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SIGNIFICANCE STATEMENT: Histologic quantification of complement C3 deposits in kidney biopsies provides prognostic information in patients with glomerulonephritis. Unfortunately, kidney biopsies are invasive procedures that cannot be performed regularly and only provide a snapshot of a small portion of one kidney at the time of sampling. We have developed a method to noninvasively detect specific C3 fragment deposition throughout both kidneys, using a monoclonal antibody targeting tissue-bound iC3b/C3d linked to a bioluminescent resonance energy transfer construct that emits near-infrared light. In a mouse model of glomerulonephritis, the probe detected iC3b/C3d in kidneys of live mice by bioluminescent imaging. This demonstrates that noninvasive imaging with an anti-iC3b/C3d probe can be used to monitor inflammation in the kidneys.
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Complemento C3b , Glomerulonefritis , Animales , Ratones , Complemento C3d , Riñón/diagnóstico por imagen , Anticuerpos MonoclonalesRESUMEN
The established role of bitter melon juice (BMJ), a natural product, in activating master metabolic regulator adenosine monophosphate-activated protein kinase in pancreatic cancer (PanC) cells served as a basis for pursuing deeper investigation into the underlying metabolic alterations leading to BMJ efficacy in PanC. We investigated the comparative metabolic profiles of PanC cells with differential KRAS mutational status on BMJ exposure. Specifically, we employed nuclear magnetic resonance (NMR) metabolomics and in vivo imaging platforms to understand the relevance of altered metabolism in PanC management by BMJ. Multinuclear NMR metabolomics was performed, as a function of time, post-BMJ treatment followed by partial least square discriminant analysis assessments on the quantitative metabolic data sets to visualize the treatment group clustering; altered glucose uptake, lactate export and energy state were identified as the key components responsible for cell death induction. We next employed PANC1 xenograft model for assessing in vivo BMJ efficacy against PanC. Positron emission tomography ([18FDG]-PET) and magnetic resonance imaging on PANC1 tumor-bearing animals reiterated the in vitro results, with BMJ-associated significant changes in tumor volumes, tumor cellularity and glucose uptake. Additional studies in BMJ-treated PanC cells and xenografts displayed a strong decrease in the expression of glucose and lactate transporters GLUT1 and MCT4, respectively, supporting their role in metabolic changes by BMJ. Collectively, these results highlight BMJ-induced modification in PanC metabolomics phenotype and establish primarily lactate efflux and glucose metabolism, specifically GLUT1 and MCT4 transporters, as the potential metabolic targets underlying BMJ efficacy in PanC.
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Intrauterine growth-restricted (IUGR) fetal sheep have increased hepatic glucose production (HGP) that is resistant to suppression during a hyperinsulinemic-isoglycemic clamp (insulin clamp). We hypothesized that the IUGR fetal liver would have activation of metabolic and signaling pathways that support HGP and inhibition of insulin-signaling pathways. To test this, we used transcriptomic profiling with liver samples from control (CON) and IUGR fetuses receiving saline or an insulin clamp. The IUGR liver had upregulation of genes associated with gluconeogenesis/glycolysis, transcription factor regulation, and cytokine responses and downregulation of genes associated with cholesterol synthesis, amino acid degradation, and detoxification pathways. During the insulin clamp, genes associated with cholesterol synthesis and innate immune response were upregulated in CON and IUGR. There were 20-fold more genes differentially expressed during the insulin clamp in IUGR versus CON. These genes were associated with proteasome activation and decreased amino acid and lipid catabolism. We found increased TRB3, JUN, MYC, and SGK1 expression and decreased PTPRD expression as molecular targets for increased HGP in IUGR. As candidate genes for resistance to insulin's suppression of HGP, expression of JUN, MYC, and SGK1 increased more during the insulin clamp in CON compared with IUGR. Metabolites were measured with 1H-nuclear magnetic resonance and support increased amino acid concentrations, decreased mitochondria activity and energy state, and increased cell stress in the IUGR liver. These results demonstrate a robust response, beyond suppression of HGP, during the insulin clamp and coordinate responses in glucose, amino acid, and lipid metabolism in the IUGR fetus.
