RESUMEN
BACKGROUND: Sarcopenia and osteoporosis frequently co-occur in the elderly and have common pathophysiological determinants. Slit guidance ligand 3 (SLIT3) has been recently discovered as a novel therapeutic factor against osteoporosis, and a SLIT3 fragment containing the second leucine-rich repeat domain (LRRD2) had a therapeutic efficacy against osteoporosis. However, a role of SLIT3 in the skeletal muscle is unknown. METHODS: Skeletal muscle mass, strength, and/or physical activity were evaluated in Slit3-/- , ovariectomized, and aged mice, based on the measurements of muscle weight and grip strength, Kondziella's inverted hanging test, and/or wheel-running test. Skeletal muscles were also histologically evaluated by haematoxylin and eosin staining and/or immunofluorescence. The ovariectomized and aged mice were intravenously injected with recombinant SLIT3 LRRD2 for 4 weeks. C2C12 cells were used to know cellular effects of SLIT3, such as in vitro myogenesis, fusion, cell viability, and proliferation, and also used to evaluate its molecular mechanisms by immunocytochemistry, immunoprecipitation, western blotting, real-time PCR, siRNA transfection, and receptor-ligand binding ELISA. RESULTS: Slit3-deficient mice exhibited decreased skeletal muscle mass, muscle strength, and physical activity. The relative masses of gastrocnemius and soleus were lower in the Slit3-/- mice (0.580 ± 0.039% and 0.033 ± 0.003%, respectively) than those in the WT littermates (0.622 ± 0.043% and 0.038 ± 0.003%, respectively) (all, P < 0.05). Gastrocnemius of Slit3-/- mice showed the reduced number of Type I and Type IIa fibres (all, P < 0.05), but not of Type IIb and Type IIx fibres. SLIT3 activated ß-catenin signalling by promoting its release from M-cadherin, thereby increasing myogenin expression to stimulate myoblast differentiation. In vitro experiments involving ROBO2 expression, knockdown, and interaction with SLIT3 indicated that ROBO2 functions as a SLIT3 receptor to aid myoblast differentiation. SLIT3 LRRD2 dissociated M-cadherin-bound ß-catenin and up-regulated myogenin expression to increase myoblast differentiation, in a manner similar to full-length SLIT3. Systemic treatment with SLIT3 LRRD2 increased skeletal muscle mass in both ovariectomized and aged mice (all, P < 0.05). The relative masses of gastrocnemius and soleus were higher in the treated aged mice (0.548 ± 0.045% and 0.033 ± 0.005%, respectively) than in the untreated aged mice (0.508 ± 0.016% and 0.028 ± 0.003%, respectively) (all, P < 0.05). SLIT3 LRRD2 treatment increased the hanging duration of the aged mice by approximately 1.7-fold (P < 0.05). CONCLUSIONS: SLIT3 plays a sarcoprotective role by activating ß-catenin signalling. SLIT3 LRRD2 can potentially be used as a therapeutic agent against muscle loss.
Asunto(s)
Desarrollo de Músculos , Músculo Esquelético , Animales , Diferenciación Celular , Proteínas de la Membrana/genética , Ratones , Atrofia Muscular , ARN Interferente Pequeño , Receptores Inmunológicos , Sarcopenia/prevención & control , TransfecciónRESUMEN
XAV939, a tankyrase inhibitor, exerts an anticancer effect in 3-dimensional (3D) cultured SW480 cells, however this is not exhibited in 2-dimensional (2D) cultured SW480 cells. In the current study, XAV939 induced a 3.7-fold increase in cellular apoptosis in 3D culture but not in the 2D culture. However, no significant changes were indicated in cell cycle distribution in the 2D or 3D culture. Based on the observation that protein expression, which was associated with the glycolytic pathway, was increased in the 3D culture, the effect of XAV939 on the patterns of glycolytic protein expression was assessed. XAV939 was revealed to decrease lactose dehydrogenase A (LDHA) expression in 3D cultured SW480 cells, but only exerted a small effect in the 2D culture. The coadministration of XAV939 with the LDHA inhibitor FX11 decreased proliferation in 3D cultured SW480 cells compared with the single administration of FX11, while there was no additive effect in the 2D culture. The lactate assay also indicated that XAV939 decreased lactate secretion in the 3D cell culture but not in the 2D culture. These results suggest that XAV939 exerts an anticancer effect through inhibition of LDHA in the 3D culture.
