RESUMEN
Molecular alterations in cell death pathways and imbalances in regulators of up- or downstream signaling pathways can lead to resistance to cell death, which is one of the hallmarks of cancer. These signaling modifications are strategies that tumor cells use to resist chemotherapy and that contribute to the high recurrence rate of head and neck squamous cell carcinoma (HNSCC). The SET oncoprotein is a PP2A inhibitor that accumulates in HNSCC and represents a promising therapeutic target. Here we report the role that SET protein plays in resistance to death of two HNSCC cell lines: Cal 27 and HN13. SET protein regulated intracellular redox balance by controlling cellular localization of APE 1 - an endonuclease that is part of the SET complex and regulates antioxidant gene transcription. SET protein knockdown (siSET) associated with tert-butyl hydroperoxide-induced oxidative stress sensitized Cal 27 and HN13 cells to apoptosis via the extrinsic and intrinsic pathways, respectively. SET protein upregulated autophagy in HNSCC cells in a PP2A-dependent manner and apparently regulated ULK1 expression. The fact that siSET lowered Bcl-2 phosphorylation levels indicated that SET protein interfered with an alternative pathway that modulated autophagy in HNSCC cells. Overall, SET protein regulated intracellular redox state and sustained autophagy in HNSCC cells, which may explain resistance to death of HNSCC cells. Altogether, the findings reported herein support SET protein as therapeutic target for HNSCC.
Asunto(s)
Autofagia , Neoplasias de Cabeza y Cuello/metabolismo , Chaperonas de Histonas/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Factores de Transcripción/metabolismo , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de Unión al ADN , Neoplasias de Cabeza y Cuello/ultraestructura , Humanos , Oxidación-Reducción , Estrés Oxidativo , Carcinoma de Células Escamosas de Cabeza y Cuello/ultraestructuraRESUMEN
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.
Asunto(s)
Dieta/efectos adversos , Metabolismo de los Lípidos , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Animales , Femenino , Factores de Crecimiento de Fibroblastos/biosíntesis , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hígado/patología , Masculino , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/etiología , Obesidad/patologíaRESUMEN
This study aims to examine the effects of a new 1,4-dihydropyridine derivative, VdiE-2N, on cell signaling pathways and mitochondrial events in head and neck squamous cell carcinoma (HNSCC) cells, and on a mice model of xenograft tumor growth/cell proliferation. Four HNSCC cell lines (HN13, HN12, HN6, and CAL27), HEK293 cells (human embryonic kidney 293 cells), and human oral healthy mucosa fibroblasts (OHMF) were used for in vitro assessment of cell viability (resazurin assay) and invasion capacity (modified Boyden chamber assay), and mitochondrial membrane potential (JC-1 fluorescence assay), morphology (transmission electron microscopy), and number of mitochondria (MitoTracker® imaging). SET and pDRP1 proteins were analyzed by immunofluorescence, and proteins involved in cell death/survival pathways were analyzed by Western blotting. HN12 xenograft tumors were established in the flank of Balb/c nude mice, and their characteristics and sensitivity to VdiE-2N were determined by immunohistochemistry and histology. VdiE-2N decreased cell viability in HNSCC cells (IC50 = 9.56 and 22.45µM for HN13 and HN12 cells, respectively) more strongly than it decreased cell viability in OHMF and HEK293 cells (IC50 = 32.90 and > 50µM, respectively). In HN13 cells, VdiE-2N dissipated mitochondrial membrane potential and altered the mitochondria size, shape, and number in a concentration-dependent manner, as well as it induced apoptosis and reduced their invasion capacity. Treatment of mice bearing xenograft tumors with VdiE-2N significantly diminished proliferation of cancer cells. Therefore, VdiE-2N induces HNSCC cell death in vitro through mitochondria-mediated apoptotic pathways and dampens tumor growth in vivo, thus supporting a potential anti-cancer effect.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/patología , Dihidropiridinas/química , Dihidropiridinas/farmacología , Neoplasias de Cabeza y Cuello/patología , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/irrigación sanguínea , Carcinoma de Células Escamosas/tratamiento farmacológico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes myc/genética , Neoplasias de Cabeza y Cuello/irrigación sanguínea , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Tamaño Mitocondrial/efectos de los fármacos , Invasividad Neoplásica , Neovascularización Patológica/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Hepatocitos/citología , Gotas Lipídicas/efectos de los fármacos , Triazenos/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Metabolismo de los Lípidos/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Biogénesis de Organelos , Ratas , Triazenos/uso terapéuticoRESUMEN
