RESUMO
Hypoxia is a prognostic biomarker of rapidly growing cancers, where the extent of hypoxia is an indication of tumor progression and prognosis; therefore, hypoxia is also used for staging while performing chemo- and radiotherapeutics for cancer. Contrast-enhanced MRI using EuII-based contrast agents is a noninvasive method that can be used to map hypoxic tumors, but quantification of hypoxia using these agents is challenging due to the dependence of signal on the concentration of both oxygen and EuII. Here, we report a ratiometric method to eliminate concentration dependence of contrast enhancement of hypoxia using fluorinated EuII/III-containing probes. We studied three different EuII/III couples of complexes containing 4, 12, or 24 fluorine atoms to balance fluorine signal-to-noise ratio with aqueous solubility. The ratio between the longitudinal relaxation time (T1) and 19F signal of solutions containing different ratios of EuII- and EuIII-containing complexes was plotted against the percentage of EuII-containing complexes in solution. We denote the slope of the resulting curves as hypoxia indices because they can be used to quantify signal enhancement from Eu, that is related to oxygen concentration, without knowledge of the absolute concentration of Eu. This mapping of hypoxia was demonstrated in vivo in an orthotopic syngeneic tumor model. Our studies significantly contribute toward improving the ability to radiographically map and quantify hypoxia in real time, which is critical to the study of cancer and a wide range of diseases.
Assuntos
Flúor , Neoplasias , Humanos , Imageamento por Ressonância Magnética/métodos , Hipóxia , OxigênioRESUMO
The availability of high-fidelity animal models for oncology research has grown enormously in recent years, enabling preclinical studies relevant to prevention, diagnosis, and treatment of cancer to be undertaken. This has led to increased opportunities to conduct co-clinical trials, which are studies on patients that are carried out parallel to or sequentially with animal models of cancer that mirror the biology of the patients' tumors. Patient-derived xenografts (PDX) and genetically engineered mouse models (GEMM) are considered to be the models that best represent human disease and have high translational value. Notably, one element of co-clinical trials that still needs significant optimization is quantitative imaging. The National Cancer Institute has organized a Co-Clinical Imaging Resource Program (CIRP) network to establish best practices for co-clinical imaging and to optimize translational quantitative imaging methodologies. This overview describes the ten co-clinical trials of investigators from eleven institutions who are currently supported by the CIRP initiative and are members of the Animal Models and Co-clinical Trials (AMCT) Working Group. Each team describes their corresponding clinical trial, type of cancer targeted, rationale for choice of animal models, therapy, and imaging modalities. The strengths and weaknesses of the co-clinical trial design and the challenges encountered are considered. The rich research resources generated by the members of the AMCT Working Group will benefit the broad research community and improve the quality and translational impact of imaging in co-clinical trials.
Assuntos
Neoplasias , Animais , Camundongos , Humanos , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Neoplasias/patologia , Modelos Animais de Doenças , Diagnóstico por ImagemRESUMO
Hypoxia is a hallmark of many diseases, including cancer, arthritis, heart and kidney diseases, and diabetes, and it is often associated with disease aggressiveness and poor prognosis. Consequently, there is a critical need for imaging hypoxia in a noninvasive and direct way to diagnose, stage, and monitor the treatment and development of new therapies for these diseases. Eu-containing contrast agents for magnetic resonance imaging have demonstrated potential for in vivo imaging of hypoxia via changes in metal oxidation state from +2 to +3, but rapid oxidation in blood limits EuII-containing complexes to studies compatible with direct injection to sites. Here, we report a new EuII-containing complex that persists in oxygenated environments and is capable of persisting in blood long enough for imaging by magnetic resonance imaging. We describe the screening of a library of ligands that led to the discovery of the complex as well as a pH-dependent mechanism that hinders oxidation to enable usefulness in vivo. These studies of the first divalent lanthanide complex that persists in oxygenated solutions open the door to the use of EuII-based contrast agents for imaging hypoxia in a wide range of diseases.
