RESUMO
B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of cancer found in children. Although the overall survival rates are now >80%, 15%-20% of pediatric patients relapse, with survival rates subsequently dropping to 5%-10%. Cmpd10357, 3-amino-5-arylamino-6-chloro-N- (diaminomethylene) pyrazine-2-carboximide, is a highly potent, cell-permeant compound recently shown to have cytotoxic effects on solid tumors, including human breast cancer and high-grade gliomas, independent of their proliferative status. Cmpd10357 demonstrated concentration-dependent cytotoxicity in two human B-ALL cell lines, JM1 and Reh, at half-maximal inhibitory concentrations (IC50) of 3.2 and 3.3 µM, respectively. Cmpd10357, at a dose of 5 mg/kg, significantly prolonged survival in our B-ALL xenograft mouse model, with a median survival time of 49.0 days compared with 45.5 days in the control group (p < 0.05). The cytotoxicity of Cmpd10357 demonstrated caspase-independent, nonapoptotic cancer cell demise associated with the nuclear translocation of apoptosis-inducing factor (AIF). The cytotoxicity of Cmpd10357 in B-ALL cells was inhibited by Necrostatin-1 but not by Necrosulfonamide. These studies suggest that an AIF-mediated, caspase-independent necrosis mechanism of Cmpd10357 in B-ALL could be used in combination with traditional apoptotic chemotherapeutic agents.
Assuntos
Antineoplásicos , Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Camundongos , Animais , Criança , Apoptose , Antineoplásicos/farmacologia , Caspases/metabolismo , Caspases/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Linfoma de Burkitt/tratamento farmacológico , Linhagem Celular TumoralRESUMO
Epidemiological studies have demonstrated that air pollution is a significant risk factor for age-related dementia, including Alzheimer's disease (AD). It has been posited that traffic-related air pollution (TRAP) promotes AD neuropathology by exacerbating neuroinflammation. To test this hypothesis, serum and hippocampal cytokines were quantified in male and female TgF344-AD rats and wildtype (WT) Fischer 344 littermates exposed to TRAP or filtered air (FA) from 1 to 15 months of age. Luminex™ rat 23-cytokine panel assays were used to measure the levels of hippocampal and serum cytokines in 3-, 6-, 10-, and 15-month-old rats (corresponding to 2, 5, 9, and 14 months of exposure, respectively). Age had a pronounced effect on both serum and hippocampal cytokines; however, age-related changes in hippocampus were not mirrored in the serum and vice versa. Age-related changes in serum cytokine levels were not influenced by sex, genotype, or TRAP exposure. However, in the hippocampus, in 3-month-old TgF344-AD and WT animals, TRAP increased IL-1ß in females while increasing TNF Éin males. In 6-month-old animals, TRAP increased hippocampal levels of M-CSF in TgF344-AD and WT females but had no significant effect in males. At 10 and 15 months of age, there were minimal effects of TRAP, genotype or sex on hippocampal cytokines. These observations demonstrate that TRAP triggers an early inflammatory response in the hippocampus that differs with sex and age and is not reflected in the serum cytokine profile. The relationship of TRAP effects on cytokines to disease progression remains to be determined.
RESUMO
Elucidating the pathogenic mechanisms of Alzheimer's disease (AD) to identify therapeutic targets has been the focus of many decades of research. While deposition of extracellular amyloid-beta plaques and intraneuronal neurofibrillary tangles of hyperphosphorylated tau have historically been the two characteristic hallmarks of AD pathology, therapeutic strategies targeting these proteinopathies have not been successful in the clinics. Neuroinflammation has been gaining more attention as a therapeutic target because increasing evidence implicates neuroinflammation as a key factor in the early onset of AD disease progression. The peripheral immune response has emerged as an important contributor to the chronic neuroinflammation associated with AD pathophysiology. In this context, the plasminogen activator system (PAS), also referred to as the vasculature's fibrinolytic system, is emerging as a potential factor in AD pathogenesis. Evolving evidence suggests that the PAS plays a role in linking chronic peripheral inflammatory conditions to neuroinflammation in the brain. While the PAS is better known for its peripheral functions, components of the PAS are expressed in the brain and have been demonstrated to alter neuroinflammation and blood-brain barrier (BBB) permeation. Here, we review plasmin-dependent and -independent mechanisms by which the PAS modulates the BBB in AD pathogenesis and discuss therapeutic implications of these observations.
