RESUMEN
Phthalates are endocrine-disrupting chemicals used in consumer products. Although phthalates are obesogens and affect metabolic function, it is unknown if chronic exposure for 6 months to a phthalate mixture alters adipose tissue phenotype in female mice. After vehicle or mixture exposure, white adipose tissue and brown adipose tissue (WAT and BAT) were analyzed for expression of adipogenesis, proliferation, angiogenesis, apoptosis, oxidative stress, inflammation, and collagen deposition markers. The mixture altered WAT morphology, leading to an increase in hyperplasia, blood vessel number, and expression of BAT markers (Adipoq and Fgf2) in WAT. The mixture increased the expression of the inflammatory markers, Il1ß, Ccl2, and Ccl5, in WAT. The mixture also increased expression of the proapoptotic (Bax and Bcl2) and antiapoptotic (Bcl2l10) factors in WAT. The mixture increased expression of the antioxidant Gpx1 in WAT. The mixture changed BAT morphology by increasing adipocyte diameter, whitening area, and blood vessel number and decreased expression of the thermogenic markers Ucp1, Pgargc1a, and Adrb3. Furthermore, the mixture increased the expression of adipogenic markers Plin1 and Cebpa, increased mast cell number, and increased Il1ß expression in BAT. The mixture also increased expression of the antioxidant markers Gpx and Nrf2 and the apoptotic marker Casp2 in BAT. Collectively, these data indicate that chronic exposure to a phthalate mixture alters WAT and BAT lipid metabolism phenotypes in female mice, leading to an apparent shift in their normal morphology. Following long-term exposure to a phthalate mixture, WAT presented BAT-like features and BAT presented WAT-like features.
Asunto(s)
Tejido Adiposo Pardo , Antioxidantes , Animales , Ratones , Femenino , Tejido Adiposo Pardo/metabolismo , Antioxidantes/metabolismo , Tejido Adiposo , Tejido Adiposo Blanco , Fenotipo , Ratones Endogámicos C57BL , Caspasa 2/metabolismoRESUMEN
BACKGROUND: CAT25 (T25mononucleotide repeat of the Caspase 2 gene), is a promising DNA marker for detecting microsatellite instability (MSI) in colorectal cancer. CAT25 has the potential to be incorporated into the Bethesda panel, a commonly used panel of DNA microsatellites, or replace it in its entirety. We aimed to develop and validate a high-resolution melting-PCR (HRM-PCR) method for CAT25 instability detection in clinical samples. METHODS: The instability of CAT25, BAT25 (a poly(A) tract occurring in c-kit) and BAT26 (a poly(A) tract localized in hMSH2) microsatellites were assessed in DNA from tumour and peripheral blood obtained from 110 patients with colorectal cancer using HRM-PCR and capillary electrophoresis. Immunohistochemistry (IHC) staining for MSH2, MSH6, MLH1, and PMS2 enzymes was performed on tumours with jigj MSI. Allelic size variation of CAT25 was analysed on peripheral blood DNA from 208 healthy volunteers. RESULTS: The HRM-PCR for CAT25 was validated in clinical samples. CAT25 showed a tight range of 64-66 base pairs. Of 110 tumours, 11 had High MSI, later confirmed by IHC. CAT25 defines MSI alone as well as when used together with BAT25 and BAT26. CAT25 results provided 100% predictive values and p < 0.0001 to classify a tumour as having high MSI. CONCLUSIONS: We developed and validated a new HRM-PCR assay to detect CAT25 instability. Our findings showed a limited allelic size variation of CAT25 and highlighted to CAT25 as a promising marker for MSI analysis.
Asunto(s)
Caspasa 2/genética , Neoplasias Colorrectales/genética , Marcadores Genéticos/genética , Repeticiones de Microsatélite/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Femenino , Humanos , Inmunohistoquímica/métodos , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Leishmaniasis is considered a serious public health problem in several regions in Brazil and worldwide. This research aimed to perform a histopathological and proteomic study of parotid, submandibular and sublingual glands of BALB/c mice infected by Leishmania (L) infantum chagasi using histological, immunohistochemical and epifluorescence techniques. Twelve isogenic BALB/c male mice, around six- to eight-weeks old, were separated into two groups: the animals of the control group were injected with 0.15 ml of NaCl, while those in the experimental group were inoculated with 5 × 10(6) amastigote forms of Leishmania (L) infantum chagasi by the ip route. After 50 days, animals were euthanized and major salivary glands were collected to perform histological, immunohistochemical and epifluorescence techniques using anti-Caspase-2, anti-Ki-67 and anti-ß-catenin antibodies, respectively. The histological and morphometric evaluation showed clusters of mononuclear inflammatory cells and a higher area and perimeter of the parotid gland. However, none of the salivary glands had morphophysiological impairment. There was no immunoreactivity to the anti-caspase-2 antibody and Ki67 expression in acinar and ductal cells in both groups. According to the immunofluorescence staining, the ß-catenin antibodies did not show nuclear expression, suggesting no uncontrolled proliferation. The data obtained in this study showed population and morphological stability of major salivary glands after 50 days post-infection by Leishmania (L) infantum chagasi.
