RESUMO
The present study was planned to improve our understanding about sex differences in the development of hepatic steatosis in cafeteria diet-induced obesity in young mice. Female (FCaf) and male (MCaf) mice fed a cafeteria diet had similar body weight gain and adiposity index, but FCaf had a more extensive steatosis than MCaf. FCaf livers exhibited a higher non-alcoholic fatty liver disease activity score, elevated lipid percentage area (+34%) in Sudan III staining and increased TG content (+25%) compared to MCaf. Steatosis in FCaf was not correlated with changes in the transcript levels of lipid metabolism-related genes, but a reduced VLDL release rate was observed. Signs of oxidative stress were found in FCaf livers, as elevated malondialdehyde content (+110%), reduced catalase activity (-36%) and increased Nrf2 and Hif1a mRNA expression compared to MCaf. Interestingly, fibroblast growth factor 21 (Fgf21) mRNA expression was found to be exclusively induced in MCaf, which also exhibited higher FGF21 serum levels (+416%) and hepatic protein abundance (+163%) than FCaf. Moreover, cafeteria diet increased Fgfr1, Fsp27 and Ucp1 mRNA expression in brown adipose tissue of males (MCaf), but not females (FCaf). FGF21 hepatic production by male mice seems to be part of a complex network of responses to the nutritional stress of the cafeteria diet, probably related to the unfolded protein response activation. Although aimed at the restoration of hepatic metabolic homeostasis, the branch involving Fgf21 upregulation seems to be impaired in females, rendering them incapable of reducing the hepatic lipid content and cellular oxidative stress.
Assuntos
Dieta/efeitos adversos , Metabolismo dos Lipídeos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Animais , Feminino , Fatores de Crescimento de Fibroblastos/biossíntese , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fígado/patologia , Masculino , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/etiologia , Obesidade/patologiaRESUMO
Intracellular long-chain acyl-CoA synthetases (ACSL) activate fatty acids to produce acyl-CoA, which undergoes ß-oxidation and participates in the synthesis of esterified lipids such as triacylglycerol (TAG). Imbalances in these metabolic routes are closely associated with the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Triacsin C is one of the few compounds that inhibit TAG accumulation into lipid droplets (LD) by suppressing ACSL activity. Here we report that treatment of primary rat hepatocytes with triacsin C at concentrations lower than the IC50 (4.1 µM) for LD formation: (i) diminished LD number in a concentration-dependent manner; (ii) increased mitochondrial amount; (iii) markedly improved mitochondrial metabolism by enhancing the ß-oxidation efficiency, electron transport chain capacity, and degree of coupling - treatment of isolated rat liver mitochondria with the same triacsin C concentrations did not affect the last two parameters; (iv) decreased the GSH/GSSG ratio and elevated the protein carbonyl level, which suggested an increased reactive oxygen species production, as observed in isolated mitochondria. The hepatocyte mitochondrial improvements were not related to either the transcriptional levels of PGC-1α or the content of mTOR and phosphorylated AMPK. Triacsin C at 10 µM induced hepatocyte death by necrosis and/or apoptosis through mechanisms associated with mitochondrial permeability transition pore opening, as demonstrated by experiments using isolated mitochondria. Therefore, triacsin C at sub-IC50 concentrations modulates the lipid imbalance by shifting hepatocytes to a more oxidative state and enhancing the fatty acid consumption, which can in turn accelerate lipid oxidation and reverse NAFLD in long-term therapies.