Assuntos
Európio , Elementos da Série dos Lantanídeos , Ligantes , Meios de Contraste , Imageamento por Ressonância Magnética/métodosRESUMO
Hypoxia in solid tumors is associated with poor prognosis, increased aggressiveness, and strong resistance to therapeutics, making accurate monitoring of hypoxia important. Several imaging modalities have been used to study hypoxia, but each modality has inherent limitations. The use of a second modality can compensate for the limitations and validate the results of any single imaging modality. In this review, we describe dual-mode imaging systems for the detection of hypoxia that have been reported since the start of the 21st century. First, we provide a brief overview of the hallmarks of hypoxia used for imaging and the imaging modalities used to detect hypoxia, including optical imaging, ultrasound imaging, photoacoustic imaging, single-photon emission tomography, X-ray computed tomography, positron emission tomography, Cerenkov radiation energy transfer imaging, magnetic resonance imaging, electron paramagnetic resonance imaging, magnetic particle imaging, and surface-enhanced Raman spectroscopy, and mass spectrometric imaging. These overviews are followed by examples of hypoxia-relevant imaging using a mixture of probes for complementary single-mode imaging techniques. Then, we describe dual-mode molecular switches that are responsive in multiple imaging modalities to at least one hypoxia-induced pathological change. Finally, we offer future perspectives toward dual-mode imaging of hypoxia and hypoxia-induced pathophysiological changes in tumor microenvironments.
Assuntos
Neoplasias , Humanos , Hipóxia/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada de Emissão de Fóton Único , Microambiente TumoralRESUMO
Millions of people a year receive magnetic resonance imaging (MRI) contrast agents for the diagnosis of conditions as diverse as fatty liver disease and cancer. Gadolinium chelates, which provide preferred T1 contrast, are the current standard but face an uncertain future due to increasing concerns about their nephrogenic toxicity as well as poor performance in high-field MRI scanners. Gadolinium-containing nanocrystals are interesting alternatives as they bypass the kidneys and can offer the possibility of both intracellular accumulation and active targeting. Nanocrystal contrast performance is notably limited, however, as their organic coatings block water from close interactions with surface Gadoliniums. Here, these steric barriers to water exchange are minimized through shape engineering of plate-like nanocrystals that possess accessible Gadoliniums at their edges. Sulfonated surface polymers promote second-sphere relaxation processes that contribute remarkable contrast even at the highest fields (r1 = 32.6 × 10-3 m Gd-1 s-1 at 9.4 T). These noncytotoxic materials release no detectable free Gadolinium even under mild acidic conditions. They preferentially accumulate in the liver of mice with a circulation half-life 50% longer than commercial agents. These features allow these T1 MRI contrast agents to be applied for the first time to the ex vivo detection of nonalcoholic fatty liver disease in mice.
Assuntos
Gadolínio , Nanopartículas , Animais , Meios de Contraste , Imageamento por Ressonância Magnética , CamundongosRESUMO
Mutations in the skeletal muscle Ca2+ release channel, the type 1 ryanodine receptor (RYR1), cause malignant hyperthermia susceptibility (MHS) and a life-threatening sensitivity to heat, which is most severe in children. Mice with an MHS-associated mutation in Ryr1 (Y524S, YS) display lethal muscle contractures in response to heat. Here we show that the heat response in the YS mice is exacerbated by brown fat adaptive thermogenesis. In addition, the YS mice have more brown adipose tissue thermogenic capacity than their littermate controls. Blood lactate levels are elevated in both heat-sensitive MHS patients with RYR1 mutations and YS mice due to Ca2+ driven increases in muscle metabolism. Lactate increases brown adipogenesis in both mouse and human brown preadipocytes. This study suggests that simple lifestyle modifications such as avoiding extreme temperatures and maintaining thermoneutrality could decrease the risk of life-threatening responses to heat and exercise in individuals with RYR1 pathogenic variants.
