RESUMO
Mesenchymal stem cells have the ability to differentiate into several cell types when exposed to determined substances, including oxysterols. Oxysterols are cholesterol products derived from its auto-oxidation by reactive species or from enzymatic action. They are present in the body in low quantities under physiological conditions and exhibit several physiological and pharmacological actions according to both the types of oxysterol and tissue. Some of them are cytotoxic while others have been shown to promote cell differentiation through the action on several different receptors, such as nuclear LXR receptors and Smoothened receptor ligands. Here, we review the main pathways by which oxysterols have been associated with cell differentiation and death of mesenchymal stem cells.
Assuntos
Células-Tronco Mesenquimais , Oxisteróis , Diferenciação Celular , Humanos , Células-Tronco Mesenquimais/citologia , Oxisteróis/farmacologiaRESUMO
Cholesterol is an essential component of mammalian plasma membranes. Alterations in sterol metabolism or oxidation have been linked to various pathological conditions, including cardiovascular diseases, cancer, and neurodegenerative disorders. Unsaturated sterols are vulnerable to oxidation induced by singlet oxygen and other reactive oxygen species. This process yields reactive sterol oxidation products, including hydroperoxides, epoxides as well as aldehydes. These oxysterols, in particular those with high electrophilicity, can modify nucleophilic sites in biomolecules and affect many cellular functions. Here, we review the generation and measurement of reactive sterol oxidation products with emphasis on cholesterol hydroperoxides and aldehyde derivatives (electrophilic oxysterols) and their effects on protein modifications.
Assuntos
Oxisteróis/metabolismo , Proteínas/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Animais , Colesterol/análogos & derivados , Colesterol/química , Colesterol/metabolismo , Humanos , Oxisteróis/química , Proteínas/químicaRESUMO
Metal-deficient Cu,Zn-superoxide dismutase (apo-SOD1) is associated with the formation of SOD1 aggregates that accumulate in ALS disease. The data supplied in this article support the accompanying publication showing SOD1 modification and aggregation induced by lipid aldehydes [1]. Here, we present the LC-MS/MS dataset on apo-SOD1 modification induced by seven different lipid aldehydes: 4-hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE), 2-hexen-1-al (HEX), 2,4-nonadienal (NON), 2,4-decadienal (DEC) or secosterol aldehydes (SECO-A or SECO-B). Modified protein samples were digested with trypsin and sequenced by a LC coupled to a Q-TOF instrument. Protein sequencing and peptide modification analysis was performed by Mascot 2.6 (Matrix Science) and further validated by manual inspection. Mass spectrometry data (RAW files) obtained in this study have been deposited to MassIVE and the observed peptide-aldehyde adducts can be used in further studies exploring SOD1 modifications in vivo.
RESUMO
Oxysterols are 27-carbon oxidation products of cholesterol metabolism. Oxysterols possess several biological actions, including the promotion of cell death. Here, we examined the ability of 7-ketocholesterol (7-KC), cholestane-3ß-5α-6ß-triol (triol), and a mixture of 5α-cholestane-3ß,6ß-diol and 5α-cholestane-3ß,6α-diol (diol) to promote cell death in a human breast cancer cell line (MDA-MB-231). We determined cell viability, after 24-h incubation with oxysterols. These oxysterols promoted apoptosis. At least part of the observed effects promoted by 7-KC and triol arose from an increase in the expression of the sonic hedgehog pathway mediator, smoothened. However, this increased expression was apparently independent of sonic hedgehog expression, which did not change. Moreover, these oxysterols led to increased expression of LXRα, which is involved in cellular cholesterol efflux, and the ATP-binding cassette transporters, ABCA1 and ABCG1. Diols did not affect these pathways. These results suggested that the sonic hedgehog and LXRα pathways might be involved in the apoptotic process promoted by 7-KC and triol.
RESUMO
The DAF-12 receptor is a ligand-activated transcription factor that in its ligand-bound form allows the expression of target genes needed to support the reproductive life cycle of the free-living nematode Caenorhabditis elegans, whereas unbound DAF-12 receptor leads to the developmentally arrested "dauer larvae", specialized for survival and dispersal. The endogenous ligands of the DAF-12 receptor are 3-keto-cholestenoic acids dubbed dafachronic acids. In a previous publication we reported that oxysterols with a shorter side chain (C24) modulate the DAF-12 receptor activity either as partial agonists or, in the case of the C24 alcohol 24-hydroxy-4-cholen-3-one, as an antagonist both in vitro and in vivo. Preliminary structure-activity relationships suggested that this activity profile could be improved with more lipophilic and less acidic functional groups at the end of the side chain. Thus, we have now synthesized two fluorine containing analogues in which the C-24 hydroxyl was replaced by a difluoromethyl group (regarded as a "lipophilic hydroxyl") or a difluoromethylidene group with similar lipophilicity but lacking the hydrogen bond donor capacity. Activity was evaluated in vitro using transactivation cell-based assays and in vivo by the effect on the development of wild-type C. elegans. The 24-difluoromethyl analogue retained the antagonist activity in vitro, being completely devoid of agonist activity and exhibited improved activity in vivo. The difluoromethylidene showed a slight antagonist tendency in vitro (statistically not significant), in the concentration range tested and was weakly active in vivo. None of the compounds were toxic, as treated worms recovered to normal development, when transferred to fresh media without added steroids.