Assuntos
Hepatócitos/citologia , Gotículas Lipídicas/efeitos dos fármacos , Triazenos/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Metabolismo dos Lipídeos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Biogênese de Organelas , Ratos , Triazenos/uso terapêuticoRESUMO
The study of selective toxicity of carbon nanotubes (CNTs) on mitochondria (CNT-mitotoxicity) is of major interest for future biomedical applications. In the current work, the mitochondrial oxygen consumption (E3) is measured under three experimental conditions by exposure to pristine and oxidized CNTs (hydroxylated and carboxylated). Respiratory functional assays showed that the information on the CNT Raman spectroscopy could be useful to predict structural parameters of mitotoxicity induced by CNTs. The in vitro functional assays show that the mitochondrial oxidative phosphorylation by ATP-synthase (or state V3 of respiration) was not perturbed in isolated rat-liver mitochondria. For the first time a star graph (SG) transform of the CNT Raman spectra is proposed in order to obtain the raw information for a nano-QSPR model. Box-Jenkins and perturbation theory operators are used for the SG Shannon entropies. A modified RRegrs methodology is employed to test four regression methods such as multiple linear regression (LM), partial least squares regression (PLS), neural networks regression (NN), and random forest (RF). RF provides the best models to predict the mitochondrial oxygen consumption in the presence of specific CNTs with R2 of 0.998-0.999 and RMSE of 0.0068-0.0133 (training and test subsets). This work is aimed at demonstrating that the SG transform of Raman spectra is useful to encode CNT information, similarly to the SG transform of the blood proteome spectra in cancer or electroencephalograms in epilepsy and also as a prospective chemoinformatics tool for nanorisk assessment. All data files and R object models are available at https://dx.doi.org/10.6084/m9.figshare.3472349 .
Assuntos
Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Nanotubos de Carbono/toxicidade , Análise Espectral Raman , Animais , Entropia , Modelos Lineares , Masculino , Mitocôndrias/ultraestrutura , Consumo de Oxigênio , Ratos , Ratos WistarRESUMO
We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidation and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer.
Assuntos
Autofagia/fisiologia , Regulação da Expressão Gênica/fisiologia , Chaperonas de Histonas/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias/fisiologia , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA , Células HEK293 , Humanos , Estresse Oxidativo/fisiologia , Proteína Desacopladora 1 , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: Maternal protein restriction in rats increases the risk of adult offspring arterial hypertension through unknown mechanisms. OBJECTIVES: The aims of the study were to evaluate the effects of a low-protein (LP) diet during pregnancy and lactation on baseline sympathetic and respiratory activities and peripheral chemoreflex sensitivity in the rat offspring. METHODS: Wistar rat dams were fed a control [normal-protein (NP); 17% protein] or an LP (8% protein) diet during pregnancy and lactation, and their male offspring were studied at 30 d of age. Direct measurements of baseline arterial blood pressure (ABP), heart rate (HR), and respiratory frequency (Rf) as well as peripheral chemoreflex activation (potassium cyanide: 0.04%) were recorded in pups while they were awake. In addition, recordings of the phrenic nerve (PN) and thoracic sympathetic nerve (tSN) activities were obtained from the in situ preparations. Hypoxia-inducible factor 1α (HIF-1α) expression was also evaluated in carotid bifurcation through a Western blotting assay. RESULTS: At 30 d of age, unanesthetized LP rats exhibited enhanced resting Rf (P = 0.001) and similar ABP and HR compared with the NP rats. Despite their similar baseline ABP values, LP rats exhibited augmented low-frequency variability (â¼91%; P = 0.01). In addition, the unanesthetized LP rats showed enhanced pressor (P = 0.01) and tachypnoeic (P = 0.03) responses to peripheral chemoreflex activation. The LP rats displayed elevated baseline tSN activity (â¼86%; P = 0.02) and PN burst frequency (45%; P = 0.01) and amplitude (53%; P = 0.001) as well as augmented sympathetic (P = 0.01) and phrenic (P = 0.04) excitatory responses to peripheral chemoreflex activation compared with the NP group. Furthermore, LP rats showed an increase of â¼100% in HIF-1α protein density in carotid bifurcation compared with NP rats. CONCLUSION: Sympathetic-respiratory overactivity and amplified peripheral chemoreceptor responses, potentially through HIF-1α-dependent mechanisms, precede the onset of hypertension in juvenile rats exposed to protein undernutrition during gestation and lactation.