Assuntos
Hipertermia Maligna/genética , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Termogênese/fisiologia , Tecido Adiposo Marrom/metabolismo , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Feminino , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Humanos , Lactente , Lactatos/sangue , Masculino , Hipertermia Maligna/etiologia , Hipertermia Maligna/mortalidade , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estudos Retrospectivos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Termogênese/genética , Proteína Desacopladora 1/genética , Adulto JovemRESUMO
Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases.
Assuntos
Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Trealose/farmacologia , Animais , Astrócitos , Autofagia/fisiologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Fibroblastos , Técnicas de Silenciamento de Genes , Células HeLa , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios , Fármacos Neuroprotetores/uso terapêutico , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trealose/uso terapêuticoRESUMO
KEY POINTS: Inhibiting Nox2 reactive oxygen species (ROS) production reduced in vivo calcium influx in dystrophic muscle. The lack of Nox2 ROS production protected against decreased in vivo muscle function in dystrophic mice. Manganese-enhanced magnetic resonance imaging (MEMRI) was able to detect alterations in basal calcium levels in skeletal muscle and differentiate disease status. Administration of Mn2+ did not affect muscle function or the health of the animal, and Mn2+ was cleared from skeletal muscle rapidly. We conclude that MEMRI may be a viable, non-invasive technique to monitor molecular alterations in disease progression and evaluate the effectiveness of potential therapies for Duchenne muscular dystrophy. ABSTRACT: Duchenne muscular dystrophy (DMD) is an X-linked progressive degenerative disease resulting from a mutation in the gene that encodes dystrophin, leading to decreased muscle mechanical stability and force production. Increased Nox2 reactive oxygen species (ROS) production and sarcolemmal Ca2+ influx are early indicators of disease pathology, and eliminating Nox2 ROS production reduces aberrant Ca2+ influx in young mdx mice, a model of DMD. Various imaging modalities have been used to study dystrophic muscle in vivo; however, they are based upon alterations in muscle morphology or inflammation. Manganese has been used for indirect monitoring of calcium influx across the sarcolemma and may allow detection of molecular alterations in disease progression in vivo using manganese-enhanced magnetic resonance imaging (MEMRI). Therefore, we hypothesized that eliminating Nox2 ROS production would decrease calcium influx in adult mdx mice and that MEMRI would be able to monitor and differentiate disease status in dystrophic muscle. Both in vitro and in vivo data demonstrate that eliminating Nox2 ROS protected against aberrant Ca2+ influx and improved muscle function in dystrophic muscle. MEMRI was able to differentiate between different pathological states in vivo, with no long-term effects on animal health or muscle function. We conclude that MEMRI is a viable, non-invasive technique to differentiate disease status and might provide a means to monitor and evaluate the effectiveness of potential therapies in dystrophic muscle.