The multifunctional SET/I2PP2A protein is known to be overexpressed in head and neck squamous cell carcinoma. However, SET has been reported to have apparently conflicting roles in promoting cancer cell survival under oxidative stress conditions and preventing invasion and metastasis, complicating efforts to understand the contribution of SET to carcinogenesis. In the present study, we overexpressed SETin a spontaneously immortalized oral keratinocyte cell line (NOK-SI SET) and demonstrated that SET upregulation alone was sufficient to transform cells. In comparison with NOK-SI cells, NOK-SI SET cells demonstrated increased levels of phosphorylated Akt, c-Myc and inactive/phosphorylated Rb, together with decreased total Rb protein levels. In addition, NOK-SI SET cells presented the following: (a) a spindle-cell shape morphology compared with the polygonal morphology of NOK-SI cells; (b) loss of mesenchymal stem cell markers CD44 and CD73, and epithelial cell markers CD71 and integrin α6/ß4; (c) the ability to form xenograft tumors in nude mice; and (d) increased mitochondrial respiration accompanied by decreased ROSlevels. Overall, our results show that SEToverexpression promotes morphological and oncogenic cell transformation of an oral keratinocyte cell.
Asunto(s)
Chaperonas de Histonas/genética , Queratinocitos/fisiología , Factores de Transcripción/genética , Carcinogénesis/genética , Carcinogénesis/metabolismo , Diferenciación Celular , Línea Celular , Proteínas de Unión al ADN , Expresión Génica , Chaperonas de Histonas/metabolismo , Humanos , Queratinocitos/ultraestructura , Dinámicas Mitocondriales , Mucosa Bucal/citología , Fenotipo , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Epigenetic modifications are essential in the control of normal cellular processes and cancer development. DNA methylation and histone acetylation are major epigenetic modifications involved in gene transcription and abnormal events driving the oncogenic process. SET protein accumulates in many cancer types, including head and neck squamous cell carcinoma (HNSCC); SET is a member of the INHAT complex that inhibits gene transcription associating with histones and preventing their acetylation. We explored how SET protein accumulation impacts on the regulation of gene expression, focusing on DNA methylation and histone acetylation. DNA methylation profile of 24 tumour suppressors evidenced that SET accumulation decreased DNA methylation in association with loss of 5-methylcytidine, formation of 5-hydroxymethylcytosine and increased TET1 levels, indicating an active DNA demethylation mechanism. However, the expression of some suppressor genes was lowered in cells with high SET levels, suggesting that loss of methylation is not the main mechanism modulating gene expression. SET accumulation also downregulated the expression of 32 genes of a panel of 84 transcription factors, and SET directly interacted with chromatin at the promoter of the downregulated genes, decreasing histone acetylation. Gene expression analysis after cell treatment with 5-aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) revealed that histone acetylation reversed transcription repression promoted by SET. These results suggest a new function for SET in the regulation of chromatin dynamics. In addition, TSA diminished both SET protein levels and SET capability to bind to gene promoter, suggesting that administration of epigenetic modifier agents could be efficient to reverse SET phenotype in cancer.
Asunto(s)
Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteínas Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Acetilación , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN , Epigénesis Genética , Perfilación de la Expresión Génica , Chaperonas de Histonas/genética , Humanos , Modelos Biológicos , Factores de Transcripción/genéticaRESUMEN
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 .