Assuntos
Células Quimiorreceptoras/metabolismo , Dieta com Restrição de Proteínas/efeitos adversos , Fenômenos Fisiológicos da Nutrição Materna , Sistema Nervoso Periférico/fisiopatologia , Pré-Hipertensão/fisiopatologia , Sistema Respiratório/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Animais , Peso ao Nascer , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Artéria Carótida Primitiva/fisiopatologia , Células Quimiorreceptoras/patologia , Feminino , Desenvolvimento Fetal , Retardo do Crescimento Fetal/etiologia , Retardo do Crescimento Fetal/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lactação , Masculino , Sistema Nervoso Periférico/patologia , Nervo Frênico/patologia , Nervo Frênico/fisiopatologia , Gravidez , Pré-Hipertensão/etiologia , Pré-Hipertensão/metabolismo , Pré-Hipertensão/patologia , Ratos Wistar , Sistema Respiratório/patologia , Sistema Nervoso Simpático/patologia , Nervos Torácicos/patologia , Nervos Torácicos/fisiopatologiaRESUMO
BACKGROUND: SET/I2PP2A is a multifunctional protein that is up-regulated in head and neck squamous cell carcinoma (HNSCC). The action of SET in HNSCC tumorigenicity is unknown. METHODS: Stable SET knockdown by shRNA (shSET) was established in three HNSCC cell lines: HN12, HN13, and Cal27. Protein expression and phosphorylated protein levels were determined by Western blotting and immunofluorescence, cell migration and invasion were measured by functional analysis, and PP2A activity was determined using a serine/threonine phosphatase assay. A real-time PCR array was used to quantify 84 genes associated with cell motility. Metalloproteinase (MMP) activity was assessed by zymographic and fluorometric assays. HN12shSET xenograft tumors (flank and tongue models) were established in Balb/c nude mice; the xenograft characteristics and cisplatin sensitivity were demonstrated by macroscopic, immunohistochemical, and histological analyses, as well as lymph node metastasis by histology. RESULTS: The HN12shSET cells displayed reduced ERK1/2 and p53 phosphorylation compared with control. ShSET reduced HN12 cell proliferation and increased the sub-G1 population of HN12 and Cal27 cells. Increased PP2A activity was also associated with shSET. The PCR array indicated up-regulation of three mRNAs in HN12 cells: vimentin, matrix metalloproteinase-9 (MMP9) and non-muscle myosin heavy chain IIB. Reduced E-cadherin and pan-cytokeratin, as well as increased vimentin, were also demonstrated as the result of SET knockdown. These changes were accompanied by an increase in MMP-9 and MMP-2 activities, migration and invasion. The HN12shSET subcutaneous xenograft tumors presented a poorly differentiated phenotype, reduced cell proliferation, and cisplatin sensitivity. An orthotopic xenograft tumor model using the HN12shSET cells displayed increased metastatic potential. CONCLUSIONS: SET accumulation has important actions in HNSCC. As an oncogene, SET promotes cell proliferation, survival, and resistance to cell death by cisplatin in vivo. As a metastasis suppressor, SET regulates invasion, the epithelial mesenchymal transition, and metastasis.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Chaperonas de Histonas/genética , Fatores de Transcrição/genética , Animais , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Cisplatino/farmacologia , Proteínas de Ligação a DNA , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Necrose/patologia , Invasividade Neoplásica , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Testosterone exerts both beneficial and harmful effects on the cardiovascular system. Considering that testosterone induces reactive oxygen species (ROS) generation and ROS activate cell death signaling pathways, we tested the hypothesis that testosterone induces apoptosis in vascular smooth muscle cells (VSMCs) via mitochondria-dependent ROS generation. Potential mechanisms were addressed. Cultured VSMCs were stimulated with testosterone (10(-7) mol/l) or vehicle (2-12 h) in the presence of flutamide (10(-5) mol/l), CCCP (10(-6) mol/l), mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 3 × 10(-5) mol/l), Z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK; 10(-5) mol/l), or vehicle. ROS were determined with lucigenin and dichlorodihydrofluorescein; apoptosis, with annexin V and calcein; O2 consumption, with a Clark-type electrode, and procaspases, caspases, cytochrome c, Bax, and