RESUMO
STUDY OBJECTIVE: Approximately 5% of emergency department patients present with altered mental status (AMS). AMS is diagnostically challenging because of the wide range of causes and is associated with high mortality. We sought to develop a clinical decision rule predicting admission risk among emergency department (ED) patients with AMS. METHODS: Using retrospective chart review of ED encounters for AMS over a 2-month period, we recorded causes of AMS and numerous clinical variables. Encounters were split into those admitted to the hospital ("cases") and those discharged from the ED ("controls"). Using the first month's data, variables correlated with hospital admission were identified and narrowed using univariate and multivariate statistics, including recursive partitioning. These variables were then organized into a clinical decision rule and validated on the second month's data. The decision rule results were also compared to 1-year mortality. RESULTS: We identified 351 encounters for AMS over a 2-month period. Significant contributors to AMS included intoxication and chronic disorder decompensation. ED data predicting hospital admission included vital sign abnormalities, select lab studies, and psychiatric/intoxicant history. The decision rule sorted patients into low, moderate, or high risk of admission (11.1%, 44.3%, and 89.1% admitted, respectively) and was predictive of 1-year mortality (low-risk group 1.8%, high-risk group 34.3%). CONCLUSIONS: We catalogued common causes for AMS among patients presenting to the ED, and our data-driven decision tool triaged these patients for risk of admission with good predictive accuracy. These methods for creating clinical decision rules might be further studied and improved to optimize ED patient care.
RESUMO
Current clinical imaging modalities do not reliably identify brain tissue regions with necrosis following radiotherapy. This creates challenges for stereotaxic biopsies and surgical-decision making. Time-resolved fluorescence spectroscopy (TRFS) provides a means to rapidly identify necrotic tissue by its distinct autofluorescence signature resulting from tissue breakdown and altered metabolic profiles in regions with radiation damage. Studies conducted in a live animal model of radiation necrosis demonstrated that necrotic tissue is characterized by respective increases of 27% and 108% in average lifetime and redox ratio, when compared with healthy tissue. Moreover, radiation-damaged tissue not visible by MRI but confirmed by histopathology, was detected by TRFS. Current results demonstrate the ability of TRFS to identify radiation-damaged brain tissue in real-time and indicates its potential to assist with surgical guidance and MRI-guided biopsy procedures.
RESUMO
Microglia are intrinsic components of the brain immune system and are activated in many central nervous system disorders. The ability to noninvasively image these cells would provide valuable information for both research and clinical applications. Today, most imaging probes for activated microglia are mainly designed for positron emission tomography (PET) and target translocator proteins that also reside on other cerebral cells. The PET images obtained are not specific for microglia-driven inflammation. Here, we describe a potential PET/MRI multimodal imaging probe that selectively targets the scavenger receptor class A (SR-A) expressed on activated microglia. These sulfated dextran-coated iron oxide (SDIO) nanoparticles are avidly taken up by microglia and appear to be nontoxic when administered intravenously in a mouse model. Intravenous administration of this SDIO demonstrated visualization by T2∗ -weighted MRI of microglia activated by intracerebral administration of tumor necrosis factor alpha (TNF-α). The contrast was significantly enhanced by SDIO, whereas there was little to no contrast change in animals treated with nontargeted nanoparticles or untreated controls. Thus, SR-A targeting represents a promising strategy to image activated microglia in the brain.