Asunto(s)
Mitocondrias/efectos de los fármacos , Modelos Biológicos , Nanotubos de Carbono/toxicidad , Espectrometría Raman , Animales , Entropía , Modelos Lineales , Masculino , Mitocondrias/ultraestructura , Consumo de Oxígeno , Ratas , Ratas WistarRESUMEN
Inducing apoptosis is an interesting therapeutic approach to develop drugs that act against helminthic parasites. Researchers have investigated how curcumin (CUR), a biologically active compound extracted from rhizomes of Curcuma longa, affects Schistosoma mansoni and several cancer cell lines. This study evaluates how CUR influences the induction of apoptosis and oxidative stress in couples of adult S. mansoni worms. CUR decreased the viability of adult worms and killed them. The tegument of the parasite suffered morphological changes, the mitochondria underwent alterations, and chromatin condensed. Different apoptotic parameters were determined in an attempt to understand how CUR affected adult S. mansoni worms. CUR induced DNA damage and fragmentation and increased the expression of SmCASP3/7 transcripts and the activity of Caspase 3 in female and male worms. However, CUR did not intensify the activity of Caspase 8 in female or male worms. Evaluation of the superoxide anion and different antioxidant enzymes helped to explore the mechanism of parasite death further. The level of superoxide anion and the activity of Superoxide Dismutase (SOD) increased, whereas the activity of Glutathione-S-Transferase (GST), Glutathione reductase (GR), and Glutathione peroxidase (GPX) decreased, which culminated in the oxidation of proteins in adult female and male worms incubated with CUR. In conclusion, CUR generated oxidative stress followed by apoptotic-like-events in both adult female and male S. mansoni worms, ultimately killing them.
Asunto(s)
Apoptosis/efectos de los fármacos , Curcumina/farmacología , Estrés Oxidativo/efectos de los fármacos , Schistosoma mansoni/metabolismo , Animales , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Femenino , Proteínas del Helminto/metabolismo , Masculino , Oxidorreductasas/metabolismo , Superóxidos/metabolismoRESUMEN
BACKGROUND: MicroRNAs (miRNAs or miRs) are post-transcriptional regulators of eukaryotic cells and knowledge of differences in miR levels may provide new approaches to diagnosis and therapy. METHODS: The present study measured the levels of nine miRs in head and neck squamous cell carcinomas (HNSCC) and determined whether clinical pathological features are associated with differences in miR levels. SET (I2PP2A) and PTEN protein levels were also measured, since their levels can be regulated by miR-199b and miR-21, respectively. Nine miRs (miR-15a, miR-21, miR-29b, miR-34c, miR-100, miR-125b, miR-137, miR-133b and miR-199b) were measured by real time qRT-PCR in HNSCC samples from 32 patients and eight resection margins. SET (I2PP2A) and PTEN protein levels were estimated by immunohistochemistry in paired HNSCC tissues and their matched resection margins. RESULTS: In HNSCC, the presence of lymph node invasion was associated with low miR-15a, miR-34c and miR-199b levels, whereas the presence of perineural invasion was associated with low miR-199b levels. In addition, miR-21 levels were high whereas miR-100 and miR-125b levels were low in HNSCC compared to the resection margins. When HNSCC line HN12, with or without knockdown of SET, were transfected with miR-34c inhibitor or miR-34c mimic, the miR-34c inhibitor increased cell invasion capacity while miR-34c mimic decreased the cell invasion. CONCLUSIONS: We showed that the levels of specific miRs in tumor tissue can provide insight into the maintenance and progression of HNSCC. GENERAL SIGNIFICANCE: MiRNAs are up- or down-regulated during cancer development and progression; they can be prognosis markers and therapeutic targets in HNSCC.