Bcl-2 levels by immunoblotting. Testosterone induced ROS generation (relative light units/mg protein, 2 h; 162.6 ± 16 vs. 100) and procaspase-3 activation [arbitrary units, (AU), 6 h; 166.2 ± 19 vs. 100]. CCCP, MnTMPyP, and flutamide abolished these effects. Testosterone increased annexin-V fluorescence (AU, 197.6 ± 21.5 vs. 100) and decreased calcein fluorescence (AU, 34.4 ± 6.4 vs. 100), and O2 consumption (nmol O2/min, 18.6 ± 2.0 vs. 34.4 ± 3.9). Testosterone also reduced Bax-to-Bcl-2 ratio but not cytochrome-c release from mitochondria. Moreover, testosterone (6 h) induced cleavage of procaspase 8 (AU, 161.1 ± 13.5 vs. 100) and increased gene expression of Fas ligand (2(ΔΔCt), 3.6 ± 1.2 vs. 0.7 ± 0.5), and TNF-α (1.7 ± 0.4 vs. 0.3 ± 0.1). CCCP, MnTMPyP, and flutamide abolished these effects. These data indicate that testosterone induces apoptosis in VSMCs via the extrinsic apoptotic pathway with the involvement of androgen receptor activation and mitochondria-generated ROS.
Assuntos
Androgênios/farmacologia , Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Testosterona/farmacologia , Antagonistas de Androgênios/farmacologia , Animais , Caspases/metabolismo , Flutamida/farmacologia , Masculino , Mitocôndrias/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismoRESUMO
SET and hnRNPK are proteins involved in gene expression and regulation of cellular signaling. We previously demonstrated that SET accumulates in head and neck squamous cell carcinoma (HNSCC); hnRNPK is a prognostic marker in cancer. Here, we postulate that SET and hnRNPK proteins interact to promote tumorigenesis. We performed studies in HEK293 and HNSCC (HN6, HN12, and HN13) cell lines with SET/hnRNPK overexpression and knockdown, respectively. We found that SET and/or hnRNPK protein accumulation increased cellular proliferation. SET accumulation up-regulated hnRNPK mRNA and total/phosphorylated protein, promoted hnRNPK nuclear location, and reduced Bcl-x mRNA levels. SET protein directly interacted with hnRNPK, increasing both its binding to nucleic acids and Bcl-xS repression. We propose that hnRNPK should be a new target of SET and that SET-hnRNPK interaction, in turn, has potential implications in cell survival and malignant transformation.
Assuntos
Proliferação de Células , Chaperonas de Histonas/metabolismo , Ácidos Nucleicos/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteína bcl-X/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Células HEK293 , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Chaperonas de Histonas/genética , Humanos , Immunoblotting , Microscopia Confocal , Ácidos Nucleicos/genética , Fosforilação , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleoproteínas/genética , Fatores de Transcrição/genética , Regulação para Cima , Proteína bcl-X/genéticaRESUMO
Daily intake of conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and to increase body metabolism; this latter effect has been often associated with the up-regulation of uncoupling proteins (UCPs). Here we addressed the effects of a CLA-supplemented murine diet (~2 % CLA mixture, cis-9, trans-10 and trans-10, cis-12 isomers; 45 % of each isomer on alternating days) on mitochondrial energetics, UCP2 expression/activity in the liver and other associated morphological and functional parameters, in C57BL/6 mice. Diet supplementation with CLA reduced both lipid accumulation in adipose tissues and triacylglycerol plasma levels, but did not augment hepatic lipid storage. Livers of mice fed a diet supplemented with CLA showed high UCP2 mRNA levels and the isolated hepatic mitochondria showed indications of UCP activity: in the presence of guanosine diphosphate, the higher stimulation of respiration promoted by linoleic acid in mitochondria from the CLA mice was almost completely reduced to the level of the stimulation from the control mice. Despite the increased generation of reactive oxygen species through oxi-reduction reactions involving NAD(+)/NADH in the Krebs cycle, no oxidative stress was observed in the liver. In addition, in the absence of free fatty acids, basal respiration rates and the phosphorylating efficiency of mitochondria were preserved. These results indicate a beneficial and secure dose of CLA for diet supplementation in mice, which induces UCP2 overexpression and UCP activity in mitochondria while preserving the lipid composition and redox state of the liver.