Asunto(s)
Leishmaniasis/patología , Glándula Parótida/patología , Glándula Sublingual/patología , Glándula Submandibular/patología , Animales , Caspasa 2/análisis , Modelos Animales de Enfermedad , Histocitoquímica , Inmunohistoquímica , Antígeno Ki-67/análisis , Leishmania infantum/crecimiento & desarrollo , Leishmaniasis/parasitología , Masculino , Ratones Endogámicos BALB C , Microscopía Fluorescente , beta Catenina/análisisRESUMEN
BACKGROUND: Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. RESULTS: The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. CONCLUSIONS: The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.
Asunto(s)
Apoptosis/fisiología , Infecciones por Ureaplasma/fisiopatología , Ureaplasma/patogenicidad , Citoesqueleto de Actina/ultraestructura , Adhesión Bacteriana , Caspasa 2/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Supervivencia Celular , Femenino , Citometría de Flujo , Expresión Génica , Gentamicinas/farmacología , Células HeLa/microbiología , Humanos , Microscopía Confocal , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Estadísticas no Paramétricas , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Ureaplasma/efectos de los fármacosRESUMEN
BACKGROUND: Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location ofUreaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversuminvasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. RESULTS: The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. CONCLUSIONS: The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.
Asunto(s)
Humanos , Femenino , Ureaplasma/patogenicidad , Infecciones por Ureaplasma/fisiopatología , Apoptosis/fisiología , Factores de Tiempo , Ureaplasma/efectos de los fármacos , Adhesión Bacteriana , Citoesqueleto de Actina/ultraestructura , Gentamicinas/farmacología , Células HeLa/microbiología , Expresión Génica , Supervivencia Celular , Factor de Necrosis Tumoral alfa/metabolismo , Estadísticas no Paramétricas , Microscopía Confocal , Caspasa 3/metabolismo , Caspasa 2/metabolismo , Caspasa 9/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Citometría de Flujo , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismoRESUMEN
PURPOSE: To analyze the influence of α-tocopherol supplementation on the levels of oxidative stress and apoptosis rates in the anal sphincter induced by orchiectomy in rats. METHODS: Forty male Wistar rats weighing 250-300 g, were divided into four groups and sacrificed 8 weeks after: I- Control: sham; II- Orchiectomy: bilateral orchiectomy; III- Pre Orchiectomy Tocopherol: α-tocopherol supplementation for 4 weeks preceding bilateral orchiectomy; IV- Orchiectomy Full Tocopherol: α-tocopherol supplementation for 4 weeks before and 8 weeks after bilateral orchiectomy. The anal sphincter was analyzed stereologically to evaluate the density of collagen and the muscle fibers. The oxidative stress and the apoptosis were determined with 8-isprostane and caspase-3, respectively. RESULTS: The collagen fibers concentration was statistically greater in Orchiectomy group than the others. The muscle fibers concentration was higher in Control and Orchiectomy Full Tocopherol than Orchiectomy and Pre Orchiectomy Tocopherol groups. Orchiectomy group showed higher 8-isoprostane concentrations compared to the other groups (p < 0.0003). Pre Orchiectomy Tocopherol and Orchiectomy Full Tocopherol groups presented caspase-3 levels lower than the Orchiectomy group (0.0072). CONCLUSION: Vitamin supplementation with α-tocopherol for 12 weeks had the highest protection against bilateral orchiectomy generation of reactive oxygen species as well as apoptosis in the muscle fibers of the anal sphincter of rats.
