RESUMO
Selenium (Se), which is a central component for the biosynthesis and functionality of selenoproteins, plays an important role in the anti-oxidative response, reproduction, thyroid hormone metabolism and the protection from infection and inflammation. However, dietary Se effects have not well been established to date and the available studies often present contradictory results. To obtain a better understanding of Se intake and its influence on the metabolism of living systems, we have utilized a metabolomics approach to gain insight into the specific metabolic alterations caused by Se deficiency in mice. Serum samples were collected from two groups of C57BL/6 mice: an experimental group which was fed a Se-deficient diet and controls consuming normal chow. The samples were analyzed by (1)H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. The resulting metabolite data were examined separately for both analytical methods and in a combined manner. By applying multivariate statistical analysis we were able to distinguish the two groups and detect a metabolite pattern associated with Se deficiency. We found that the concentrations of 15 metabolites significantly changed in serum samples collected from Se-deficient mice when compared to the controls. Many of the perturbed biological pathways pointed towards compensatory mechanisms during Se deficiency and were associated with amino acid metabolism. Our findings show that a metabolomics approach may be applied to identify the metabolic impact of Se and reveal the most impaired biological pathways as well as induced regulatory mechanisms during Se deficiency.
Assuntos
Selênio/deficiência , Aminoácidos/metabolismo , Animais , Antioxidantes/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Piruvatos/metabolismo , Selênio/metabolismo , Selenoproteínas/metabolismoRESUMO
Although various lines of evidence suggest that oxidative stress plays a role in human prostate cancer initiation and progression, there is a paucity of direct evidence for its role in tumor initiation. To begin to address this issue, we developed a novel tumorigenesis model by reducing the expression of multiple selenoproteins (SPs) in mouse prostatic epithelium. This was accomplished via the prostate-specific deletion of Trsp, a gene that encodes a transfer RNA (Sec tRNA) required for the insertion of selenocysteine residues into SPs during their translation. By 6 weeks of age, Trsp-deficient mice exhibited widespread prostatic intraepithelial neoplasia lesions in all prostatic lobes, which then progressed to high-grade dysplasia and microinvasive carcinoma by 24 weeks. In contrast to other murine prostate cancer models, Trsp-deficient mice required neither the deletion of a tumor suppressor nor the transgenic introduction of an oncogene for prostatic intraepithelial neoplasia lesion development. In keeping with the antioxidant functions of several SPs, we found increases in lipid peroxidation markers in Trsp-deficient epithelial cells. This novel model of prostate neoplasia provides evidence for the existence of a selenoprotein or selenoproteins capable of acting as a tumor suppressor in the murine prostate.
Assuntos
Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/genética , RNA de Transferência Aminoácido-Específico/genética , Animais , Progressão da Doença , Epitélio/patologia , Deleção de Genes , Genes Supressores de Tumor , Humanos , Masculino , Camundongos , Estresse Oxidativo , Próstata/patologia , Neoplasia Prostática Intraepitelial/genética , Neoplasias da Próstata/patologia , Selenoproteínas/genéticaRESUMO
Infection with helminth parasites triggers strong and stereotypic immune responses in humans and mice, which can protect against specific experimentally-induced autoimmune diseases. We have shown that infection with the rat tapeworm, Hymenolepis diminuta, confers a protective effect on FCA-induced joint inflammation. Here, we investigated the effect of a prophylactic infection with H. diminuta on the K/BxN-serum model of polyarthritis in BALB/c mice. Mice were infected with 10 cysticercoids of H. diminuta by oral gavage and 8 days later arthritis was induced by i.p. injection of K/BxN arthritogenic serum. Joint swelling and pain measurements were recorded throughout a 13 day time course. At necropsy, joints and blood serum were collected. K/BxN-treated mice developed joint inflammation in the front paws, hind paws and knees as shown by increased swelling, mechanical allodynia and myeloperoxidase activity. Mice infected with H. diminuta had more severe disease, with increased eosinophil peroxidase activity in their paws and greater inflammatory infiltrate and synovitis in the knee joints. Hymenolepis diminuta-infected mice displayed significant increases in serum levels of C5a and mast cell protease-1 compared with K/BxN-serum only treatment, the latter being indicative of mast cell activation. In contrast to the protective effect of infection with H. diminuta in FCA-induced monoarthritis, infection with this helminth exacerbated K/BxN serum-induced polyarthritis in BALB/c mice. This correlated with increases in C5a and mast cell activation: factors critical in the development of K/BxN-induced arthritis. Thus, while data accumulate from animal models showing that infection with helminth parasites may be beneficial for a variety of auto-inflammatory diseases, our findings demonstrate the potential for helminths to exacerbate disease. Hence care is needed when helminth therapy is translated into a clinical setting.