RESUMEN
The antioxidant enzyme human extracellular superoxide dismutase (SOD3) is a promising biopharmaceutical candidate for the treatment of various diseases. To support the early development of SOD3 as a biopharmaceutical, a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry procedure was developed and validated for the determination of SOD3 levels in the plasma of ICR mice. After purification with Ni-NTA magnetic beads and digestion with trypsin, SOD3 signature peptides and internal standard signature peptide (ISP) were separated via high performance liquid chromatography using a Zorbax C18 column (2.1 × 50 mm, 3.5 µm) and a mobile phase consisting of 10 mM ammonium formate, 0.1% formic acid, and acetonitrile. The analyte and ISP were detected via a tandem mass spectrometer in electrospray ionization and multiple reaction monitoring modes to select both the signature peptide for SOD3 at m/z 669 to 969 and the ISP at m/z 655 to 941 in the positive ion mode. The calibration curves were linear (r > 0.99) between 5 and 1000 µg/mL with a lower limit of quantification of 5 µg/mL. The relative standard deviation ranged from 3.08 to 8.84% while the relative error ranged from -0.13 to -9.56%. This method was successfully applied to a preclinical pharmacokinetic study of SOD3 in male ICR mice.
Asunto(s)
Productos Biológicos/farmacocinética , Fraccionamiento Químico/métodos , Superóxido Dismutasa/farmacocinética , Animales , Productos Biológicos/sangre , Fraccionamiento Químico/instrumentación , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Células HEK293 , Humanos , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos ICR , Modelos Animales , Péptidos/sangre , Péptidos/aislamiento & purificación , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/sangre , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacocinética , Estándares de Referencia , Reproducibilidad de los Resultados , Organismos Libres de Patógenos Específicos , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masa por Ionización de Electrospray/métodos , Superóxido Dismutasa/administración & dosificación , Superóxido Dismutasa/sangre , Superóxido Dismutasa/aislamiento & purificación , Espectrometría de Masas en Tándem/instrumentación , Espectrometría de Masas en Tándem/métodosRESUMEN
1. We characterized the pharmacokinetics of tafamidis, a novel drug to treat transthyretin-related amyloidosis, in rats after intravenous and oral administration at doses of 0.3-3 mg/kg. In vitro Caco-2 cell permeability and liver microsomal stability, as well as in vivo tissue distribution and plasma protein binding were also examined. 2. After intravenous injection, systemic clearance (CL), volumes of distribution at steady state (Vss) and half-life (T½) remained unaltered as a function of dose, with values in the ranges of 6.41-7.03 mL/h/kg, 270-354 mL/kg and 39.5-46.9 h, respectively. Following oral administration, absolute bioavailability was 99.7-104% and was independent of doses from 0.3 to 3 mg/kg. In the urine and faeces, 4.36% and 48.9% of tafamidis, respectively, were recovered. 3. Tafamidis was distributed primarily in the liver and not in the brain, kidney, testis, heart, spleen, lung, gut, muscle, or adipose tissue. Further, tafamidis was very stable in rat liver microsomes, and its plasma protein binding was 99.9%. 4. In conclusion, tafamidis showed dose-independent pharmacokinetics with intravenous and oral doses of 0.3-3 mg/kg. Tafamidis undergoes minimal first-pass metabolism, distributes mostly in the liver and plasma, and appears to be eliminated primarily via biliary excretion.