Asunto(s)
Canal Anal/metabolismo , Antioxidantes/administración & dosificación , Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , alfa-Tocoferol/administración & dosificación , Animales , Caspasa 2/metabolismo , Dinoprost/análogos & derivados , Dinoprost/sangre , Masculino , Microscopía Fluorescente , Fibras Musculares Esqueléticas/metabolismo , Orquiectomía , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Testosterona/sangreRESUMEN
Canine distemper virus (CDV) may induce multifocal demyelination in the central nervous system of infected dogs. The present work investigated apoptosis in white and gray matter (granular layer) in the cerebellum of naturally infected dogs by the analysis of the expression of the pro-apoptotic antigens caspase 2 and 3 , b(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL-staining) positivity, annexin-V immunodetection, and the presence of the anti-apoptotic antigens, BCl-2 and p53. Cerebellum specimens were obtained from the Laboratory of Animal Pathology, from 1995 to 2009, and the 5-µm thick fragments were stained both with hematoxylin-eosin and Shorr. All samples were diagnosed as positive for CDV genome by reverse transcriptase polymerase chain reaction targeting the nucleocapsid gene. The anti-apoptotic pathways evidenced in this study were BCl-2 and p53 proteins that were intensively detected in cerebellum of CDV positive slides (40-80% of labeled cells/mm2). In addition, the apoptosis markers annexin-V and TUNEL are directly correlated among the same samples (80 and 40% of labeled cells, respectively). This is the first description of p53 and annexin-V expression, characterized as anti-apoptotic and apoptotic proteins, involvement in canine natural cases of CDV infections. (AU)
Asunto(s)
Animales , Virus del Moquillo Canino/aislamiento & purificación , Caspasa 2/análisis , Caspasa 2 , Caspasa 3/análisis , Caspasa 3 , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor , Anexina A5/biosíntesis , Hematoxilina , Eosina Amarillenta-(YS) , Coloración y Etiquetado/métodos , Coloración y Etiquetado/veterinariaRESUMEN
Since the regression of the corpus luteum (CL) occurs via a tightly controlled apoptotic process, studies were designed to determine if local administration of the antiapoptotic agent sphingosine 1-phosphate (S1P) effectively blocks the luteolytic action of prostaglandin F-2alpha (PGF-2alpha). On day 19 of pregnancy, 2 hr before systemic PGF-2alpha administration, rats were injected intrabursa with either S1P or vehicle (control). The activity of four caspases, which contribute to the initial (caspase-2, -8, and -9) and final (caspase-3) events in apoptosis was measured in pooled CL from four individual ovaries at 0 and 4 hr after PGF-2alpha injection. The expression of the phosphorylated form of AKT (pAKT) and tumor necrosis factor-alpha (TNF-alpha) was analyzed by ELISA. In addition, cell death was evaluated by electronic microscopy (EM) in CL 4 and 36 hr after PGF-2alpha injection. The activity of caspase-2, -3, and -8 was significantly greater by 4 hr after PGF-2alpha, but not caspase-9 activity. In contrast, expression of pAKT and TNF-alpha decreased significantly. Administration of S1P suppressed (P < 0.05) these effects, decreasing caspase activities and increasing pAKT and TNF-alpha expression. The administration of S1P also significantly decreased the percentage of luteal apoptotic cells induced by PGF-2alpha. PGF-2alpha treatment increased the prevalence of luteal cells with advanced signs of apoptosis (i.e., multiple nuclear fragments, chromatin condensation, or apoptotic bodies). S1P treatment suppressed these changes and increased the blood vessel density. These results suggest that S1P blocks the luteolytic effect of the PGF-2alpha by decreasing caspase-2, -3, and -8 activities and increasing AKT phosphorylation and TNF-alpha expression.
Asunto(s)
Dinoprost/metabolismo , Luteólisis , Lisofosfolípidos/farmacología , Esfingosina/análogos & derivados , Animales , Caspasa 2/metabolismo , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Cuerpo Lúteo/metabolismo , Cuerpo Lúteo/ultraestructura , Femenino , Luteólisis/efectos de los fármacos , Luteólisis/fisiología , Lisofosfolípidos/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Embarazo , Progesterona/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Esfingosina/metabolismo , Esfingosina/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Apoptosis is associated with the regression of the corpus luteum (CL) in many species. Since caspases play a central role in apoptosis, we studied several initiators (-2, -8, and -9) and the main effector (-3) caspase in the CL during the estrous cycle of the rat. Two different populations of CL (old and new) were identified on ovaries at estrus and diestrus II (DII). Diminished (P < 0.05) luteal progesterone content and P450scc levels suggested that functional luteolysis occurred between the new CL at DII and old CL at estrus, whereas the decline (P < 0.05) in luteal weight indicated that structural regression was occurring between old CL at estrus to DII. Immunostaining for caspase-2 in luteal and endothelial cells appeared to increase as the luteal phase progressed, peaking at DII in the old CL. However, caspase-8 and -9 immunostaining showed little change with a slight increase at estrus in the old population. Notably, caspase-3 staining appeared to peak at DII in the new CL. Enzyme activity of caspase-9 increased (P < 0.05) in the new CL at DII, followed by that of caspase-2 and -3 in old CL at estrus. Caspase-8 activity did not change at any stage. The number of apoptotic cells increased at DII in the old CL. These results suggest an important role for this protease family during early events of luteolysis in the rat estrous cycle.