Assuntos
Suplementos Nutricionais , Endopeptidases/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Ácidos Linoleicos Conjugados/farmacologia , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Animais , Masculino , Camundongos , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteases Específicas de UbiquitinaRESUMO
Nitrosyl ruthenium complexes are promising NO donor agents with numerous advantages for the biologic applications of NO. We have characterized the NO release from the nitrosyl ruthenium complex [Ru(NO(2))(bpy)(2)(4-pic)](+) (I) and the reactive oxygen/nitrogen species (ROS/RNS)-mediated NO actions on isolated rat liver mitochondria. The results indicated that oxidation of mitochondrial NADH promotes NO release from (I) in a manner mediated by NO(2) formation (at neutral pH) as in mammalian cells, followed by an oxygen atom transfer mechanism (OAT). The NO released from (I) uncoupled mitochondria at low concentrations/incubation times and inhibited the respiratory chain at high concentrations/incubation times. In the presence of ROS generated by mitochondria NO gave rise to peroxynitrite, which, in turn, inhibited the respiratory chain and oxidized membrane protein-thiols to elicit a Ca(2+)-independent mitochondrial permeability transition; this process was only partially inhibited by cyclosporine-A, almost fully inhibited by the thiol reagent N-ethylmaleimide (NEM) and fully inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). These actions correlated with the release of cytochrome c from isolated mitochondria as detected by Western blotting analysis. These events, typically involved in cell necrosis and/or apoptosis denote a potential specific action of (I) and analogs against tumor cells via mitochondria-mediated processes.
Assuntos
Complexos de Coordenação/farmacocinética , Mitocôndrias Hepáticas/metabolismo , NADP/metabolismo , Doadores de Óxido Nítrico/farmacocinética , Óxido Nítrico/farmacocinética , Rutênio/farmacocinética , Análise de Variância , Animais , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Citocromos c/metabolismo , Concentração de Íons de Hidrogênio , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Oxirredução , Ratos , Ratos Wistar , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rutênio/química , Rutênio/metabolismo , Compostos de SulfidrilaRESUMO
SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 µM tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Chaperonas de Histonas/metabolismo , Estresse Oxidativo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Apoptose , Western Blotting , Caspases/metabolismo , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular , Citoplasma/efeitos dos fármacos , Proteínas de Ligação a DNA , Imunofluorescência , Glutationa/metabolismo , Células HEK293 , Chaperonas de Histonas/genética , Humanos , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Tempo , Fatores de Transcrição/genética , Transfecção , Regulação para Cima , terc-Butil Hidroperóxido/farmacologiaRESUMO
Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.
Assuntos
Micélio/fisiologia , Oxirredutases/biossíntese , Paracoccidioides/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Antifúngicos/farmacologia , Antimicina A/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Humanos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Oxirredução , Estresse Oxidativo , Paracoccidioides/citologia , Paracoccidioides/crescimento & desenvolvimento , Proteínas de Plantas , Cianeto de Potássio/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação para CimaRESUMO
The frequency of opportunistic fungal infection has increased drastically, mainly in patients who are immunocompromised due to organ transplant, leukemia or HIV infection. In spite of this, only a few classes of drugs with a limited array of targets, are available for antifungal therapy. Therefore, more specific and less toxic drugs with new molecular targets is desirable for the treatment of fungal infections. In this context, searching for differences between mitochondrial mammalian hosts and fungi in the classical and alternative components of the mitochondrial respiratory chain may provide new potential therapeutic targets for this purpose.