Assuntos
Cálcio/metabolismo , Glicoproteínas de Membrana/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , NADPH Oxidases/genética , Espécies Reativas de Oxigênio/metabolismo , Animais , Imageamento por Ressonância Magnética/métodos , Manganês/farmacocinética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/diagnóstico por imagem , Distrofia Muscular de Duchenne/diagnóstico por imagem , Distrofia Muscular de Duchenne/genética , NADPH Oxidase 2 , NADPH Oxidases/metabolismoRESUMO
In many human diseases, the presence of inflammation is associated with an increase in the level of reactive oxygen species (ROS). The resulting state of oxidative stress is highly detrimental and can initiate a cascade of events that ultimately lead to cell death. Thus, many therapeutic attempts have been focused on either modulating the immune system to lower inflammation or reducing the damaging caused by ROS. Berlin et al. reported the development of a novel nanoantioxidant known as poly(ethylene glycol)-functionalized-hydrophilic carbon clusters (PEG-HCCs). They showed that PEG-HCCs could be targeted to cancer cells, utilized as a drug delivery vector, and can even be visualized ex vivo. Our work here furthers this work and characterizes Gd-DTPA conjugated PEG-HCCs and explores the potential for in vivo tracking of T cells in live mice. We utilized a mouse model of delayed-type hypersensitivity (DTH) to assess the immunomodulatory effects of PEG-HCCs. The T1 -agent Gd-DTPA was then conjugated to the PEG-HCCs and T1 measurements, and T1 -weighted MRI of the modified PEG-HCCs was done to assess their relaxivity. We then assessed if PEG-HCCs could be visualized both ex vivo and in vivo within the mouse lymph node and spleen. Mice treated with PEG-HCCs showed significant improvements in the DTH assay as compared to the vehicle (saline)-treated control. Flow cytometry demonstrated that splenic T cells are capable of internalizing PEG-HCCs whereas fluorescent immunohistochemistry showed that PEG-HCCs are detectable within the cortex of lymph nodes. Finally, our nanoantioxidants can be visualized in vivo within the lymph nodes and spleen of a mouse after addition of the Gd-DTPA. PEG-HCCs are internalized by T cells in the spleen and can reduce inflammation by suppression of a recall immune response. PEG-HCCs can be modified to allow for both in vitro and in vivo visualization using MRI. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.
Assuntos
Antioxidantes/administração & dosagem , Imunidade Inata/imunologia , Memória Imunológica/imunologia , Imageamento por Ressonância Magnética/métodos , Nanopartículas/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Animais , Antioxidantes/química , Rastreamento de Células/métodos , Células Cultivadas , Feminino , Gadolínio DTPA/química , Imunidade Inata/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Nanoconjugados/administração & dosagem , Nanoconjugados/química , Nanopartículas/química , Espécies Reativas de Oxigênio/imunologia , Linfócitos T/citologiaRESUMO
BACKGROUND & AIMS: Pediatric liver cancer is a rare but serious disease whose incidence is rising, and for which the therapeutic options are limited. Development of more targeted, less toxic therapies is hindered by the lack of an experimental animal model that captures the heterogeneity and metastatic capability of these tumors. METHODS: Here we established an orthotopic engraftment technique to model a series of patient-derived tumor xenograft (PDTX) from pediatric liver cancers of all major histologic subtypes: hepatoblastoma, hepatocellular cancer and hepatocellular malignant neoplasm. We utilized standard (immuno) staining methods for histological characterization, RNA sequencing for gene expression profiling and genome sequencing for identification of druggable targets. We also adapted stem cell culturing techniques to derive two new pediatric cancer cell lines from the xenografted mice. RESULTS: The patient-derived tumor xenografts recapitulated the histologic, genetic, and biological characteristics-including the metastatic behavior-of the corresponding primary tumors. Furthermore, the gene expression profiles of the two new liver cancer cell lines closely resemble those of the primary tumors. Targeted therapy of PDTX from an aggressive hepatocellular malignant neoplasm with the MEK1 inhibitor trametinib and pan-class I PI3 kinase inhibitor NVP-BKM120 resulted in significant growth inhibition, thus confirming this PDTX model as a valuable tool to study tumor biology and patient-specific therapeutic responses. CONCLUSIONS: The novel metastatic xenograft model and the isogenic xenograft-derived cell lines described in this study provide reliable tools for developing mutation- and patient-specific therapies for pediatric liver cancer. LAY SUMMARY: Pediatric liver cancer is a rare but serious disease and no experimental animal model currently captures the complexity and metastatic capability of these tumors. We have established a novel animal model using human tumor tissue that recapitulates the genetic and biological characteristics of this cancer. We demonstrate that our patient-derived animal model, as well as two new cell lines, are useful tools for experimental therapies.