Asunto(s)
Caspasas/metabolismo , Cuerpo Lúteo/enzimología , Ciclo Estral/fisiología , Animales , Apoptosis , Western Blotting/métodos , Caspasa 2/análisis , Caspasa 3/análisis , Caspasa 8/análisis , Caspasa 9/análisis , Caspasas/análisis , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/análisis , Femenino , Inmunohistoquímica/métodos , Etiquetado Corte-Fin in Situ , Progesterona/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
A variety of stimuli can induce cells to undergo apoptosis, with one of the most reproducible inducers being mild oxidative stress following exposure to anticancer agents. Apoptosis involves events mediated by cysteine proteases (caspases) that are classified as initiators (-8, -9 and -12) or executors (-2, -3, -6 and -7). In this study, we examined the mechanisms of apoptosis induced by dehydrocrotonin (DHC), a diterpene lactone isolated from the Amazonian plant Croton cajucara, and its synthetic derivative, dimethylamide-crotonin (DCR), in human HL60 promyelocytic leukemia cells. Flow cytometric analysis of HL60 cells after treatment for 72 h showed that DCR- and DHC-induced apoptosis, with maximum cell death at a concentration of 250 microM for both compounds. DCR and DHC were effective in triggering the activation of caspases-2, -6 and -9. The level of reduced glutathione (GSH) decreased, whereas there was an increase in thiobarbituric acid-reactive substance (TBARS) production and in mitochondrial swelling. These effects on mitochondrial swelling, GSH content and lipid peroxidation were abolished by cyclosporine A, an inhibitor of the membrane permeability transition. The cytotoxicity of DHC and DCR was prevented by a high concentration of GSH (15 mM) in the culture medium. These results indicate that DCR and DHC produced apoptosis partly by oxidative stress-induced lipid peroxidation, which triggered the caspase cascade, that lead to apoptotic cell death in HL60 cells. Based on the pattern of caspase activation, on the increase in mitochondrial swelling and on the inhibitory action of cyclosporine A, we conclude that DCR and DHC triggered apoptosis in HL60 cells probably through cytochrome c release and apoptosome formation.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Diterpenos de Tipo Clerodano/farmacología , Diterpenos/farmacología , Lactonas/farmacología , Peroxidación de Lípido/efectos de los fármacos , Anexina A5 , Caspasa 2 , Caspasa 6 , Caspasa 9 , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Fluoresceína-5-Isotiocianato , Células HL-60 , Humanos , Mitocondrias/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Corteza de la Planta/química , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Violacein, a pigment isolated from Chromobacterium violaceum, has been reported to have multiple biological activities including in vitro antitumor effects. Certain anticancer agents are known to induce apoptosis in human tumor cell lines. In this work, our aim was to investigate the effectiveness of violacein/beta-cyclodextrin (beta-CD)-containing systems to produce lethal effects in the human promyelocytic leukemia cell line HL60. Using the MTT tetrazolium reduction test, IC(50) for the inclusion complexes (1:1 and 1:2 violacein:beta-CD molar ratios) and violacein alone were less than 1 microM. Violacein and violacein/beta-CD complexes were able to induce NBT reduction. Moreover, by using the Feulgen reaction, all the compounds were found to trigger apoptosis in HL60 cells, inducing around 35% of DNA fragmentation, as analyzed through the diphenylamine assay. In addition, caspases seem to play an important role in the activation of the executioner phase of apoptosis induced by violacein and its derivatives.
Asunto(s)
Apoptosis/efectos de los fármacos , Indoles/toxicidad , Colorantes de Rosanilina , Tripanocidas/toxicidad , beta-Ciclodextrinas , Caspasa 2 , Caspasa 6 , Caspasa 9 , Caspasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colorantes , Ciclodextrinas/química , Fragmentación del ADN/efectos de los fármacos , Células HL-60 , Humanos , Indicadores y Reactivos , Indoles/química , Nitroazul de Tetrazolio , Tripanocidas/químicaRESUMEN
The aim of this study was to evaluate the participation of the Jak-1 and STAT-1 proteins in sodium butyrate-induced apoptosis in 2C4 cells derived from human fibrosarcoma. Making use of Jak-1 or STAT-1 deficient cell lines, we demonstrated that the apoptotic process induced by butyrate is independent of the presence of these proteins. In addition, this work showed that, although the constitutive expression of pro-caspases-2 and -3 is reduced in STAT-1 cells, the activity of caspase-3 is preserved in both Jak-1 and STAT-1 deficient cells and is similar to that seen in 2C4 parental cells. In conclusion, we demonstrated that the absence of functionally active Jak-1 or STAT-1 protein directly affects the TNF-alpha-induced apoptosis, but does not alter the sodium butyrate-induced apoptosis in cells derived from human fibrosarcoma.