RESUMEN
Clinical conditions associated with obesity can be improved by daily intake of conjugated linoleic acid (CLA) or extra virgin olive oil (EVOO). Here we investigated whether dietary supplementation with CLA and EVOO, either alone or in combination, changes body metabolism associated with mitochondrial energetics. Male C57Bl/6 mice were divided into one of four groups: CLA (1:1 cis-9, trans-11:trans-10, cis-12; 18:2 isomers), EVOO, CLA plus EVOO or control (linoleic acid). Each mouse received 3 g/kg body weight of the stated oil by gavage on alternating days for 60 days. Dietary supplementation with CLA, alone or in combination with EVOO: (a) reduced the white adipose tissue gain; (b) increased body VO2 consumption, VCO2 production and energy expenditure; (c) elevated uncoupling protein (UCP)-2 expression and UCP activity in isolated liver mitochondria. This organelle, when energized with NAD(+)-linked substrates, produced high amounts of H2O2 without inducing oxidative damage. Dietary supplementation with EVOO alone did not change any metabolic parameter, but supplementation with CLA itself promoted insulin resistance and elevated weight, lipid content and acetyl-CoA carboxylase-1 expression in liver. Interestingly, the in vivo antioxidant therapy with N-acetylcysteine abolished the CLA-induced rise of body metabolism and liver UCP expression and activity, while the in vitro antioxidant treatment with catalase mitigated the CLA-dependent UCP-2 expression in hepatocytes; these findings suggest the participation of an oxidative-dependent pathway. Therefore, this study clarifies the mechanisms by which CLA induces liver UCP expression and activity, and demonstrates for the first time the beneficial effects of combined CLA and EVOO supplementation.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Hipertrofia/prevención & control , Resistencia a la Insulina , Ácidos Linoleicos Conjugados/farmacología , Hígado/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Aceite de Oliva/farmacología , Animales , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Bcr-Abl tyrosine kinase protein, which confers resistance to apoptosis in leukemic cells. Tyrosine kinase inhibitors (TKIs) are effectively used to treat CML; however, CML patients in the advanced (CML-AP) and chronic (CML-CP) phases of the disease are usually resistant to TKI therapy. Thus, it is necessary to seek for novel agents to treat CML, such as the enzyme l-amino acid oxidase from Calloselasma rhodostoma (CR-LAAO) snake venom. We examined the antitumor effect of CR-LAAO in Bcr-Abl(+) cell lines and peripheral blood mononuclear cells (PBMC) from healthy subjects and CML patients. CR-LAAO was more cytotoxic towards Bcr-Abl(+) cell lines than towards healthy subjects' PBMC. The H2O2 produced during the enzymatic action of CR-LAAO mediated its cytotoxic effect. The CR-LAAO induced apoptosis in Bcr-Abl(+) cells, as detected by caspases 3, 8, and 9 activation, loss of mitochondrial membrane potential, and DNA damage. CR-LAAO elicited apoptosis in PBMC from CML-CP patients without TKI treatment more strongly than in PBMC from healthy subjects and TKI-treated CML-CP and CML-AP patients. The antitumor effect of CR-LAAO against Bcr-Abl(+) cells makes this toxin a promising candidate to CML therapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Venenos de Crotálidos/enzimología , Proteínas de Fusión bcr-abl/metabolismo , Peróxido de Hidrógeno/metabolismo , L-Aminoácido Oxidasa/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Adulto , Antineoplásicos/uso terapéutico , Caspasas/metabolismo , Línea Celular Tumoral , Daño del ADN , Interacciones Farmacológicas , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , L-Aminoácido Oxidasa/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Células Quimiorreceptoras/metabolismo , Dieta con Restricción de Proteínas/efectos adversos , Fenómenos Fisiologicos Nutricionales Maternos , Sistema Nervioso Periférico/fisiopatología , Prehipertensión/fisiopatología , Sistema Respiratorio/fisiopatología , Sistema Nervioso Simpático/fisiopatología , Animales , Peso al Nacer , Arteria Carótida Común/metabolismo , Arteria Carótida Común/patología , Arteria Carótida Común/fisiopatología , Células Quimiorreceptoras/patología , Femenino , Desarrollo Fetal , Retardo del Crecimiento Fetal/etiología , Retardo del Crecimiento Fetal/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lactancia , Masculino , Sistema Nervioso Periférico/patología , Nervio Frénico/patología , Nervio Frénico/fisiopatología , Embarazo , Prehipertensión/etiología , Prehipertensión/metabolismo , Prehipertensión/patología , Ratas Wistar , Sistema Respiratorio/patología , Sistema Nervioso Simpático/patología , Nervios Torácicos/patología , Nervios Torácicos/fisiopatologíaRESUMEN
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.