Assuntos
Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Colenos/síntese química , Colenos/farmacologia , Halogenação , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Técnicas de Química Sintética , Colenos/química , Células HEK293 , Humanos , Ligação de HidrogênioRESUMO
7-Ketocholesterol (7-KC) is a cholesterol oxidation product with several biological functions. 7-KC has the capacity to cause cell death depending on the concentration and specific cell type. Mesenchymal stem cells (MSCs) are multipotent cells with the ability to differentiate into various types of cells, such as osteoblasts and adipocytes, among others. MSCs contribute to the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases, such as leukemia, to a yet unknown extent. Here, we describe the effect of 7-KC on the death of bone marrow MSCs from patients with acute myeloid leukemia (LMSCs). LMSCs were less susceptible to the death-promoting effect of 7-KC than other cell types. 7-KC exposure triggered the extrinsic pathway of apoptosis with an increase in activated caspase-8 and caspase-3 activity. Mechanisms other than caspase-dependent pathways were involved. 7-KC increased ROS generation by LMSCs, which was related to decreased cell viability. 7-KC also led to disruption of the cytoskeleton of LMSCs, increased the number of cells in S phase, and decreased the number of cells in the G1/S transition. Autophagosome accumulation was also observed. 7-KC downregulated the SHh protein in LMSCs but did not change the expression of SMO. In conclusion, oxiapoptophagy (OXIdative stress + APOPTOsis + autophagy) seems to be activated by 7-KC in LMSCs. More studies are needed to better understand the role of 7-KC in the death of LMSCs and the possible effects on the SHh pathway.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cetocolesteróis/farmacologia , Leucemia Mieloide Aguda/patologia , Células-Tronco Mesenquimais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Autofagossomos/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Hedgehog/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor Smoothened/metabolismoRESUMO
Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (ß-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.
Assuntos
Aspergillus oryzae/metabolismo , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Receptores de Esteroides/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Ergosterol/metabolismo , Expressão Gênica , Hidroxicolesteróis/metabolismo , Cetocolesteróis/metabolismo , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Estigmasterol/metabolismoRESUMO
Oxysterols are 27-carbon oxidation products of cholesterol metabolism. Oxysterols possess several biological actions, including the promotion of cell death. Here, we examined the ability of several oxysterols to induce short-term death in cancerous (human breast cancer and mouse skin melanoma cells) and non-cancerous (human endothelial cells and lung fibroblasts) cell lines. We determined cell viability, Ki67 expression, cell cycle regulation, and apoptosis after 24-h incubations with oxysterols. We found that different oxysterols had different effects on the studied parameters. Moreover, the effects depended on cell type and oxysterol concentration. Three cytotoxic oxysterols (7-ketocholesterol, cholestane-3ß-5α-6ß-triol, and 5α-cholestane-3ß,6ß-diol) inhibited the S phase and stimulated the G0/G1 or G2/M phases. These oxysterols promoted apoptosis, determined with Annexin V and propidium iodide assays. These results showed that different oxysterols have cytotoxic effects depending on the cell line. The findings suggest a potential pharmacological utility of cytotoxic oxysterols.