Asunto(s)
Neuropatías Amiloides Familiares , Benzoxazoles/farmacología , Benzoxazoles/farmacocinética , Encéfalo/metabolismo , Hígado/metabolismo , Neuropatías Amiloides Familiares/tratamiento farmacológico , Neuropatías Amiloides Familiares/metabolismo , Neuropatías Amiloides Familiares/patología , Animales , Encéfalo/patología , Células CACO-2 , Humanos , Hígado/patología , Masculino , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/patología , Especificidad de Órganos , Ratas , Ratas Sprague-DawleyRESUMEN
TALLYHO/Jng (TH) mice reveal hypercholesterolemia at an early age before their plasma glucose levels have increased. The increased plasma cholesterol should be related to bile acids (BAs) metabolism, because cholesterol is the precursor of BAs and BAs change cholesterol metabolism in a feedback manner. We analyzed the BAs pool size, BAs composition, and expression levels of several proteins that have key roles in BAs synthesis, excretion, and reabsorption and compared them to those of C57BL/6 (B6) mice to study BAs metabolism in TH mice. TH mice exhibited an increased total BAs pool size, increased BAs content in the cecum feces, and an increased ratio of muricholic acid (MCA)/cholic acid (CA). The mRNA and protein levels of cholesterol 7 alpha-hydroxylase (Cyp7a1) and the ATP-binding cassette sub-family G member 5 (Abcg5) were elevated in the liver but not in the apical sodium bile acid transporter (Asbt) and organic solute transporters (Osts) in the ileum. These results indicate that synthesis and the excretion of BAs from the liver to the gallbladder might be elevated, but the reabsorption rate of BAs in the ileum might be reduced. The declined expression of fibroblast growth factor 15 (Fgf15) and fibroblast growth factor receptor 4 (Fgfr4) was respectively identified in the ileum and the liver, indicating the disrupted feedback inhibition of Cyp7a1. Consequently, hypercholesterolemia in TH mice might increase the BAs amounts via the interrupted Fxr/Fgf15/Fgfr4-mediated feedback regulation of Cyp7a1.
Asunto(s)
Colesterol 7-alfa-Hidroxilasa/genética , Diabetes Mellitus Experimental/genética , Retroalimentación Fisiológica , Hipercolesterolemia/genética , Receptores Citoplasmáticos y Nucleares/genética , Animales , Ácidos y Sales Biliares/metabolismo , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilasa/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Retroalimentación Fisiológica/fisiología , Hipercolesterolemia/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
As populations continue to age worldwide, sarcopenic obesity has heightened interest due to its medical importance. Although much evidence now indicates that n-3 fatty acids (FAs) may have beneficial effects on body composition including fat and muscle, their exact mechanisms have not yet been elucidated. Because free FA receptor 4 (FFA4) has been reported to be a receptor for n-3 FAs, we hypothesized that the protective role of n-3 FAs on body composition could be mediated by FFA4. To test this possibility, we generated mice overexpressing n-3 FAs but lacking FFA4 by crossing fat-1 transgenic (fat-1 Tg+) and FFA4 knockout (Ffar4 -/-) mice. Because fat-1 Tg+ mice, in which n-6 is endogenously converted into n-3 FAs, contain high n-3 FA levels, they could be a good animal model for studying the effects of n-3 FAs in vivo. Male and female littermates were included in high-fat-diet- (HFD) and ovariectomy-induced models, respectively. In the HFD model, male fat-1 Tg+ mice had a lower percentage of fat mass and a higher percentage of lean mass than their wild-type littermates only when they had the Ffar4 +/+ not the Ffar4 -/- background. Female fat-1 Tg+ mice showed less increase of fat mass percentage and less decrease of lean mass percentage after ovariectomy than wild-type littermates. However, these effects on body composition were attenuated in the Ffar4 -/- background. Taken together, our results indicate that the beneficial effects of n-3 FAs on body composition were mediated by FFA4 and thus suggest that FFA4 may be a potential therapeutic target for modulating sarcopenic obesity.