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Glucemia/metabolismo , Metabolismo Energético , Retardo del Crecimiento Fetal/metabolismo , Técnica de Clampeo de la Glucosa , Resistencia a la Insulina , Insulina/sangre , Hígado/metabolismo , Animales , Biomarcadores/sangre , Western Blotting , Modelos Animales de Enfermedad , Metabolismo Energético/genética , Femenino , Retardo del Crecimiento Fetal/sangre , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/fisiopatología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Edad Gestacional , Resistencia a la Insulina/genética , Metabolismo de los Lípidos/genética , Hígado/embriología , Embarazo , Espectroscopía de Protones por Resonancia Magnética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Oveja Doméstica , TranscriptomaRESUMEN
LEVEL OF EVIDENCE: 5 Technical Efficacy: Stage 5 J. Magn. Reson. Imaging 2019.
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Imagen por Resonancia Magnética/métodos , Imagen por Resonancia Magnética/normas , Antropometría , Mama/diagnóstico por imagen , Toma de Decisiones , Aprendizaje Profundo , Diseño de Equipo , Femenino , Humanos , Interpretación de Imagen Asistida por Computador/métodos , Procesamiento de Imagen Asistido por Computador , Masculino , Fantasmas de Imagen , Medicina de Precisión , Radiología Intervencionista , Estándares de Referencia , Valores de Referencia , Reproducibilidad de los Resultados , Robótica , Programas InformáticosRESUMEN
Feraheme (ferumoxytol), a negatively charged, carboxymethyl dextran-coated ultrasmall superparamagnetic iron oxide nanoparticle (USPIO, 30 nm, -16 mV), is clinically approved as an iron supplement and is used off-label for magnetic resonance imaging (MRI) of macrophage-rich lesions, but the mechanism of recognition is not known. We investigated mechanisms of uptake of Feraheme by various types of macrophages in vitro and in vivo. The uptake by mouse peritoneal macrophages was not inhibited in complement-deficient serum. In contrast, the uptake of larger and less charged SPIO nanoworms (60 nm, -5 mV; 120 nm, -5 mV, respectively) was completely inhibited in complement deficient serum, which could be attributed to more C3 molecules bound per nanoparticle than Feraheme. The uptake of Feraheme in vitro was blocked by scavenger receptor (SR) inhibitor polyinosinic acid (PIA) and by antibody against scavenger receptor type A I/II (SR-AI/II). Antibodies against other SRs including MARCO, CD14, SR-BI, and CD11b had no effect on Feraheme uptake. Intraperitoneally administered PIA inhibited the peritoneal macrophage uptake of Feraheme in vivo. Nonmacrophage cells transfected with SR-AI plasmid efficiently internalized Feraheme but not noncharged ultrasmall SPIO of the same size (26 nm, -6 mV), suggesting that the anionic carboxymethyl groups of Feraheme are responsible for the SR-AI recognition. The uptake by nondifferentiated bone marrow derived macrophages (BMDM) and by BMDM differentiated into M1 (proinflammatory) and M2 (anti-inflammatory) types was efficiently inhibited by PIA and anti-SR-AI/II antibody. Interestingly, all BMDM types expressed similar levels of SR-AI/II. In conclusion, Feraheme is efficiently recognized via SR-AI/II but not via complement by different macrophage types. The recognition by the common phagocytic receptor has implications for specificity of imaging of macrophage subtypes.
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Óxido Ferrosoférrico/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Receptores Depuradores de Clase A/metabolismo , Animales , Células Cultivadas , Femenino , Hematínicos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.
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Proteínas de la Ataxia Telangiectasia Mutada/genética , Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica , Glucosafosfato Deshidrogenasa/genética , Leucemia Mieloide Aguda/genética , Tirosina Quinasa 3 Similar a fms/genética , Animales , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Benzotiazoles/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quimioterapia Combinada , Femenino , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucosafosfato Deshidrogenasa/metabolismo , Humanos , Hidrazinas/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Ratones Endogámicos NOD , Persona de Mediana Edad , Oxidación-Reducción , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
INTRODUCTION: Fetal tracheal occlusion (TO) is currently an experimental approach to drive accelerated lung growth. It is stimulated by mechanotransduction that results in increased cellular proliferation and growth. However, it is currently unknown how TO affects the metabolic landscape of fetal lungs. MATERIALS AND METHODS: TO or sham was performed on fetal rabbits at 26 days followed by lung harvest on day 30. Mass spectrometry was performed to evaluate global metabolic changes. Fluorescence lifetime intensity microscopy (FLIM) was performed to estimate local free/bound NADH relative ratio as an indicator of aerobic glycolysis versus oxidative phosphorylation (glycolysis/OXPHOS). RESULTS: TO results in a metabolic shift from tricarboxylic acid cycle towards glycolysis. FLIM reveals uniform structures in control lungs characterized by similar ratios of free/bound NADH indicating a homogenous topological pattern. Similar uniform structures are observed in shams with some variability in the glycolysis/OXPHOS ratio. In contrast, lungs following TO demonstrate different types of unique distinct topological zones: one with enlarged alveoli and a shift towards glycolysis; the other maintains balance between glycolysis/OXPHOS similar to control lungs. CONCLUSION: We demonstrate for the first time a unique variable topological pattern of metabolism in fetal lungs following TO with a wide variation of metabolism between zones.