Assuntos
Apoptose/efeitos dos fármacos , Oxisteróis/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Relação Estrutura-AtividadeRESUMO
Background: Oxysterols are bioactive lipids that control cellular cholesterol synthesis, uptake, and exportation besides mediating inflammation and cytotoxicity that modulate the development of atherosclerosis. Aerobic exercise training (AET) prevents and regresses atherosclerosis by the improvement of lipid metabolism, reverse cholesterol transport (RCT) and antioxidant defenses in the arterial wall. We investigated in dyslipidemic mice the role of a 6-week AET program in the content of plasma and aortic arch cholesterol and oxysterols, the expression of genes related to cholesterol flux and the effect of the exercise-mimetic AICAR, an AMPK activator, in macrophage oxysterols concentration. Methods: Sixteen-week old male apo E KO mice fed a chow diet were included in the protocol. Animals were trained in a treadmill running, 15 m/min, 5 days/week, for 60 min (T; n = 29). A control group was kept sedentary (S; n = 32). Plasma lipids and glucose were determined by enzymatic techniques and glucometer, respectively. Cholesterol and oxysterols in aortic arch and macrophages were measured by gas chromatography/mass spectrometry. The expression of genes involved in lipid metabolism was determined by RT-qPCR. The effect of AMPK in oxysterols metabolism was determined in J774 macrophages treated with 0.25 mM AICAR. Results: Body weight and plasma TC, TG, HDL-c, glucose, and oxysterols were similar between groups. As compared to S group, AET enhanced 7ß-hydroxycholesterol (70%) and reduced cholesterol (32%) in aorta. In addition, exercise increased Cyp27a1 (54%), Cd36 (75%), Cat (70%), Prkaa1 (40%), and Prkaa2 (51%) mRNA. In macrophages, the activation of AMPK followed by incubation with HDL2 increased Abca1 (52%) and Cd36 (220%) and decrease Prkaa1 (19%), Cyp27a1 (47%) and 7α-hydroxycholesterol level. Conclusion: AET increases 7ß-hydroxycholesterol in the aortic arch of dyslipidemic mice, which is related to the enhanced expression of Cd36. In addition, the increase and reduction of Cyp27a1 and Cyp7b1 in trained mice may contribute to enhance levels of 27-OH C. Both oxysterols may act as an alternative pathway for the RCT contributing to the reduction of cholesterol in the aortic arch preventing atherogenesis.
RESUMO
Oxysterols are oxidized products of cholesterol that play several roles in various pathophysiological processes, including the control of lipid metabolism, immunological processes, and cytotoxicity. Mesenchymal stem cells are multipotent cells with properties of self-renewal and the ability to differentiate into other cell types, including osteoblasts and adipocytes. Here, we review the literature regarding the effects of oxysterols on mesenchymal stem cell differentiation and the main signaling pathways involved in this process.
Assuntos
Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Oxisteróis/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Humanos , Oxisteróis/química , Oxisteróis/metabolismoRESUMO
Oxysterols are cholesterol oxidation products formed through enzymatic or autoxidation mechanisms. 7-ketocholeterol (7KC) is one of most abundant oxysterols found in atherosclerotic lesions. Its role in atherosclerosis pathogenesis has been broadly studied in a variety of models. The arterial microenvironment is a multicellular dynamic compartment that, among other systemic factors, is continuously stimulated by 7KC. Endothelial cells have a key role on that environment, being in intimate contact with both the blood stream and the vessel wall, the site of disease origin. 7KC has been shown to promote endothelial cell death and/or dysfunction, depending on its concentration. However, its contribution to the cell microenvironment through cell stimulation has not received much attention. Here we applied mass spectrometry-based proteomics followed by bioinformatics workflow to analyze the effect of a non-toxic 7KC concentration on endothelial cell protein expression and secretion in vitro. Trypsin digests were prepared from the secretome of the endothelial cells and from the total cell pellet after 24h exposure to 7KC. All samples were analyzed by high resolution and accurate mass nano-LC MS/MS. After database search and statistical analysis, differentially expressed proteins were selected for further studies. Our workflow identified 1805 secreted proteins and 2203 intracellular proteins, and of these, 48 and 53, respectively, were regulated. Regulated proteins upon 7KC exposure are involved in unfolded protein response, vascular homeostasis, and reduced control of angiogenesis. Moreover, blood coagulation was another main pathway regulated through Tissue Factor Pathway Inhibitor (TFPI), an antithrombotic agent associated with coronary disease that we found to be more than 2 times downregulated. Taken together, these data show differential endothelial protein regulation and secretion upon 7KC exposure for short time periods under non-toxic conditions. Herewith, these data support the role of 7KC in atherosclerosis pathophysiology and thus reinforce the deleterious effect of endothelial cells stress in the arterial microenvironment.
Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Cetocolesteróis/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteômica , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Biologia Computacional , Relação Dose-Resposta a Droga , Humanos , Espectrometria de Massas , Agregação Plaquetária/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation capabilities. Oxysterols comprise a very heterogeneous group derived from cholesterol through enzymatic and non-enzymatic oxidation. Potent effects in cell death processes, including cytoxicity and apoptosis induction, were described in several cell lines. Very little is known about the effects of oxysterols in MSCs. 7-ketocholesterol (7-KC), one of the most important oxysterols, was shown to be cytotoxic to human adipose tissue-derived MSCs. Here, we describe the short-term (24h) cytotoxic effects of cholestan-3α-5ß-6α-triol, 3,5 cholestan-7-one, (3α-5ß-6α)- cholestane-3,6-diol, 7-oxocholest-5-en-3ß-yl acetate, and 5ß-6ß epoxy-cholesterol, on MSCs derived from human adipose tissue. MSCs were isolated from adipose tissue obtained from three young, healthy women. Oxysterols, with the exception of 3,5 cholestan-7-one and 7-oxocholest-5-en-3ß-yl acetate, led to a complex mode of cell death that include apoptosis, necrosis and autophagy, depending on the type of oxysterol and concentration, being cholestan-3α-5ß-6α-triol the most effective. Inhibition of proliferation was also promoted by these oxysterols, but no changes in cell cycle were observed.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Oxisteróis/farmacologia , Actinas/metabolismo , Adulto , Apoptose , Autofagia , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Colestanos/farmacologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Cetocolesteróis/farmacologia , Potenciais da Membrana , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Necrose , OxirreduçãoRESUMO
A novel microwave-assisted direct saponification method for the simultaneous determination of cholesterol and cholesterol oxides in shrimp was developed and validated. Optimal saponification conditions, determined by means of an experimental design, were achieved using 500mg of sample and 20mL of 1mol/L KOH ethanol solution for 16min at 45°C at maximum power at 200W and magnetic stirring at 120rpm. Higher extraction of cholesterol oxides in a reduced saponification time (â¼75 times) was achieved in comparison with the direct cold saponification method. The new method showed low detection (≤0.57µg/mL) and quantification (≤1.73µg/mL) limits, good repeatability (≤10.50% intraday and ≤8.56% interday) and low artifact formation (evaluated by using a deuterated cholesterol-D6 standard). Raw, salted and dried-salted shrimps were successfully analyzed by the validated method. The content of cholesterol oxides increased after salting and decreased after drying.
Assuntos
Colesterol/análise , Crustáceos/química , Análise de Alimentos/métodos , Micro-Ondas , Óxidos/análise , Animais , Artefatos , Colesterol/química , Cromatografia Gasosa , Cinética , Limite de Detecção , Magnetismo , Óxidos/química , Reprodutibilidade dos Testes , Sais/química , TemperaturaRESUMO
Non-enzymatic lipid peroxidation may change biomembrane structure and function. Here, we employed molecular dynamics simulations to study the effects of either phospholipid or cholesterol peroxidation individually, as well as the combined peroxidation of both components. When lipids were peroxidized, the generated OOH groups migrated to the membrane surface and engaged in H-bonds with each other and the phospholipid carbonyl ester groups. It caused the sn-2 acyl chains of phospholipid hydroperoxides to bend and the whole sterol backbone of cholesterol hydroperoxides to tilt. When phospholipids were kept intact, peroxidation of the sterol backbone led to a partial degradation of its condensing and ordering properties, independently of the position and isomerism of the OOH substitution. However, even in massively peroxidized membranes in which all phospholipids and cholesterol were peroxidized, the condensing and ordering properties of the sterol backbone were still significant. The possible implications for the formation of membrane lateral domains were discussed. Cholesterol peroxyl radicals were also investigated and we found that the OO groups did not migrate to the headgroups region.
Assuntos
Colesterol/química , Peroxidação de Lipídeos , Membranas Artificiais , Simulação de Dinâmica Molecular , Fosfolipídeos/químicaRESUMO
BACKGROUND: In previous studies, the extract from Anadenanthera colubrina was active against Alternaria alternata in vitro and reduced the disease caused by this fungus on Murcott tangor fruits to levels that have been obtained using commercial fungicides. Therefore, the goal of the present work was to isolate and identify the active substances in this extract and identify in silico their protein target in the fungus. RESULTS: The bioguided fractionation of the methanol extract from the fruits of A. colubrina resulted in the isolation of ß-sitosterol and ß-sitosteryl linoleate, which had minimal inhibitory concentrations (MICs) of 250 and 500 µg mL(-1) , respectively, against A. alternata. Under the same conditions, the MICs for two commercial fungicides were 1250 and 19 µg mL(-1) . In silico studies showed that these steroidal substances bind well to oxysterol-binding proteins from Saccharomyces cerevisiae. CONCLUSION: ß-Sitosterol and ß-sitosteryl linoleate, produced by A. colubrina, are active against A. alternata. In silico studies suggest that these substances may act by binding to oxysterol-binding proteins. Therefore, both substances and these proteins have potential use in the development of new steroidal structures and analogues to control the disease caused by A. alternata.
Assuntos
Alternaria/efeitos dos fármacos , Alternaria/crescimento & desenvolvimento , Fabaceae/química , Fungicidas Industriais/farmacologia , Doenças das Plantas/prevenção & controle , Extratos Vegetais/farmacologia , Sitosteroides/farmacologia , Alternaria/ultraestrutura , Citrus/microbiologia , Simulação por Computador , Extratos Vegetais/uso terapêutico , Receptores de Esteroides/metabolismo , Sitosteroides/isolamento & purificaçãoRESUMO
7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorpórate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.