Assuntos
Artrite/patologia , Doenças Autoimunes/patologia , Himenolepíase/patologia , Hymenolepis diminuta/imunologia , Animais , Artrite/induzido quimicamente , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/complicações , Modelos Animais de Doenças , Himenolepíase/complicações , Articulações/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , DorRESUMO
Techniques for visualizing and quantifying the microvasculature of tumors are essential not only for studying angiogenic processes but also for monitoring the effects of anti-angiogenic treatments. Given the relatively limited information that can be gleaned from conventional 2-D histological analyses, there has been considerable interest in methods that enable the 3-D assessment of the vasculature. To this end, we employed a polymerizing intravascular contrast medium (Microfil) and micro-computed tomography (micro-CT) in combination with a maximal spheres direct 3-D analysis method to visualize and quantify ex-vivo vessel structural features, and to define regions of hypoperfusion within tumors that would be indicative of necrosis. Employing these techniques we quantified the effects of a vascular disrupting agent on the tumor vasculature. The methods described herein for quantifying whole tumor vascularity represent a significant advance in the 3-D study of tumor angiogenesis and evaluation of novel therapeutics, and will also find potential application in other fields where quantification of blood vessel structure and necrosis are important outcome parameters.
Assuntos
Angiografia , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Microtomografia por Raio-X , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Humanos , Imageamento Tridimensional , Camundongos , Necrose , Neoplasias/diagnóstico por imagemRESUMO
Loss of the PTEN tumor suppressor is a common occurrence in human prostate cancer, particularly in advanced disease. In keeping with its role as a pivotal upstream regulator of the phosphatidylinositol 3-kinase signaling pathway, experimentally-induced deletion of Pten in the murine prostate invariably results in neoplasia. However, and unlike humans where prostate tumorigenesis likely evolves over decades, disease progression in the constitutively Pten deficient mouse prostate is relatively rapid, culminating in invasive cancer within several weeks post-puberty. Given that the prostate undergoes rapid androgen-dependent growth at puberty, and that Pten excisions during this time might be especially tumorigenic, we hypothesized that delaying prostate-specific Pten deletions until immediately after puberty might alter the pace of tumorigenesis. To this end we generated mice with a tamoxifen-inducible Cre recombinase transgene enabling temporal control over prostate-specific gene alterations. This line was then interbred with mice carrying floxed Pten alleles. Despite evidence of increased Akt/mTOR/S6K axis activity at early time points in Pten-deficient epithelial cells, excisions induced in the post-pubertal (6 wk-old) prostate yielded gradual acquisition of a range of lesions. These progressed from pre-malignant changes (nuclear atypia, focal hyperplasia) and low grade prostatic intraepithelial neoplasia (PIN) at 16-20 wks post-tamoxifen exposure, to overtly malignant lesions by approximately 1 yr of age, characterized by high-grade PIN and microinvasive carcinoma. In contrast, when Pten excisions were triggered in the pre-pubertal (2 week-old) prostate, neoplasia evolved over a more abbreviated time-frame, with a spectrum of premalignant lesions, as well as overt PIN and microinvasive carcinoma by 10-12 wks post-tamoxifen exposure. These results indicate that the developmental stage at which Pten deletions are induced dictates the pace of PIN development.