Assuntos
Trifosfato de Adenosina/biossíntese , Citocromos c/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Fungos/fisiologia , Mitocôndrias/fisiologia , Micoses/tratamento farmacológico , Descoberta de Drogas , Transporte de Elétrons/fisiologia , Humanos , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 1RESUMO
In addition to adenosine triphosphate (ATP) production, mitochondria have been implicated in the regulation of several physiological responses in plants, such as programmed cell death (PCD) activation. Salicylic acid (SA) and reactive oxygen species (ROS) are essential signaling molecules involved in such physiological responses; however, the mechanisms by which they act remain unknown. In non-photosynthesizing tissues, mitochondria appear to serve as the main source of ROS generation. Evidence suggests that SA and ROS could regulate plant PCD through a synergistic mechanism that involves mitochondria. Herein, we isolate and characterize the mitochondria from non-photosynthesizing cell suspension cultures of Rubus fruticosus. Furthermore, we assess the primary site of ROS generation and the effects of SA on isolated organelles. Mitochondrial Complex III was found to be the major source of ROS generation in this model. In addition, we discovered that SA inhibits the electron transport chain by inactivating the semiquinone radical during the Q cycle. Computational analyses confirmed the experimental data, and a mechanism for this action is proposed.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Rosaceae/metabolismo , Ácido Salicílico/farmacologia , Mitocôndrias/efeitos dos fármacos , Oxirredução , Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rosaceae/química , Ácido Salicílico/metabolismoRESUMO
Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 µM) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca²âº efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP+ transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Benzofenonas/farmacologia , Membranas Mitocondriais/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Trifosfato de Adenosina/análise , Animais , Benzofenonas/farmacocinética , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Células Hep G2 , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , NAD/análise , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with several reported pharmacological actions. We have assessed the protective action of GA on iron-induced neuronal cell damage by employing the PC12 cell line and primary culture of rat cortical neurons (PCRCN). A strong protection by GA, assessed by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT) assay, was revealed, with IC(50) values <1 µM. GA also inhibited Fe(3+)-ascorbate reduction, iron-induced oxidative degradation of 2-deoxiribose, and iron-induced lipid peroxidation in rat brain homogenate, as well as stimulated oxygen consumption by Fe(2+) autoxidation. Absorption spectra and cyclic voltammograms of GA-Fe(2+)/Fe(3+) complexes suggest the formation of a transient charge transfer complex between Fe(2+) and GA, accelerating Fe(2+) oxidation. The more stable Fe(3+) complex with GA would be unable to participate in Fenton-Haber Weiss-type reactions and the propagation phase of lipid peroxidation. The results show a potential of GA against neuronal diseases associated with iron-induced oxidative stress.
Assuntos
Benzofenonas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Ácido Ascórbico/química , Ácido Ascórbico/toxicidade , Benzofenonas/química , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Desoxirribose/metabolismo , Embrião de Mamíferos , Compostos Férricos/química , Compostos Férricos/toxicidade , Quelantes de Ferro/química , Quelantes de Ferro/farmacologia , Cinética , Malondialdeído/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Oxidantes/química , Oxidantes/toxicidade , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Ratos , Ratos WistarRESUMO
The oxidation of critical cysteines/related thiols of adenine nucleotide translocase (ANT) is believed to be an important event of the Ca(2+)-induced mitochondrial permeability transition (MPT), a process mediated by a cyclosporine A/ADP-sensitive permeability transition pores (PTP) opening. We addressed the ANT-Cys(56) relative mobility status resulting from the interaction of ANT/surrounding cardiolipins with Ca(2+) and/or ADP by means of computational chemistry analysis (Molecular Interaction Fields and Molecular Dynamics studies), supported by classic mitochondrial swelling assays. The following events were predicted: (i) Ca(2+) interacts preferentially with the ANT surrounding cardiolipins bound to the H4 helix of translocase, (ii) weakens the cardiolipins/ANT interactions and (iii) destabilizes the initial ANT-Cys(56) residue increasing its relative mobility. The binding of ADP that stabilizes the conformation "m" of ANT and/or cardiolipin, respectively to H5 and H4 helices, could stabilize their contacts with the short helix h56 that includes Cys(56), accounting for reducing its relative mobility. The results suggest that Ca(2+) binding to adenine nucleotide translocase (ANT)-surrounding cardiolipins in c-state of the translocase enhances (ANT)-Cys(56) relative mobility and that this may constitute a potential critical step of Ca(2+)-induced PTP opening.