Assuntos
Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Criança , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Transplante de Neoplasias , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Accumulation and deposition of ß-amyloid peptides (Aß) in the brain is a central event in the pathogenesis of Alzheimer's disease (AD). Besides the parenchymal pathology, Aß is known to undergo active transport across the blood-brain barrier and cerebral amyloid angiopathy (CAA) is a prominent feature in the majority of AD. Although impaired cerebral blood flow (CBF) has been implicated in faulty Aß transport and clearance, and cerebral hypoperfusion can exist in the pre-clinical phase of Alzheimer's disease (AD), it is still unclear whether it is one of the causal factors for AD pathogenesis, or an early consequence of a multi-factor condition that would lead to AD at late stage. To study the potential interaction between faulty CBF and amyloid accumulation in clinical-relevant situation, we generated a new amyloid precursor protein (APP) knock-in allele that expresses humanized Aß and a Dutch mutation in addition to Swedish/London mutations and compared this line with an equivalent knock-in line but in the absence of the Dutch mutation, both crossed onto the PS1M146V knock-in background. RESULTS: Introduction of the Dutch mutation results in robust CAA and parenchymal Aß pathology, age-dependent reduction of spatial learning and memory deficits, and CBF reduction as detected by fMRI. Direct manipulation of CBF by transverse aortic constriction surgery on the left common carotid artery caused differential changes in CBF in the anterior and middle region of the cortex, where it is reduced on the left side and increased on the right side. However these perturbations in CBF resulted in the same effect: both significantly exacerbate CAA and amyloid pathology. CONCLUSIONS: Our study reveals a direct and positive link between vascular and parenchymal Aß; both can be modulated by CBF. The new APP knock-in mouse model recapitulates many symptoms of AD including progressive vascular and parenchymal Aß pathology and behavioral deficits in the absence of APP overexpression.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/irrigação sanguínea , Angiopatia Amiloide Cerebral/patologia , Circulação Cerebrovascular/fisiologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Animais , Comportamento Animal , Encéfalo/metabolismo , Encéfalo/patologia , Angiopatia Amiloide Cerebral/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Técnicas de Introdução de Genes , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , CamundongosRESUMO
AIM: We report a magneto-fluorescent theranostic nanocomplex targeted to neutrophil gelatinase-associated lipocalin (NGAL) for imaging and therapy of pancreatic cancer. MATERIALS & METHODS: Gold nanoshells resonant at 810 nm were encapsulated in silica epilayers doped with iron oxide and the near-infrared (NIR) dye indocyanine green, resulting in theranostic gold nanoshells (TGNS), which were subsequently conjugated with antibodies targeting NGAL in AsPC-1-derived xenografts in nude mice. RESULTS: Anti-NGAL-conjugated TGNS specifically targeted pancreatic cancer cells in vitro and in vivo providing contrast for both NIR fluorescence and T2-weighted MRI with higher tumor contrast than can be obtained using long-circulating, but nontargeted, PEGylated nanoparticles. The nanocomplexes also enabled highly specific cancer cell death via NIR photothermal therapy in vitro. CONCLUSION: TGNS with embedded NIR and magnetic resonance contrasts can be specifically targeted to pancreatic cancer cells with expression of early disease marker NGAL, and enable molecularly targeted imaging and photothermal therapy.