Assuntos
Deleção de Genes , Genes Supressores de Tumor , PTEN Fosfo-Hidrolase/genética , Neoplasia Prostática Intraepitelial/enzimologia , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteína de Ligação a Androgênios/genética , Animais , Apoptose , Arrestinas/metabolismo , Proliferação de Células , Cruzamentos Genéticos , Progressão da Doença , Epitélio/enzimologia , Epitélio/patologia , Feminino , Humanos , Integrases/metabolismo , Masculino , Camundongos , Invasividade Neoplásica , PTEN Fosfo-Hidrolase/deficiência , Fosfatidilinositol 3-Quinases/metabolismo , Lesões Pré-Cancerosas/tratamento farmacológico , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Neoplasia Prostática Intraepitelial/tratamento farmacológico , Neoplasia Prostática Intraepitelial/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Ratos , Proteína S6 Ribossômica/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/uso terapêutico , Fatores de Tempo , Regulação para Cima , beta-ArrestinasRESUMO
It is well established that misfolded forms of cellular prion protein (PrP [PrPC]) are crucial in the genesis and progression of transmissible spongiform encephalitis, whereas the function of native PrPC remains incompletely understood. To determine the physiological role of PrPC, we examine the neurophysiological properties of hippocampal neurons isolated from PrP-null mice. We show that PrP-null mouse neurons exhibit enhanced and drastically prolonged N-methyl-D-aspartate (NMDA)-evoked currents as a result of a functional upregulation of NMDA receptors (NMDARs) containing NR2D subunits. These effects are phenocopied by RNA interference and are rescued upon the overexpression of exogenous PrPC. The enhanced NMDAR activity results in an increase in neuronal excitability as well as enhanced glutamate excitotoxicity both in vitro and in vivo. Thus, native PrPC mediates an important neuroprotective role by virtue of its ability to inhibit NR2D subunits.
RESUMO
It is well established that misfolded forms of cellular prion protein (PrP [PrP(C)]) are crucial in the genesis and progression of transmissible spongiform encephalitis, whereas the function of native PrP(C) remains incompletely understood. To determine the physiological role of PrP(C), we examine the neurophysiological properties of hippocampal neurons isolated from PrP-null mice. We show that PrP-null mouse neurons exhibit enhanced and drastically prolonged N-methyl-d-aspartate (NMDA)-evoked currents as a result of a functional upregulation of NMDA receptors (NMDARs) containing NR2D subunits. These effects are phenocopied by RNA interference and are rescued upon the overexpression of exogenous PrP(C). The enhanced NMDAR activity results in an increase in neuronal excitability as well as enhanced glutamate excitotoxicity both in vitro and in vivo. Thus, native PrP(C) mediates an important neuroprotective role by virtue of its ability to inhibit NR2D subunits.
Assuntos
Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Proteínas PrPC/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Morte Celular/fisiologia , Células Cultivadas , Eletrofisiologia , Agonistas de Aminoácidos Excitatórios/metabolismo , Feminino , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , N-Metilaspartato/metabolismo , Neurônios/citologia , Proteínas PrPC/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Employing the Hprt locus as the site for targeted transgenesis we have developed mice expressing the tamoxifen-inducible Cre-ER(T2) fusion protein under the control of the ARR2-rat probasin promoter. This system enables external control over the timing of prostate epithelial cell-specific gene alterations. Using both the ROSA26-lacZ and ROSA26-EYFP reporter strains to monitor recombinase activity, Cre-ER(T2) was found to be specifically expressed in the prostatic epithelium and was strictly tamoxifen dependent. This strain thus allows precise control over the timing of gene alterations in the mouse prostate, enabling analyses of the phenotypic consequences of gene alterations in mice of any age. It also provides an ideal platform to study the impact of environmental, hormonal, and age-related factors on prostate tumorigenesis. This latter feature will be of particular value given the paucity of murine models that accurately mimic the late onset and prolonged natural history of human prostate cancer.