Assuntos
Encefalite/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/uso terapêutico , Tomografia por Emissão de Pósitrons/métodos , Animais , Compostos Férricos , Nanopartículas de Magnetita/química , Camundongos , Microglia/química , Microglia/metabolismo , Imagem Multimodal/métodos , Receptores Depuradores Classe A/análiseRESUMO
High grade gliomas (HGGs) are primary CNS cancers with more than 95% of patients experiencing tumor recurrence following radiation therapy, chemotherapy, and/or an anti-angiogenic therapy. Populations of glioma 'stem-like' cells (GSCs) exist in both proliferative and non-proliferative states and are capable of tumor regrowth. These GSCs survive within hypoxic tumor regions and avascular tumor margins, while retaining the capability to regenerate. Successful treatment of HGGs depends on therapeutic targeting of GSCs to avert tumor regeneration. Here, we review novel intracellular mechanisms by which 3-amino-5-arylamino-6-chloro-N-(diaminomethylene) pyrazine-2-carboximide (UCD38B) and the much more potent 5'-substituted arylamino compounds (cmpd 10357) irreversibly kill GSCs utilizing caspase-independent, programmed necrotic cell death. Drug-induced relocation of a subset of endosomes to perinuclear mitochondria triggers the mitochondrial release and nuclear translocation of apoptosis inducible factor (AIF) that is followed by nuclear condensation and cancer cell demise. This drug-induced endosomal 'mis-trafficking' affects a subset of endosomes containing proteins belonging to the urokinase plasminogen activator system (uPAS) and guided by lipoprotein receptor protein type 1 (LRP-1). UCD38B and congeners act intracellularly and bind to intracellular urokinase plasminogen activator (uPA) to disrupt uPA binding to PAI-1 and the endosomal LRP-1 guidance protein. These small molecules are cytotoxic to persistently hypoxic and acidotic HGG cell lines and to high grade gliomas from patient derived xenografts (PDX). Immunodeficient mice with intracerebral PDX glial tumors demonstrate drug-specific, AIF- mediated necrosis after 24h of treatment. The propensity of these small molecules to kill non-proliferating and proliferating hypoxic GSCs, suggests a potential synergistic therapeutic role with radiotherapy, anti-mitotic and anti-angiogenic therapies.
Assuntos
Amilorida/farmacologia , Antineoplásicos/farmacologia , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Endossomos/efeitos dos fármacos , Glioma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Amilorida/análogos & derivados , Amilorida/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Neoplasias do Sistema Nervoso Central/metabolismo , Neoplasias do Sistema Nervoso Central/patologia , Endossomos/metabolismo , Endossomos/patologia , Glioma/metabolismo , Glioma/patologia , Humanos , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
5-Benzylglycinyl-amiloride (UCD38B) is the parent molecule of a class of anticancer small molecules that kill proliferative and nonproliferative high-grade glioma cells by programmed necrosis. UCD38B intracellularly triggers endocytosis, causing 40-50% of endosomes containing proteins of the urokinase plasminogen activator system (uPAS) to relocate to perinuclear mitochondrial regions. Endosomal "mis-trafficking" caused by UCD38B in human glioma cells corresponds to mitochondrial depolarization with the release and nuclear translocation of apoptotis-inducing factor (AIF) followed by irreversible caspase-independent cell demise. High-content quantification of immunocytochemical colocalization studies identified that UCD38B treatment increased endocytosis of the urokinase plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) into the early and late endosomes by 4- to 5-fold prior to AIF nuclear translocation and subsequent glioma demise. PAI-1 was found to comparably relocate with a subset of early and late endosomes in four different human glioma cell lines after UCD38B treatment, followed by caspase-independent, nonapoptotic cell death. Following UCD38B treatment, the receptor guidance protein LRP-1, which is required for endosomal recycling of the uPA receptor to the plasmalemma, remained abnormally associated with PAI-1 in early and late endosomes. The resultant aberrant endosomal recycling increased the total cellular content of the uPA-PAI-1 protein complex. Reversible inhibition of cellular endocytosis demonstrated that UCD38B bypasses the plasmalemmal uPAS complex and directly acts intracellularly to alter uPAS endocytotic trafficking. UCD38B represents a class of small molecules whose anticancer cytotoxicity is a consequence of causing the mis-trafficking of early and late endosomes containing uPAS cargo and leading to AIF-mediated necrotic cell death.