Assuntos
Cálcio/metabolismo , Cardiolipinas/metabolismo , Cisteína/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Simulação por Computador , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Masculino , Mitocôndrias Hepáticas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial/efeitos dos fármacos , Modelos Químicos , Modelos Moleculares , Estresse Oxidativo , Conformação Proteica , Ratos , Ratos Wistar , Ácido Succínico/farmacologiaRESUMO
Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca(2+)-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys(56) relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca(2+)-induced ANT conformational change and mitochondrial swelling. ANT-Pro(61) isomerization increased ANT-Cys(56) relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca(2+) induced ANT "c" conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca(2+)-induced ANT "c" conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca(2+) induces the ANT "c" conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.
Assuntos
Cálcio/metabolismo , Cisteína/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Prolina/metabolismo , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Cálcio/farmacologia , Humanos , Isomerismo , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
A new nitrosyl ruthenium complex [Ru(NH.NHq)(terpy)NO](3+) nitric oxide donor was recently developed and due to its excellent vasodilator activity, it has been considered as a potential drug candidate. Drug metabolism is one of the main parameters that should be evaluated in the early drug development, so the biotransformation of this complex by rat hepatic microsomes was investigated. In order to perform the biotransformation study, a simple, sensitive and selective HPLC method was developed and carefully validated. The parameters evaluated in the validation procedure were: linearity, recovery, precision, accuracy, selectivity and stability. Except for the stability study, all the parameters evaluated presented values below the recommended by FDA guidelines. The stability study showed a time-dependent degradation profile. After method validation, the biotransformation study was accomplished and the kinetic parameters were determined. The biotransformation study obeyed the Michaelis-Menten kinetics. The V(max) and K(m) were, respectively, 0.1625+/-0.010 micromol/mg protein/min and 79.97+/-11.52 microM. These results indicate that the nitrosyl complex is metabolized by CYP450.
Assuntos
Microssomos Hepáticos/metabolismo , Doadores de Óxido Nítrico/metabolismo , Compostos Organometálicos/metabolismo , Rutênio/metabolismo , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Cinética , Masculino , NADP/metabolismo , Doadores de Óxido Nítrico/química , Dinâmica não Linear , Compostos Organometálicos/química , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Rutênio/químicaRESUMO
The ruthenium nitrosyl complex trans-[Ru(NO)(NH(3))(4)(py)](PF(6))(3) (pyNO), a nitric oxide (NO) donor, was studied in regard to the release of NO and its impact both on isolated mitochondria and HepG2 cells. In isolated mitochondria, NO release from pyNO was concomitant with NAD(P)H oxidation and, in the 25-100 microM range, it resulted in dissipation of mitochondrial membrane potential, inhibition of state 3 respiration, ATP depletion and reactive oxygen species (ROS) generation. In the presence of Ca(2+), mitochondrial permeability transition (MPT), an unspecific membrane permeabilization involved in cell necrosis and some types of apoptosis, was elicited. As demonstrated by externalization of phosphatidylserine and activation of caspase-9 and caspase-3, pyNO (50-100 microM) induced HepG2 cell death, mainly by apoptosis. The combined action of the NO itself, the peroxynitrite yielded by NO in the presence of reactive oxygen species (ROS) and the oxidative stress generated by the NAD(P)H oxidation is proposed to be involved in cell death by pyNO, both via respiratory chain inhibition and ROS levels increase, or even via MPT, if Ca(2+) is present.