Assuntos
Ouro/uso terapêutico , Nanoconchas/uso terapêutico , Pâncreas/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/terapia , Proteínas de Fase Aguda/metabolismo , Animais , Linhagem Celular Tumoral , Meios de Contraste/química , Meios de Contraste/uso terapêutico , Sistemas de Liberação de Medicamentos , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/uso terapêutico , Ouro/química , Humanos , Hipertermia Induzida , Lipocalina-2 , Lipocalinas/metabolismo , Imageamento por Ressonância Magnética , Imãs/química , Camundongos Nus , Nanoconchas/química , Proteínas Oncogênicas/metabolismo , Imagem Óptica , Neoplasias Pancreáticas/patologia , FototerapiaRESUMO
A major limitation of exogenous vitamin D3 administration for the treatment of prostate cancer is the marginal, if any, clinical efficacy. We dissected the basis for the resistance to the vitamin D3 antitumor properties and specifically examined the effect of its major catabolic enzyme, CYP24A1, in prostate cancer. Local CYP24A1 expression levels and the effect of selective modulation were analyzed using tissue microarrays from needle core biopsy specimens and xenograft-bearing mouse models. CYP24A1 mRNA was elevated in malignant human prostate tissues compared to benign lesions. High CYP24A1 protein levels were seen in poorly differentiated and highly advanced stages of prostate cancer and correlated with parallel increase in the tumor proliferation rate. The use of CYP24A1 RNAi enhanced the cytostatic effects of vitamin D3 in human prostate cancer cells. Remarkably, subcutaneous and orthotopic xenografts of prostate cancer cells harboring CYP24A1 shRNA resulted in a drastic reduction in tumor volume when mice were subjected to vitamin D3 supplementation. CYP24A1 may be a predictive marker of vitamin D3 clinical efficacy in patients with advanced prostate cancer. For those with up-regulated CYP24A1, combination therapy with RNAi targeting CYP24A1 could be considered to improve clinical responsiveness to vitamin D3.
Assuntos
Colecalciferol/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Esteroide Hidroxilases/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroide Hidroxilases/genética , Vitamina D3 24-Hidroxilase , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The transcription factor NFκB is an important mediator of cell survival and inflammation in the immune system. In the central nervous system (CNS), NFκB signaling has been implicated in regulating neuronal survival following acute pathologic damage such as traumatic brain injury (TBI) and stroke. NFκB is normally bound by the principal inhibitory protein, IκBα, and sequestered in the cytoplasm. Activation of NFκB requires the degradation of IκBα, thereby freeing NFκB to translocate to the nucleus and activate the target genes. Mice deficient in IκBα display deregulated and sustained NFκB activation and early postnatal lethality, highlighting a critical role of IκBα in NFκB regulation. RESULTS: We investigated the role of IκBα in regulating NFκB activity in the brain and the effects of the NFκB/IκBα pathway in mediating neuroinflammation under both physiological and brain injury conditions. We report that astrocytes, but not neurons, exhibit prominent NFκB activity, and that basal NFκB activity in astrocytes is elevated in the absence of IκBα. By generating mice with brain-specific deletion of IκBα, we show that IκBα deficiency does not compromise normal brain development. However, basal neuroinflammation detected by GFAP and Iba1 immunoreactivity is elevated. This leads to impaired inflammatory responses following TBI and worsened brain damage including higher blood brain barrier permeability, increased injury volumes and enlarged ventricle volumes. CONCLUSIONS: We conclude that, in the CNS, astrocyte is the primary cell type subject to NFκB regulation. We further demonstrate that IκBα plays an important role in regulating NFκB activity in the brain and a robust NFκB/IκBα-mediated neuroinflammatory response immediately following TBI is beneficial.
Assuntos
Astrócitos/metabolismo , Lesões Encefálicas/metabolismo , Encéfalo/metabolismo , Proteínas I-kappa B/deficiência , Recuperação de Função Fisiológica/fisiologia , Animais , Barreira Hematoencefálica/fisiologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Lesões Encefálicas/patologia , Proteína Glial Fibrilar Ácida , Inflamação/metabolismo , Camundongos , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Generation of reactive oxygen species (ROS) causes cellular oxidative damage and has been implicated in the etiology of Alzheimer's disease (AD). In contrast, multiple lines of evidence indicate that ROS can normally modulate long-term potentiation (LTP), a cellular model for memory formation. We recently showed that decreasing the level of superoxide through the overexpression of mitochondrial superoxide dismutase (SOD-2) prevents memory deficits in the Tg2576 mouse model of AD. In the current study, we explored whether AD-related LTP impairments could be prevented when ROS generation from mitochondria was diminished either pharmacologically or via genetic manipulation. In wild-type hippocampal slices treated with exogenous amyloid ß peptide (Aß1-42) and in slices from APP/PS1 mutant mice that model AD, LTP was impaired. The LTP impairments were prevented by MitoQ, a mitochondria-targeted antioxidant, and EUK134, an SOD and catalase mimetic. In contrast, inhibition of NADPH oxidase either by diphenyliodonium (DPI) or by genetically deleting gp91(phox), the key enzymatic component of NADPH oxidase, had no effect on Aß-induced LTP blockade. Moreover, live staining with MitoSOX Red, a mitochondrial superoxide indicator, combined with confocal microscopy, revealed that Aß-induced superoxide production could be blunted by MitoQ, but not DPI, in agreement with our electrophysiological findings. Finally, in transgenic mice overexpressing SOD-2, Aß-induced LTP impairments and superoxide generation were prevented. Our data suggest a causal relationship between mitochondrial ROS imbalance and Aß-induced impairments in hippocampal synaptic plasticity.