Assuntos
Amilorida/análogos & derivados , Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Glioma/enzimologia , Glicina/análogos & derivados , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Amilorida/farmacologia , Fator de Indução de Apoptose/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Glioma/patologia , Glicina/farmacologia , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mitocôndrias/metabolismo , Necrose , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Transporte Proteico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Neuropathic pain is a maladaptive immune response to peripheral nerve injury that causes a chronic painful condition refractory to most analgesics. Nitric oxide (NO), which is produced by nitric oxide synthases (NOSs), has been implicated as a key factor in the pathogenesis of neuropathic pain. ß-Carbolines are a large group of natural and synthetic indole alkaloids, some of which block activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), a predominant transcriptional regulator of NOS expression. Here, we characterize the inhibitory effects of a novel 6-chloro-8-(glycinyl)-amino-ß-carboline (8-Gly carb) on NO formation and NF-κB activation in macrophages. 8-Gly carb was significantly more potent than the NOS inhibitor NG-nitro-L-arginine methyl ester in inhibiting constitutive and inducible NO formation in primary rat macrophages. 8-Gly carb interfered with NF-κB-mediated gene expression in differentiated THP1-XBlue cells, a human NF-κB reporter macrophage cell line, but only at concentrations severalfold higher than needed to significantly inhibit NO production. 8-Gly carb also had no effect on tumor necrosis factor α (TNFα)-induced phosphorylation of the p38 mitogen-activated protein kinase in differentiated THP1 cells, and did not inhibit lipopolysaccharide- or TNFα-stimulated expression of TNFα and interleukin-1ß. These data demonstrate that relative to other carbolines and pharmacologic inhibitors of NOS, 8-Gly carb exhibits a unique pharmacological profile by inhibiting constitutive and inducible NO formation independent of NF-κB activation and cytokine expression. Thus, this novel carboline derivative holds promise as a parent compound, leading to therapeutic agents that prevent the development of neuropathic pain mediated by macrophage-derived NO without interfering with cytokine expression required for neural recovery following peripheral nerve injury.
Assuntos
Carbolinas/farmacologia , Interleucina-1beta/biossíntese , Macrófagos/metabolismo , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Animais , Carbolinas/química , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Solid lipid nanoparticles (SLNs) have recently emerged as nontoxic, versatile alternatives to traditional carriers (liposomes, polymeric nanoparticles) for drug delivery. Because SLNs are composed of a solid lipid core, they offer significant protection against chemical degradation of their drug cargo and offer the potential for controlled release. SLNs also hold promise for use as targeted agents and multimodal imaging agents. Here we report the synthesis and characterization of SLNs loaded with gadolinium (1,4,7,10-tetraazacyclododecane)-1,4,7,10-tetraacetate (Gd-DOTA) in order to produce a new category of stable T1-weighted (T1w) magnetic resonance imaging (MRI) contrast agents. Systematically varying components in the SLN synthesis, we demonstrated an increase in Gd-DOTA incorporation and an increase in longitudinal relaxivity (r1) through optimizing the amount of surfactant (Span 80) in the "oil" phase. These highly monodisperse SLNs confirm stable loading of Gd-DOTA and a stable size distribution (â¼150 nm) over time in aqueous solution. Relaxivity measurements (1.4T, 37 °C) demonstrate that the r1 of Gd-DOTA does not strongly decrease following encapsulation in SLNs, demonstrating an advantage over liposomes. These Gd-loaded SLNs demonstrate enhanced contrast in vivo at 7T using T1w MRI and in the future can be loaded with other cargo (hydrophilic or hydrophobic) to enable functionality with other imaging modalities such as optical or positron emission tomography.