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Hernias Diafragmáticas Congénitas/metabolismo , Pulmón/metabolismo , Estenosis Traqueal/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Hernias Diafragmáticas Congénitas/diagnóstico por imagen , Pulmón/diagnóstico por imagen , Imagen por Resonancia Magnética , Embarazo , Conejos , Estenosis Traqueal/diagnóstico por imagenRESUMEN
BACKGROUND: Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1). METHODS: RNA sequencing, quantitative polymerase chain reaction,13C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U-13C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD+ ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice. RESULTS: We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD+ ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated proliferation, and suppressed macrophage numbers and remodeling in the distal pulmonary vasculature. CONCLUSIONS: CtBP1 is a critical factor linking changes in cell metabolism to cell phenotype in hypoxic and other forms of PH and a therapeutic target.
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Oxidorreductasas de Alcohol/metabolismo , Proteínas de Unión al ADN/metabolismo , Hipertensión Pulmonar Primaria Familiar/metabolismo , Fibroblastos/metabolismo , Hipertensión Pulmonar/metabolismo , Adventicia/metabolismo , Adventicia/patología , Oxidorreductasas de Alcohol/genética , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Hipertensión Pulmonar Primaria Familiar/genética , Hipertensión Pulmonar Primaria Familiar/patología , Fibroblastos/patología , Humanos , Hipertensión Pulmonar/patología , Ratones , FenotipoRESUMEN
Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis, and metabolic changes in human PCa, LNCaP, and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity, and endothelial cells tube formation by hypoxic (1% O2 ) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity, and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative 1 H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN, and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. © 2016 Wiley Periodicals, Inc.
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Antineoplásicos/administración & dosificación , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lipogénesis/efectos de los fármacos , Metabolómica/métodos , Neovascularización Patológica/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Silimarina/administración & dosificación , Animales , Antineoplásicos/farmacología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Masculino , Ratones , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Transducción de Señal/efectos de los fármacos , Silibina , Silimarina/farmacología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To assess the feasibility of acquiring vessel size imaging (VSI) metrics using ferumoxytol injections and stock pulse sequences in a multicenter Phase I trial of a novel therapy in patients with advanced metastatic disease. METHODS: Scans were acquired before, immediately after, and 48 h after injection, at screening and after 2 weeks of treatment. ΔR2 , ΔR2*, vessel density (Q), and relative vascular volume fractions (VVF) were estimated in both normal tissue and tumor, and compared with model-derived theoretical and experimental estimates based on preclinical murine xenograft data. RESULTS: R2 and R2* relaxation rates were still significantly elevated in tumors and liver 48 h after ferumoxytol injection; liver values returned to baseline by week 2. Q was relatively insensitive to changes in ΔR2*, indicating lack of dependence on contrast agent concentration. Variability in Q was higher among human tumors compared with xenografts and was mostly driven by ΔR2 . Relative VVFs were higher in human tumors compared with xenografts, while values in muscle were similar between species. CONCLUSION: Clinical ferumoxytol-based VSI is feasible using standard MRI techniques in a multicenter study of patients with lesions outside of the brain. Ferumoxytol accumulation in the liver does not preclude measurement of VSI parameters in liver metastases. Magn Reson Med 77:814-825, 2017. © 2016 International Society for Magnetic Resonance in Medicine.