Assuntos
Peptídeos beta-Amiloides/fisiologia , Hipocampo/efeitos dos fármacos , Mitocôndrias/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Oxidantes/metabolismo , Superóxidos/metabolismo , Sinapses/efeitos dos fármacos , Peptídeos beta-Amiloides/genética , Animais , Antioxidantes/farmacologia , Compostos de Bifenilo/farmacologia , Fenômenos Eletrofisiológicos , Humanos , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , NADPH Oxidases/fisiologia , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/farmacologiaRESUMO
Nanoparticle-based therapeutics with local delivery and external electromagnetic field modulation holds extraordinary promise for soft-tissue cancers such as breast cancer; however, knowledge of the distribution and fate of nanoparticles in vivo is crucial for clinical translation. Here we demonstrate that multiple diagnostic capabilities can be introduced in photothermal therapeutic nanocomplexes by simultaneously enhancing both near-infrared fluorescence and magnetic resonance imaging (MRI). We track nanocomplexes in vivo, examining the influence of HER2 antibody targeting on nanocomplex distribution over 72 h. This approach provides valuable, detailed information regarding the distribution and fate of complex nanoparticles designed for specific diagnostic and therapeutic functions.
Assuntos
Neoplasias da Mama/terapia , Nanopartículas , Animais , Linhagem Celular Tumoral , Campos Eletromagnéticos , Feminino , Fluorescência , Humanos , Imageamento por Ressonância Magnética , Camundongos , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
Overexpression of the human epidermal growth factor receptor (HER) family has been implicated in ovarian cancer because of its participation in signaling pathway regulating cellular proliferation, differentiation, motility, and survival. Currently, effective diagnostic and therapeutic schemes are lacking for treating ovarian cancer, and consequently ovarian cancer has a high mortality rate. Although HER2 receptor expression does not usually affect the survival rates of ovarian cancer to the same extent as in breast cancer, it can be used as a docking site for directed nanotherapies in cases with de novo or acquired chemotherapy resistance. In this study, we have exploited a novel gold nanoshell-based complex (nanocomplex) for targeting, dual modal imaging, and photothermal therapy of HER2-overexpressing and drug-resistant ovarian cancer OVCAR3 cells in vitro. The nanocomplexes are engineered to simultaneously provide contrast as fluorescence optical imaging probe and a magnetic resonance imaging agent. Immunofluorescence staining and magnetic resonance imaging successfully show that nanocomplex-anti-HER2 conjugates specifically bind to OVCAR3 cells as opposed to the control, MDA-MB-231 cells, which have low HER2 expression. In addition, nanocomplexes targeted to OVCAR3 cells, when irradiated with near-IR laser, result in selective destruction of cancer cells through photothermal ablation. We also show that near-IR light therapy and the nanocomplexes by themselves are noncytotoxic in vitro. To the best of our knowledge, this is the first successful integration of dual modal bioimaging with photothermal cancer therapy for treatment of ovarian cancer. Based on their efficacy in vitro, these nanocomplexes are highly promising for image-guided photothermal therapy of ovarian cancer, as well as other HER2-overexpressing cancers. Mol Cancer Ther; 9(4); 1028-38. (c)2010 AACR.