Assuntos
Meios de Contraste/administração & dosagem , Compostos Heterocíclicos/administração & dosagem , Lipídeos/química , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Compostos Organometálicos/administração & dosagem , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Contraste/toxicidade , Compostos Heterocíclicos/toxicidade , Lipídeos/toxicidade , Camundongos , Nanopartículas/toxicidade , Compostos Organometálicos/toxicidadeRESUMO
Amiloride is a potassium-sparing diuretic that has been used as an anti-kaliuretic for the chronic management of hypertension and heart failure. Several studies have identified a potential anti-cancer role for amiloride, however the mechanisms underlying its anti-tumor effects remain to be fully delineated. Our group previously demonstrated that amiloride triggers caspase-independent cytotoxic cell death in human glioblastoma cell lines but not in primary astrocytes. To delineate the cellular mechanisms underlying amiloride's anti-cancer cytotoxicity, cell permeant and cell impermeant derivatives of amiloride were synthesized that exhibit markedly different potencies in cancer cell death assays. Here we compare the cytotoxicities of 5-benzylglycinyl amiloride (UCD38B) and its free acid 5-glycinyl amiloride (UCD74A) toward human breast cancer cells. UCD74A exhibits poor cell permeability and has very little cytotoxic activity, while UCD38B is cell permeant and induces the caspase-independent death of proliferating and non-proliferating breast cancer cells. UCD38B treatment of human breast cancer cells promotes autophagy reflected in LC3 conversion, and induces the dramatic swelling of the endoplasmic reticulum, however these events do not appear to be the cause of cell death. Surprisingly, UCD38B but not UCD74A induces efficient AIF translocation from the mitochondria to the nucleus, and AIF function is necessary for the efficient induction of cancer cell death. Our observations indicate that UCD38B induces programmed necrosis through AIF translocation, and suggest that its cytosolic accessibility may facilitate drug action.
Assuntos
Amilorida/análogos & derivados , Amilorida/farmacologia , Fator de Indução de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Permeabilidade da Membrana Celular , Glicina/análogos & derivados , Amilorida/química , Amilorida/toxicidade , Cálcio/metabolismo , Calpaína/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Feminino , Glicina/química , Glicina/farmacologia , Glicina/toxicidade , Humanos , Transporte ProteicoRESUMO
5'-Βenzylglycinyl-amiloride (UCD38B) and glycinyl-amiloride (UCD74A) are cell-permeant and cell-impermeant derivatives of amiloride, respectively, and used here to identify the cellular mechanisms of action underlying their antiglioma effects. UCD38B comparably kills proliferating and nonproliferating gliomas cells when cell cycle progression is arrested either by cyclin D1 siRNA or by acidification. Cell impermeant UCD74A inhibits plasmalemmal urokinase plasminogen activator (uPA) and the type 1 sodium-proton exchanger with potencies analogous to UCD38B, but is cytostatic. In contrast, UCD38B targets intracellular uPA causing mistrafficking of uPA into perinuclear mitochondria, reducing the mitochondrial membrane potential, and followed by the release of apoptotic inducible factor (AIF). AIF nuclear translocation is followed by a caspase-independent necroptotic cell death. Reduction in AIF expression by siRNA reduces the antiglioma cytotoxic effects of UCD38B, while not activating the caspase pathway. Ultrastructural changes shortly following treatment with UCD38B demonstrate dilation of endoplasmic reticulum (ER) and mitochondrial swelling followed by nuclear condensation within hours consistent with a necroptotic cell death differing from apoptosis and from autophagy. These drug mechanism of action studies demonstrate that UCD38B induces a cell cycle-independent, caspase-independent necroptotic glioma cell death that is mediated by AIF and independent of poly (ADP-ribose) polymerase and H2AX activation.