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Medios de Contraste/metabolismo , Óxido Ferrosoférrico/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Neoplasias/diagnóstico por imagen , Animales , Humanos , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias Experimentales/diagnóstico por imagenRESUMEN
While dietary folate deficiency is associated with increased risk for birth defects and other diseases, evidence suggests that supplementation with folic acid can contribute to predisposition to some diseases, including immune dysfunction and cancer. Herein, we show that diets supplemented with folic acid both below and above the recommended levels led to significantly altered metabolism in multiple tissues in mice. Surprisingly, both low and excessive dietary folate induced similar metabolic changes, which were particularly evident for nucleotide biosynthetic pathways in B-progenitor cells. Diet-induced metabolic changes in these cells partially phenocopied those observed in mice treated with anti-folate drugs, suggesting that both deficiency and excessive levels of dietary folic acid compromise folate-dependent biosynthetic pathways. Both folate deficiency and excessive dietary folate levels compromise hematopoiesis, resulting in defective cell cycle progression, persistent DNA damage, and impaired production of lymphocytes. These defects reduce the reconstitution potential in transplantation settings and increase radiation-induced mortality. We conclude that excessive folic acid supplementation can metabolically mimic dietary folate insufficiency, leading to similar functional impairment of hematopoiesis.
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Suplementos Dietéticos/efectos adversos , Deficiencia de Ácido Fólico/metabolismo , Ácido Fólico/farmacología , Hematopoyesis/efectos de los fármacos , Animales , Ácido Fólico/metabolismo , Ácido Fólico/uso terapéutico , Metabolismo/efectos de los fármacos , Ratones , Nucleótidos/biosíntesis , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/metabolismoRESUMEN
In severe pulmonary hypertension (SPH), prior studies have shown an increase in right ventricle (RV) uptake of glucose, but it is unclear whether there is a change in the relative utilization of fatty acids. We hypothesized that in the RV in SPH, as in left ventricular (LV) failure, there is altered substrate utilization, with increased glucose uptake and decreased fatty acid uptake. SPH was induced in rats by treatment with the VEGF receptor inhibitor SU5416 and 3 wk of hypoxia (10% FiO2 ), followed by an additional 4 wk of normoxia (SU-Hx group). Control rats were treated with carboxymethylcellulose vehicle and 7 wk of normoxia (CMC-Nx group). The rodents then underwent positron emission tomography with sequential administration of two radiotracers, 2-deoxy-2-[(18)F]fluoroglucose ((18)F-FDG) and 14-(R,S)-[(18)F]fluoro-6-thia-heptadecanoic acid ((18)F-FTHA), analogs of glucose and fatty acid, respectively. Five CMC-Nx and 3 SU-Hx rats completed the entire experimental protocol. In the RV, there was a mild increase in (18)F-FDG uptake (1.35-fold, P = 0.085) and a significant decrease in (18)F-FTHA uptake (-2.1-fold, P < 0.05) in the SU-Hx rats relative to the CMC-Nx rats. In the LV, SU-Hx rats had less uptake of both radiotracers compared with CMC-Nx rats. Less RV fatty acid uptake in SPH was corroborated by decreased fatty acid transporters and enzymes in the RV tissue, and specifically a decrease in lipoprotein lipase. In the RV in rats with SPH, there is a major shift in metabolic substrate preference, largely due to decreased fatty acid uptake.
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Ácidos Grasos/metabolismo , Glucosa/metabolismo , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Hipertensión Pulmonar/fisiopatología , Hipoxia/fisiopatología , Animales , Transporte Biológico , Proteínas de Transporte de Ácidos Grasos/metabolismo , Femenino , Indoles/farmacología , Lipoproteína Lipasa/metabolismo , Oxidación-Reducción , Tomografía de Emisión de Positrones , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Radiofármacos , Ratas , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Glioblastoma multiforme (GBM) is an untreatable malignancy. Existing therapeutic options are insufficient, and adversely affect functional and non-cancerous cells in the brain impairing different functions of the body. Therefore, there is an urgent need for additional preventive and therapeutic non-toxic drugs against GBM. Asiatic acid (AsA; 2,3,23-trihydroxy-12-ursen-28-oic acid, C30 H48 O5 ) is a natural small molecule widely used to treat various neurological disorders, and the present research investigates AsA's efficacy against GBM both in vitro and in vivo. Results showed that AsA treatment (10-100 µM) decreased the human GBM cell (LN18, U87MG, and U118MG) viability, with better efficacy than temozolomide at equimolar doses. Orally administered AsA (30 mg/kg/d) strongly decreased tumor volume in mice when administered immediately after ectopic U87MG xenograft implantation (54% decrease, P ≤ 0.05) or in mice with established xenografts (48% decrease, P ≤ 0.05) without any apparent toxicity. Importantly, AsA feeding (30 mg/kg/twice a day) also decreased the orthotopic U87MG xenografts growth in nude mice as measured by magnetic resonance imaging. Using LC/MS-MS methods, AsA was detected in mice plasma and brain tissue, confirming that AsA crosses blood-brain barrier. Mechanistic studies showed that AsA induces apoptotic death by modulating the protein expression of several apoptosis regulators (caspases, Bcl2 family members, and survivin) in GBM cells. Furthermore, AsA induced ER stress (increased GRP78 and Calpain, and decreased Calnexin and IRE1α expression), enhanced free intra-cellular calcium, and damaged cellular organization in GBM cells. These experimental results demonstrate that AsA is effective against GBM, and advocate further pre-clinical and clinical evaluations of AsA against GBM.