Asunto(s)
Composición Corporal/fisiología , Ácidos Grasos Omega-3/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Dieta Alta en Grasa , Femenino , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , OvariectomíaRESUMEN
We characterized the pharmacokinetics of enzalutamide, a novel anti-prostate cancer drug, in rats after intravenous and oral administration in the dose range 0.5-5 mg/kg. Tissue distribution, liver microsomal stability, and plasma protein binding were also examined. After intravenous injection, systemic clearance, volumes of distribution at steady state (Vss), and half-life (T½) remained unaltered as a function of dose, with values in the ranges of 80.4-86.3 mL/h/kg, 1020-1250 mL/kg, and 9.13-10.6 h, respectively. Following oral administration, absolute oral bioavailability was 89.7 % and not dose-dependent. The recoveries of enzalutamide in urine and feces were 0.0620 and 2.04 %, respectively. Enzalutamide was distributed primarily in 10 tissues (brain, liver, kidneys, testis, heart, spleen, lungs, gut, muscle, and adipose) and tissue-to-plasma ratios of enzalutamide ranged from 0.406 (brain) to 10.2 (adipose tissue). Further, enzalutamide was stable in rat liver microsomes, and its plasma protein binding was 94.7 %. In conclusion, enzalutamide showed dose-independent pharmacokinetics at intravenous and oral doses of 0.5-5 mg/kg. Enzalutamide distributed primarily to 10 tissues and appeared to be eliminated primarily by metabolism.
Asunto(s)
Antineoplásicos/farmacocinética , Microsomas Hepáticos/metabolismo , Feniltiohidantoína/análogos & derivados , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Benzamidas , Disponibilidad Biológica , Relación Dosis-Respuesta a Droga , Semivida , Inyecciones Intravenosas , Masculino , Nitrilos , Feniltiohidantoína/administración & dosificación , Feniltiohidantoína/farmacocinética , Neoplasias de la Próstata/tratamiento farmacológico , Unión Proteica , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Extracellular superoxide dismutase (EC-SOD) overexpression modulates cellular responses such as tumor cell suppression and is induced by IFNγ. Therefore, we examined the role of EC-SOD in IFNγ-mediated tumor cell suppression. We observed that the dominant-negative protein kinase C delta (PKCδ) suppresses IFNγ-induced EC-SOD expression in both keratinocytes and melanoma cells. Our results also showed that PKCδ-induced ECSOD expression was reduced by pretreatment with a PKCspecific inhibitor or a siRNA against PKCδ. PKCδ-induced ECSOD expression suppressed cell proliferations by the up-regulation of p21 and Rb, and the downregulation of cyclin A and D. Finally, we demonstrated that increased expression of EC-SOD drastically suppressed lung melanoma proliferation in an EC-SOD transgenic mouse via p21 expression. In summary, our findings suggest that IFNγ-induced EC-SOD expression occurs via activation of PKCδ. Therefore, the upregulation of EC-SOD may be effective for prevention of various cancers, including melanoma, via cell cycle arrest.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Melanoma/patología , Proteína Quinasa C-delta/metabolismo , Neoplasias Cutáneas/patología , Superóxido Dismutasa/metabolismo , Animales , Antivirales/farmacología , Western Blotting , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Células Cultivadas , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/genética , Regulación hacia ArribaRESUMEN
Target-specific antibodies can be rapidly enriched and identified from an antibody library using phage display. Large, naïve antibody libraries derived from synthetic or unimmunized sources can yield antibodies against virtually any antigen, whereas libraries from immunized sources tend to be smaller and are used exclusively against the antigen of immunization. In this study, 25 scFv libraries made from the spleens of immunized rabbits, each with a size ranging from 10(8) to higher than 10(9), were combined into a single large library with > 10(10) individual clones. Panning of this combined library yielded target-specific rabbit scFv clones against many non-immunizing antigens, including proteins, peptides, and a small molecule. Notably, specific scFv clones against a rabbit self-antigen (rabbit serum albumin) and a phosphorylated protein (epidermal growth factor receptor pTyr1173) could be isolated from the library. These results suggest that the immune library contained a significant number of unimmunized clones and that a sufficiently large immune library can be utilized similarly to a naïve library, i.e., against various non-immunizing antigens to yield specific antibodies.