Asunto(s)
Autofagia/fisiología , Regulación de la Expresión Génica/fisiología , Chaperonas de Histonas/metabolismo , Canales Iónicos/metabolismo , Mitocondrias/fisiología , Dinámicas Mitocondriales/fisiología , Proteínas Mitocondriales/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN , Células HEK293 , Humanos , Estrés Oxidativo/fisiología , Proteína Desacopladora 1 , Regulación hacia Arriba/fisiologíaRESUMEN
Three main types of Cuban propolis directly related to their secondary metabolite composition have been identified: brown, red and yellow propolis; the former is majoritarian and is characterized by the presence of nemorosone. In this study, brown Cuban propolis extracts were found cytotoxic against HepG2 cells and primary rat hepatocytes, in close association with the nemorosone contents. In mitochondria isolated from rat liver the extracts displayed uncoupling activity, which was demonstrated by the increase in succinate-supported state 4 respiration rates, dissipation of mitochondrial membrane potential, Ca(2+) release from Ca(2+)-loaded mitochondria, and a marked ATP depletion. As in cells, the degree of such mitotoxic events was closely correlated to the nemorosone content. The propolis extracts that do not contain nemorosone were neither cytotoxic nor mitotoxic, except R-29, whose detrimental effect upon cells and mitochondria could be mediated by its isoflavonoids and chalcones components, well known mitochondrial uncouplers. Our results at least partly unravel the cytotoxic mechanism of Cuban propolis, particularly regarding brown propolis, and raise concerns about the toxicological implication of Cuban propolis consumption.
Asunto(s)
Benzofenonas/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Própolis/química , Própolis/farmacología , Desacopladores/farmacología , Animales , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Cuba , Relación Dosis-Respuesta a Droga , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Ratas , Relación Estructura-Actividad , Desacopladores/químicaRESUMEN
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.
Asunto(s)
Andrógenos/farmacología , Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Testosterona/farmacología , Antagonistas de Andrógenos/farmacología , Animales , Caspasas/metabolismo , Flutamida/farmacología , Masculino , Mitocondrias/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Wistar , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
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.
Asunto(s)
Proliferación Celular , Chaperonas de Histonas/metabolismo , Ácidos Nucleicos/metabolismo , Ribonucleoproteínas/metabolismo , Factores de Transcripción/metabolismo , Proteína bcl-X/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Células HEK293 , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo K , Chaperonas de Histonas/genética , Humanos , Immunoblotting , Microscopía Confocal , Ácidos Nucleicos/genética , Fosforilación , Unión Proteica , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas/genética , Factores de Transcripción/genética , Regulación hacia Arriba , Proteína bcl-X/genéticaRESUMEN
Clusianone is a member of the polycyclic polyprenylated acylphloroglucinol family of natural products; its cytotoxic mechanism is unknown. Clusianone is a structural isomer of nemorosone, which is a mitochondrial uncoupler and a well-known cytotoxic anti-cancer agent; thus, we addressed clusianone action at the mitochondria and its potential cytotoxic effects on cancer cells. In the HepG2 hepatocarcinoma cell line, clusianone induced mitochondrial membrane potential dissipation, ATP depletion and phosphatidyl serine externalization; this later event is indicative of apoptosis induction. In isolated mitochondria from rat liver, clusianone promoted protonophoric mitochondrial uncoupling. This was evidenced by the dissipation of mitochondrial membrane potential, an increase in resting respiration, an inhibition of Ca(2+) influx, stimulation of Ca(2+) efflux in Ca(2+)-loaded mitochondria, a decrease in ATP and NAD(P)H levels, generation of ROS, and swelling of valinomycin-treated organelles in hyposmotic potassium acetate media. The cytotoxic and uncoupling actions of clusianone were appreciably less than those of nemorosone, likely due to the presence of an intra-molecular hydrogen bond with the juxtaposed carbonyl group at the C15 position. Therefore, clusianone is capable of pharmacologically increasing the leakage of protons from the mitochondria and with favorable cytotoxicity in relation to nemorosone.
Asunto(s)
Benzofenonas/química , Productos Biológicos/química , Productos Biológicos/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Desacopladores/química , Adenosina Trifosfato/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Benzofenonas/farmacología , Benzoquinonas , Transporte Biológico/efectos de los fármacos , Compuestos Bicíclicos con Puentes/química , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Respiración de la Célula/efectos de los fármacos , Células Hep G2 , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , NAD/metabolismo , Presión Osmótica/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Desacopladores/farmacologíaRESUMEN
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.
Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Chaperonas de Histonas/genética , Factores de Transcripción/genética , Animales , Antineoplásicos/farmacología , Western Blotting , Línea Celular Tumoral , Cisplatino/farmacología , Proteínas de Unión al ADN , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Metástasis Linfática/genética , Metástasis Linfática/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Necrosis/patología , Invasividad Neoplásica , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Carcinoma de Células Escamosas de Cabeza y Cuello , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ischemic stroke cascade is composed of several pathophysiological events, providing multiple targets for pharmacological intervention. JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine) is a novel hybrid molecule, in which a benzodiazepine portion is covalently linked to a dihydropyridine ring, forming a new chemical entity with potential multisite neuroprotective activity. In the present study, JM-20 prevented PC-12 cell death induced either by glutamate, hydrogen peroxide or KCN-mediated chemical hypoxia. This molecule also protected cerebellar granule neurons from glutamate or glutamate plus pentylenetetrazole-induced damage at very low micromolar concentrations. In rat liver mitochondria, JM-20, at low micromolar concentrations, prevented the Ca2+-induced mitochondrial permeability transition, as assessed by mitochondrial swelling, membrane potential dissipation and organelle release of the pro-apoptotic protein cytochrome c. JM-20 also inhibited the mitochondrial hydrolytic activity of F1F0-ATP synthase and Ca2+ influx. Therefore, JM-20 may be a multi-target neuroprotective agent, promoting reductions in neuronal excitotoxic injury and the protection of the mitochondria from Ca2+-induced impairment as well as the preservation of cellular energy balance.
Asunto(s)
Benzodiazepinas/química , Benzodiazepinas/farmacología , Isquemia Encefálica/patología , Dihidropiridinas/química , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Niacina/análogos & derivados , Animales , Isquemia Encefálica/complicaciones , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Cerebelo/citología , Citocromos c/metabolismo , Ácido Glutámico/farmacología , Peróxido de Hidrógeno/farmacología , Hidrólisis/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Dilatación Mitocondrial/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Niacina/química , Niacina/farmacología , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Pentilenotetrazol/farmacología , Fosfatos/metabolismo , Cianuro de Potasio/farmacología , Ratas , Accidente Cerebrovascular/complicacionesRESUMEN
INTRODUCTION: Some fatty acids may play an important role in regulating metabolism through PPARs activation. Conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and increase body metabolism; this effect has been associated with up-regulation of mitochondrial uncoupling proteins (UCPs) and PPARalfa activation. Oleic acid has shown beneficial effects on health, decreasing oxidative stress and improving clinical conditions related to obesity. Therefore, in this work, we addressed the effects of a oleic plus CLA-supplemented murine diet on body metabolism, mitochondrial energetics and oxidative stress in the liver, as well as on other associated morphological and functional parameters in C57BL/6 mice. METHOD: The diet was supplemented with 2% CLA mixture (cis-9, trans-10 and trans-10, cis-12 isomers; 45% of each isomer) and/or 0.7% olive oil on alternating days (60 days) by gavage. RESULTS: The results showed that diet supplementation with CLA increases body metabolism and reduces lipid accumulation in adipose tissues. Groups that received oleic acid (oleic and CLA oleic) showed decreased levels of total cholesterol and cholesterol non-HDL, and increased levels of HDL-cholesterol. Livers of mice fed a diet supplemented with CLA showed high levels UCP2 mRNA, and the isolated hepatic mitochondria showed indications of UCP activity and increased ROS generation. Oleic acid partially reversed the lower lipid accumulation increasing PPARgamma content, reversed the higher ROS generation by liver mitochondria and improved liver oxidative status. CONCLUSIONS: These results indicate a beneficial and secure dose of CLA and oleic acid for diet supplementation in mice, which increases body metabolism inducing UCP2 overexpression/activity in liver while preserving the redox state of the liver. Therefore, diet supplementation with CLA associated to oleic acid may be regarded as a potential strategy for controlling obesity and oxidative stress. Supported by FAPESP.