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Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Triterpenos Pentacíclicos/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Chaperón BiP del Retículo Endoplásmico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Temozolomida , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefined during lactation. Spot14-null mice produce milk deficient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When [(13)C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed.
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Acido Graso Sintasa Tipo I/metabolismo , Lactancia/fisiología , Glándulas Mamarias Animales/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Catálisis , Acido Graso Sintasa Tipo I/genética , Femenino , Ratones , Ratones Mutantes , Proteínas Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
Microbubbles (MBs) combined with focused ultrasound (FUS) has emerged as a promising noninvasive technique to permeabilize the blood-brain barrier (BBB) for drug delivery into the brain. However, the safety and biological consequences of BBB opening (BBBO) remain incompletely understood. This study aims to investigate the effects of two parameters mediating BBBO: microbubble volume dose (MVD) and mechanical index (MI). High-resolution MRI-guided FUS was employed in mouse brains to assess BBBO by manipulating these two parameters. Afterward, the sterile inflammatory response (SIR) was studied 6 h post-FUS treatment. Results demonstrated that both MVD and MI significantly influenced the extent of BBBO, with higher MVD and MI leading to increased permeability. Moreover, RNA sequencing revealed upregulation of major inflammatory pathways and immune cell infiltration after BBBO, indicating the presence and extent of SIR. Gene set enrichment analysis identified 12 gene sets associated with inflammatory responses that were significantly upregulated at higher MVD or MI. A therapeutic window was established between therapeutically relevant BBBO and the onset of SIR, providing operating regimes to avoid damage from stimulation of the NFκB pathway via TNFÉ signaling to apoptosis. These results contribute to the optimization and standardization of BBB opening parameters for safe and effective drug delivery to the brain and further elucidate the underlying molecular mechanisms driving sterile inflammation.
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Barrera Hematoencefálica , Inflamación , Microburbujas , Barrera Hematoencefálica/metabolismo , Animales , Ratones , Inflamación/metabolismo , Sistemas de Liberación de Medicamentos , Imagen por Resonancia Magnética , Encéfalo/metabolismo , Encéfalo/patología , MasculinoRESUMEN
Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.
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Regulación Neoplásica de la Expresión Génica , Glioma , Factor B de Elongación Transcripcional Positiva , Humanos , Glioma/radioterapia , Glioma/genética , Glioma/metabolismo , Glioma/patología , Animales , Factor B de Elongación Transcripcional Positiva/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Ratones , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Transcripción Genética/efectos de la radiación , Apoptosis/efectos de la radiación , Apoptosis/genética , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Reparación del ADN/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: There are no effective treatment strategies for children with highest-risk posterior fossa group A ependymoma (PFA). Chromosome 1q gains (1q+) are present in approximately 25% of newly diagnosed PFA tumors, and this number doubles at recurrence. Seventy percent of children with chromosome 1q+ PFA will die because of the tumor, highlighting the urgent need to develop new therapeutic strategies for this population. EXPERIMENTAL DESIGN: In this study, we utilize 1q+ PFA in vitro and in vivo models to test the efficacy of combination radiation and chemotherapy in a preclinical setting. RESULTS: 5-fluorouracil (5FU) enhances radiotherapy in 1q+ PFA cell lines. Specifically, 5FU increases p53 activity mediated by the extra copy of UCK2 located on chromosome 1q in 1q+ PFA. Experimental downregulation of UCK2 resulted in decreased 5FU sensitivity in 1q+ PFA cells. In in vitro studies, a combination of 5FU, retinoid tretinoin (ATRA), and radiation provided the greatest reduction in cellular proliferation and greatest increase in markers of apoptosis in 1q+ PFA cell lines compared with other treatment arms. Similarly, in vivo experiments demonstrated significant enhancement of survival in mice treated with combination radiation and 5FU and ATRA. CONCLUSIONS: These results are the first to identify a chromosome 1q+ specific therapy approach in 1q+ PFA. Existing phase I studies have already established single-agent pediatric safety and dosages of 5FU and ATRA, allowing for expedited clinical application as phase II trials for children with high-risk PFA.