Assuntos
Sondas Moleculares , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/terapia , Temperatura , Técnicas de Ablação , Morte Celular , Linhagem Celular Tumoral , Meios de Contraste , Feminino , Humanos , Imageamento por Ressonância Magnética , Microscopia de Fluorescência , Nanoconchas , Neoplasias Ovarianas/patologia , Receptor ErbB-2/metabolismoRESUMO
Gadonanotubes (GNTs), which are powerful new T(1)-weighted MRI contrast agents, were derivatized with serine amino acid substituents to produce water-soluble (2 mg ml(-1)) ser-gadonanotubes (ser-GNs) as magnetic nanoprobes for intracellular labeling. The ser-GNTs were used to efficiently label MCF-7 human breast cancer cells (1.5 x 10(9) Gd(3+) ions/cell) with no observable cytotoxicity. Cell pellets derived from the ser-GNT labeled cells give bright T(1)-weighted MR images, confirming that the ser-GNTs are a promising new nanoprobe technology for magnetic cell labeling and possibly for in vivo cellular trafficking.
Assuntos
Gadolínio/metabolismo , Espaço Intracelular/metabolismo , Magnetismo , Sondas Moleculares/metabolismo , Nanotubos/química , Serina/metabolismo , Coloração e Rotulagem/métodos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Forma Celular , Feminino , Humanos , Imageamento por Ressonância Magnética , Microscopia Eletrônica de Transmissão , Imagem Molecular/métodos , Nanotubos/ultraestrutura , Imagens de FantasmasRESUMO
BACKGROUND: Previous studies have shown that beta amyloid (Abeta) peptide triggers the activation of several signal transduction cascades in the hippocampus, including the extracellular signal-regulated kinase (ERK) cascade. In this study we sought to characterize the cellular localization of phosphorylated, active ERK in organotypic hippocampal cultures after acute exposure to either Abeta (1-42) or nicotine. RESULTS: We observed that Abeta and nicotine increased the levels of active ERK in distinct cellular localizations. We also examined whether phospho-ERK was regulated by redox signaling mechanisms and found that increases in active ERK induced by Abeta and nicotine were blocked by inhibitors of NADPH oxidase. CONCLUSION: Our findings indicate that NADPH oxidase-dependent redox signaling is required for Abeta-induced activation of ERK, and suggest a similar mechanism may occur during early stages of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , NADPH Oxidases/metabolismo , Técnicas de Cultura de Órgãos , Acetofenonas/farmacologia , Aconitina/análogos & derivados , Aconitina/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , Nicotina/farmacologia , Oniocompostos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7RESUMO
The Cdk7/cyclin H/ménage-à-trois 1 (MAT1) heterotrimer has proposed functions in transcription as the kinase component of basal transcription factor TFIIH and is activated in adult hearts by Gq-, calcineurin-, and biomechanical stress-dependent pathways for hypertrophic growth. Using cardiac-specific Cre, we have ablated MAT1 in myocardium. Despite reduced Cdk7 activity, MAT1-deficient hearts grew normally, but fatal heart failure ensued at 6-8 weeks. By microarray profiling, quantitative RT-PCR, and western blotting at 4 weeks, genes for energy metabolism were found to be suppressed selectively, including targets of peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1). Cardiac metabolic defects were substantiated in isolated perfused hearts and isolated mitochondria. In culture, deleting MAT1 with Cre disrupted PGC-1 function: PGC-1alpha failed to activate PGC-1-responsive promoters and nuclear receptors, GAL4-PGC-1alpha was functionally defective, and PGC-1beta was likewise deficient. PGC-1 bound to both MAT1 and Cdk7 in coprecipitation assays. Thus, we demonstrate a requirement for MAT1 in the operation of PGC-1 coactivators that control cell metabolism.