrations. This reduction of osteoblast viability was associated with lysosomal membrane damage caused by OxLDL as demonstrated by acridine orange relocalization. Accordingly, chloroquine, an inhibitor of lysosomal activity, accentuated cell death induced by OxLDL. Finally, we demonstrate that osteoblasts have the capacity to oxidize LDL and thereby potentially increase the local concentration of OxLDL. Overall, the current study confirms the potential role of OxLDL in the development of osteoporosis given its influence on osteoblastic viability.     

Long-lasting hypotensive action of stable preparations of dinitrosyl-iron complexes with thiol-containing ligands in conscious normotensive and hypertensive rats

Previously we established the hypotensive action of nitric oxide donors, dinitrosyl-iron complexes (DNIC) with thiol-containing ligands, stored in frozen solution at 77K. In the present study, we tested recently designed water soluble dry powder preparations of DNICs keeping their characteristics in dry air for a long time. The complexes dissolved in PBS were injected intravenously into normotensive Wistar and spontaneously hypertensive SHR rats. The average arterial pressure (AAP) was recorded through preliminary implanted catheter in a carotid artery. The initial hypotensive action of DNIC with cysteine (DNIC-cys) was comparable to action of nitroprusside (SNP) but, in contrast to the latter, lasted for 20-120min depending on a doze. The blood DNIC content as detected by electronic paramagnetic resonance steadily decreased at this time. The hypotensive action of S-nitrosocysteine was similar to SNP while binding of iron in DNIC by batophenantroline-disulphonate prevented its hypotensive effect. These data suggest that long-lasting hypotensive action of DNICs may be caused by stable protein-bound DNICs forming in the process of transfer of Fe(+)(NO(+))(2) moieties from low-molecular DNICs to thiol protein ligands. The relative initial dose-dependent effect of DNIC-cys was similar in Wistar and SHR but secondary AAP reduction was more profound in SHR. A substitution of cysteine in DNIC by thiosulphate resulted in markedly less initial AAP reduction while long-lasting effect was similar and substitution by glutathione smoothed initial AAP decline and stabilized AAP level in the second phase. Prolonged AAP reduction induced by DNIC-cys was considerably shortened in narcotized rats. Thus, dry preparations of DNICs preserve prolonged hypotensive activity.     

Possible therapeutic effects of myxobacterial metabolites on type I Gaucher disease

Gaucher disease (GD) is the most prevalent lysosomal storage disorder caused by an inherited deficiency of glucocerebrosidase. In the present study, we aimed to determine whether myxobacterial metabolites exhibit a potential therapeutic effect in the cells from a patient with type I GD. We screened 288 bioactive compounds of myxobacteria in the skin fibroblasts from a patient with type I GD. MTT assays were performed to determine their effects on cell viability. The expression levels of Bcl-2-associated X protein (Bax), ATP-citrate synthase (ATP-CS), E3-binding protein (E3BP), and acetyl-coenzyme A acetyltransferase 1 (ACAT1) were determined by western blotting to understand the molecular mechanisms of myxobacterial metabolites in cells. Thin-layer chromatography (TLC) was carried out to measure changes in glucosylceramide levels in the cultured fibroblasts. This screening process identified 4 compounds that increased cell viability more than 1.45 times. After exposure to these compounds, the expression level of Bax decreased, whereas those of ATP-CS, E3BP, and ACAT1 increased. TLC revealed reduced amounts of intracellular glucosylceramides in patient cells. Here we suggest that myxobacterial metabolites can relieve the stress due to glucosylceramide accumulation, and that it may be utilized as a new therapeutic approach.