Asunto(s)
Ependimoma , Neoplasias Infratentoriales , Niño , Humanos , Animales , Ratones , Neoplasias Infratentoriales/genética , Neoplasias Infratentoriales/patología , Neoplasias Infratentoriales/terapia , Resultado del Tratamiento , Ependimoma/genética , Ependimoma/terapia , Fluorouracilo , Cromosomas/metabolismoRESUMEN
BACKGROUND: This study further evaluated the safety and efficacy of the combination of alisertib and sapanisertib in an expansion cohort of patients, including a subset of patients with refractory pancreatic adenocarcinoma, with further evaluation of the pharmacodynamic characteristics of combination therapy. METHODS: Twenty patients with refractory solid tumors and 11 patients with pancreatic adenocarcinoma were treated at the recommended phase 2 dose of alisertib and sapanisertib. Adverse events and disease response were assessed. Patients in the expansion cohort were treated with a 7-day lead-in of either alisertib or sapanisertib prior to combination therapy, with tumor tissue biopsy and serial functional imaging performed for correlative analysis. RESULTS: Toxicity across treatment groups was overall similar to prior studies. One partial response to treatment was observed in a patient with ER positive breast cancer, and a patient with pancreatic cancer experienced prolonged stable disease. In an additional cohort of pancreatic cancer patients, treatment response was modest. Correlative analysis revealed variability in markers of apoptosis and immune cell infiltrate according to lead-in therapy and response. CONCLUSIONS: Dual targeting of Aurora A kinase and mTOR resulted in marginal clinical benefit in a population of patients with refractory solid tumors, including pancreatic adenocarcinoma, though individual patients experienced significant response to therapy. Correlatives indicate apoptotic response and tumor immune cell infiltrate may affect clinical outcomes.
RESUMEN
In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.
Asunto(s)
Nanomedicina , Humanos , Portadores de Fármacos/química , Liposomas/química , Nanopartículas/química , Estados UnidosRESUMEN
INTRODUCTION: Pharmacological agents that block beta-adrenergic receptors have been associated with improved outcome in burn injury. It has been hypothesized that injuries leading to a hypermetabolic state, such as septic shock, may also benefit from beta-blockade; however, outcome data in experimental models have been contradictory. Thus, we investigated the effect of beta-blockade with propranolol on survival, hemodynamics, lung heat shock protein (HSP) expression, metabolism and inflammatory markers in a rat cecal ligation and puncture (CLP) model of sepsis. METHODS: Sprague-Dawley rats receiving either repeated doses (30 minutes pre-CLP and every 8 hours for 24 hours postoperatively) of propranolol or control (normal saline), underwent CLP and were monitored for survival. Additionally, lung and blood samples were collected at 6 and 24 hours for analysis. Animals also underwent monitoring to evaluate global hemodynamics. RESULTS: Seven days following CLP, propranolol improved survival versus control (P < 0.01). Heart rates in the propranolol-treated rats were approximately 23% lower than control rats (P < 0.05) over the first 24 hours, but the mean arterial blood pressure was not different between groups. Metabolic analysis of lung tissue demonstrated an increase in lung ATP/ADP ratio and NAD+ content and a decreased ratio of polyunsaturated fatty acids to monounsaturated fatty acids (PUFA/MUFA). Cytokine analysis of the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) demonstrated decreased expression of TNF-alpha in both lung and plasma at 24 hours post CLP induced sepsis. Finally, propranolol led to a significant increase in lung hemeoxygenase-1 expression, a key cellular protective heat shock protein (HSP) in the lung. Other lung HSP expression was unchanged. CONCLUSIONS: These results suggest that propranolol treatment may decrease mortality during sepsis potentially via a combination of improving metabolism, suppressing aspects of the inflammatory response and enhancing tissue protection.