Assuntos
Amilorida/análogos & derivados , Amilorida/farmacologia , Fator de Indução de Apoptose/metabolismo , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Glioma/tratamento farmacológico , Apoptose/efeitos dos fármacos , Apoptose/genética , Fator de Indução de Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Caspases/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Morte Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Histonas/genética , Histonas/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , RNA Interferente Pequeno/genética , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The relative non-toxicity of the diuretic amiloride, coupled with its selective inhibition of the protease urokinase plasminogen activator (uPA), makes this compound class attractive for structure-activity studies. Herein we substituted the C(2)-acylguanidine of C(5)-glycyl-amiloride with amidine and amidoxime groups. The data show the importance of maintaining C(5)-hydrophobicity. The C(5)-benzylglycine analogs containing either C(2)-acylguanidine or amidine inhibited uPA with an IC(50) ranging from 3 to 7 µM and were cytotoxic to human U87 malignant glioma cells.
Assuntos
Amidinas/síntese química , Amilorida/análogos & derivados , Amilorida/síntese química , Antineoplásicos/síntese química , Glicina/análogos & derivados , Glicina/síntese química , Inibidores de Serina Proteinase/síntese química , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Amidinas/farmacologia , Amilorida/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Glicina/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
We demonstrate for the first time the application of an endoscopic fluorescence lifetime imaging microscopy (FLIM) system to the intraoperative diagnosis of glioblastoma multiforme (GBM). The clinically compatible FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system with a fiber-bundle (fiber image guide of 0.5 mm diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, and 4 mm field of view) to provide intraoperative access to the surgical field. Experiments conducted in three patients undergoing craniotomy for tumor resection demonstrate that FLIM-derived parameters allow for delineation of tumor from normal cortex. For example, at 460±25-nm wavelength band emission corresponding to NADH/NADPH fluorescence, GBM exhibited a weaker fluorescence intensity (35% less, p-value<0.05) and a longer lifetime τGBM-Amean=1.59±0.24 ns than normal cortex τNC-Amean=1.28±0.04 ns (p-value<0.005). Current results demonstrate the potential use of FLIM as a tool for image-guided surgery of brain tumors.
Assuntos
Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/cirurgia , Glioblastoma/diagnóstico , Glioblastoma/cirurgia , Microscopia de Fluorescência/métodos , Cirurgia Assistida por Computador/métodos , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/instrumentação , NAD/metabolismo , NADP/metabolismo , Fenômenos Ópticos , Projetos Piloto , Cirurgia Assistida por Computador/instrumentaçãoRESUMO
Malignant glioma cells maintain an elevated intracellular pH (pH(i)) within hypoxic-ischemic tumor microenvironments through persistent activation of sodium-proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pH(i) of malignant gliomas by inhibiting isoform 1 of sodium-proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium-calcium exchange. Amiloride's glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and of Na(+)-dependent calcium efflux by isoform 1.1 of the sodium-calcium exchanger (NCX1.1), which increases [Ca(2+)](i) and initiates glioma cell demise. As a result of persistent NHE1 activity, cytosolic free levels of sodium ([Na(+)](i)) in U87 and C6 glioma cells are elevated 3-fold, as compared with normal astrocytes. Basal cytosolic free calcium levels ([Ca(2+)](i)) also are increased 5-fold. 2', 4'-dichlorobenzamil (DCB) inhibits the sodium-dependent calcium transporter (NCX1.1) much more potently than NHE1. DCB was employed in a concentration-dependent fashion in glioma cells to selectively inhibit the forward mode of NCX1.1 at ≤1µM, while dually inhibiting both NHE1 and NCX1.1 at ≥20µM. DCB (1µM) was not cytotoxic to glioma cells, while DCB (20µM) further increased basal elevated levels of [Ca(2+)](i) in glioma cells that was followed by cell demise. Cariporide and SEA0400 are more selective inhibitors of NHE1 and NCX1.1 than amiloride or DCB, respectively. Individually, Cariporide and SEA0400 are not cytotoxic, but in combination induced glioma cell death. Like amiloride, the combination of Cariporide and SEA0400 produced glioma cell death in the absence of demonstrable caspase activation.