Asunto(s)
Antígenos/química , Biblioteca de Péptidos , Proteínas Recombinantes/biosíntesis , Anticuerpos de Cadena Única/biosíntesis , Secuencia de Aminoácidos , Animales , Extractos Celulares/química , Receptores ErbB/química , Escherichia coli/genética , Vectores Genéticos , Datos de Secuencia Molecular , Fosfoproteínas/química , Conejos , Proteínas Recombinantes/química , Anticuerpos de Cadena Única/químicaRESUMEN
Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects cells and tissues from extracellular damage by eliminating superoxide anion radicals produced during metabolism. Two different forms of EC-SOD exist, and their different enzyme activities are a result of different disulfide bond patterns. Although only two folding variants have been discovered so far, five folding variants are theoretically possible. Therefore, we constructed five different mutant EC-SOD expression vectors by substituting cysteine residues with serine residues and evaluated their expression levels and enzyme activities. The mutant EC-SODs were expressed at lower levels than that of wild-type EC-SOD, and all of the mutants exhibited inhibited extracellular secretion, except for C195S ECSOD. Finally, we demonstrated that co-expression of wild-type EC-SOD and any one of the mutant EC-SODs resulted in reduced secretion of wild-type EC-SOD. We speculate that mutant EC-SOD causes malfunctions in systems such as antioxidant systems and sensitizes tissues to ROS-mediated diseases.
Asunto(s)
Superóxido Dismutasa/metabolismo , Sustitución de Aminoácidos , Cisteína/química , Disulfuros/química , Células HEK293 , Humanos , Mutagénesis Sitio-Dirigida , Superóxido Dismutasa/genética , Superóxido Dismutasa/fisiología , Superóxidos/químicaRESUMEN
Synthesis of a novel series of DPPIV inhibitors with 1,2,4- and 1,3,4-oxadiazolyl ketone derivatives and its structure-activity relationships are discussed. Compound 18h showed good inhibitory activity against DPPIV and favorable pharmacokinetic properties. In vivo pharmacodynamic efficacy and co-crystal structure of compound 18h with DPPIV is also described.
Asunto(s)
Inhibidores de la Dipeptidil-Peptidasa IV , Cetonas/química , Cetonas/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/síntesis química , Animales , Cristalografía por Rayos X , Dipeptidil Peptidasa 4/metabolismo , Perros , Haplorrinos , Cetonas/farmacología , Cinética , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of apoptosis in normal hepatocytes, and acquiring resistance to TGF-beta1 may be a critical step in the development of hepatocellular carcinoma (HCC). In this study, we investigated the possible involvement of c-Src in the regulation of TGF-beta1-induced apoptosis. TGF-beta1 induced transient activation of c-Src and its subsequent caspase-mediated degradation concomitant with cell death in FaO hepatoma cells, which are sensitive to TGF-beta1. In response to TGF-beta1, activated c-Src was translocated into the cytoplasmic membrane, then relocated to the nuclei of apoptotic cells during its cleavage. In TGF-beta1-induced apoptotic cells, c-Src maintained its tight association with p85 FAK fragment cleaved by caspases, possibly contributing to focal adhesion disassembly. TGF-beta1-induced apoptosis was enhanced by either inhibition of c-Src activity using PP1 or PP2, or by overexpression of dominant-negative c-Src. In contrast, overexpression of constitutively active c-Src inhibited apoptosis suppressing TGF-beta1-induced activation of p38, JNK and caspases. In many HCC cell lines resistant to TGF-beta1, enhanced c-Src activity was detected. We hypothesize that activated c-Src in HCC may contribute to resistance against the apoptotic and/ or antiproliferative properties of TGF-beta1.