Assuntos
Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Cálcio/metabolismo , Morte Celular/fisiologia , Glioma/metabolismo , Sódio/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Compostos de Anilina/farmacologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citosol/metabolismo , Glioma/patologia , Guanidinas/farmacologia , Humanos , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Ionomicina/farmacologia , Ionóforos/farmacologia , Espaço Pessoal , Éteres Fenílicos/farmacologia , Prótons , Ratos , Ratos Sprague-Dawley , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonas/farmacologia , Microambiente Tumoral/fisiologiaRESUMO
This study investigated the contributions of the reverse mode of the sodium-calcium exchanger (NCX) and the type 1 sodium-proton antiporter (NHE-1) to acute astrocyte and neuronal pathology in the hippocampus following fluid percussion traumatic brain injury (TBI) in the rat. KB-R7943, EIPA, or amiloride, which respectively inhibit NCX, NHE-1, or NCX, NHE-1, and ASIC1a (acid-sensing ion channel type 1a), was infused intraventricularly over a 60-min period immediately prior to TBI. Astrocytes were immunostained for glial fibrillary acidic protein (GFAP), and degenerating neurons were identified by Fluoro-Jade staining at 24 h after injury. Stereological analysis of the CA2/3 sub-regions of the hippocampus demonstrated that higher doses of KB-R7943 (2 and 20 nmoles) significantly reduced astrocyte GFAP immunoreactivity compared to vehicle-treated animals. EIPA (2-200 nmoles) did not alter astrocyte GFAP immunoreactivity. Amiloride (100 nmoles) significantly attenuated the TBI-induced acute reduction in astrocyte GFAP immunoreactivity. Of the three compounds examined, only amiloride (100 nmoles) reduced hippocampal neuronal degeneration assessed with Fluoro-Jade. The results provide additional evidence of acute astrocyte pathology in the hippocampus following TBI, while suggesting that activation of NHE-1 and the reverse mode of NCX contribute to both astrocyte and neuronal pathology following experimental TBI.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Bloqueadores dos Canais de Cálcio/farmacologia , Hipocampo/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais Iônicos Sensíveis a Ácido , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Biomarcadores/análise , Biomarcadores/metabolismo , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/tratamento farmacológico , Gliose/metabolismo , Gliose/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Injeções Intraventriculares , Masculino , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
The human glioma cell lines, U87 and T98G, were evaluated for their ability to survive and form colonies in an acidic environment of pH(ext) 6.0. In contrast to U87, which showed an 80-90% survival rate, only 40% of T98G cells survived 6 days at pH(ext) 6.0 and lost their colony forming ability when returned to a normocidic environment. Although both U87 and T98G cells maintain an intracellular pH (pH(i)) of 7.0 at pH(ext) 6.0 and arrest mostly in G1 phase of the cell cycle, only T98G demonstrated a major loss of cyclin D1 that was prevented by the proteasome inhibitor MG132. Colony forming ability was restored by stably transfecting T98G cells with a cyclin D1-expressing plasmid. Both U87 and T98G cells demonstrated increased cytoplasmic localization of cyclin D1 during exposure at pH(ext) 6.0. Upon prolonged (24 h) incubation at pH(ext) 6.0, nuclear cyclin D1 was nearly absent in T98G in contrast to U87 cells. Thus, an acidic environment triggers cytoplasmic localization and proteasomal degradation of cyclin D1.
Assuntos
Ácidos/metabolismo , Neoplasias Encefálicas/metabolismo , Ciclina D1/metabolismo , Glioma/metabolismo , Ácidos/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina D1/genética , Citoplasma/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/metabolismo , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , RNA Interferente Pequeno/farmacologia , Ensaio Tumoral de Célula-TroncoRESUMO
Tyrosine hydroxylase's catalysis of tyrosine to dihydroxyphenylalanine (DOPA) is the highly regulated, rate-limiting step catalyzing the synthesis of the catecholamine neurotransmitter dopamine. Phosphorylation, cofactor-mediated regulation, and the cell's redox status, have been shown to regulate the enzyme's activity. This paper incorporates these regulatory mechanisms into an integrated dynamic model that is capable of demonstrating relative rates of dopamine synthesis under various physiological conditions. Most of the kinetic equations and substrate parameters used in the model correspond with published experimental data, while a few which were not available in literature have been optimized based on explicit assumptions. This kinetic pathway model permits a comparison of the relative regulatory contributions made by variations in substrate, phosphorylation, and redox status on enzymatic activity and permits predictions of potential disease states. For example, the model correctly predicts the recent observation that individuals with haemochromatosis and having excessive iron accumulation are at increased risk for acquiring Parkinsonism, a defect in neuronal dopamine synthesis (Bartzokis et al., 2004; Costello et al., 2004). Alpha synuclein mediated regulation of tyrosine hydroxylase has also been incorporated in the model, allowing an insight into the overexpression and aggregation of alpha synuclein in Parkinson's disease.
Assuntos
Dopamina/metabolismo , Modelos Biológicos , Dinâmica não Linear , Tirosina 3-Mono-Oxigenase/fisiologia , Humanos , Ferro/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Valor Preditivo dos Testes , alfa-Sinucleína/metabolismoRESUMO
Traditionally, astrocytes have been considered less susceptible to injury than neurons. Yet, we have recently shown that astrocyte death precedes neuronal death in a rat model of traumatic brain injury (TBI) (Zhao et al.: Glia 44:140-152, 2003). A main mechanism hypothesized to contribute to cellular injury and death after TBI is elevated intracellular calcium ([Ca2+]i). Since calcium regulation is also influenced by regulation of intracellular sodium ([Na+]i), we used an in vitro model of strain-induced traumatic injury and live-cell fluorescent digital imaging to investigate alterations in [Na+]i in cortical astrocytes after injury. Changes in [Na+]i, or [Ca2+]i were monitored after mechanical injury or L-glutamate exposure by ratiometric imaging of sodium-binding benzofuran isophthalate (SBFI-AM), or Fura-2-AM, respectively. Mechanical strain injury or exogenous glutamate application produced increases in [Na+]i that were dependent on the severity of injury or concentration. Injury-induced increases in [Na+]i were significantly reduced, but not completely eliminated, by inhibition of glutamate uptake by DL-threo-beta-benzyloxyaspartate (TBOA). Blockade of sodium-dependent calcium influx through the sodium-calcium exchanger with 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate (KB-R7943) reduced [Ca2+]i after injury. KB-R7943 also reduced astrocyte death after injury. These findings suggest that in astrocytes subjected to mechanical injury or glutamate excitotoxicity, increases in intracellular Na+ may be a critical component in the injury cascade and a therapeutic target for reduction of lasting deficits after traumatic brain injury.
Assuntos
Astrócitos/metabolismo , Líquido Intracelular/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Estresse Mecânico , Animais , Astrócitos/patologia , Transporte Biológico Ativo , Calibragem , Morte Celular , Células Cultivadas , Corantes Fluorescentes , Ácido Glutâmico/metabolismo , Microscopia de Fluorescência , Ratos , Ratos Sprague-DawleyRESUMO
Inhibition of the sodium-proton exchanger (NHE) plays an important role in reducing tissue damage during ischemic reperfusion injury; however, pharmacological inhibitors of NHE have restricted access to acutely ischemic tissues because of severely compromised tissue perfusion. We describe the syntheses, characterization, and NHE inhibitory activities of a novel class of amiloride derivatives where peptides are conjugated to the amiloride C(5) amino group. These new peptide-C(5)-amiloride conjugates are inactive; however, peptide residues were chosen such that selective cleavage by neutral endopeptidase 24.11 (enkephalinase) liberates an amino acid-C(5)-amiloride conjugate that inhibits NHE in a glial cell line. These results confirm the feasibility of using peptide-amiloride conjugates as NHE inhibitor prodrugs. We envision the design of analogous peptide-amiloride prodrugs that can be administered prior to ischemic events and subsequently activated by endopeptidases selectively expressed by ischemic tissues.