RESUMEN
BACKGROUND: Dantrolene binds to the Leu601-Cys620 region of the N-terminal domain of cardiac ryanodine receptor (RyR2), which corresponds to the Leu590-Cys609 region of the skeletal ryanodine receptor, and suppresses diastolic Ca2+ leakage through RyR2. OBJECTIVE: We investigated whether the chronic administration of dantrolene prevented left ventricular (LV) remodeling and ventricular tachycardia (VT) after myocardial infarction (MI) by the same mechanism with the mutation V3599K of RyR2, which indicated that the inhibition of diastolic Ca2+ leakage occurred by enhancing the binding affinity of calmodulin (CaM) to RyR2. METHODS AND RESULTS: A left anterior descending coronary artery ligation MI model was developed in mice. Wild-type (WT) were divided into four groups: sham-operated mice (WT-Sham), sham-operated mice treated with dantrolene (WT-Sham-DAN), MI mice (WT-MI), and MI mice treated with dantrolene (WT-MI-DAN). Homozygous V3599K RyR2 knock-in (KI) mice were divided into two groups: sham-operated mice (KI-Sham) and MI mice (KI-MI). The mice were followed for 12 weeks. Survival was significantly higher in the WT-MI-DAN (73%) and KI-MI groups (70%) than the WT-MI group (40%). Echocardiography, pathological tissue, and epinephrine-induced VT studies showed that LV remodeling and VT were prevented in the WT-MI-DAN and KI-MI groups compared to the WT-MI group. An increase in diastolic Ca2+ spark frequency and a decrease in the binding affinity of CaM to the RyR2 were observed at 12 weeks after MI in the WT-MI group, although significant improvements in these values were observed in the WT-MI-DAN and KI-MI groups. CONCLUSIONS: Pharmacological or genetic stabilization of RyR2 tetrameric structure improves survival after MI by suppressing LV remodeling and proarrhythmia.
Asunto(s)
Insuficiencia Cardíaca , Infarto del Miocardio , Taquicardia Ventricular , Ratones , Animales , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Dantroleno/farmacología , Remodelación Ventricular , Miocitos Cardíacos/metabolismo , Taquicardia Ventricular/etiología , Taquicardia Ventricular/genética , Arritmias Cardíacas/metabolismo , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Calmodulina/metabolismo , Infarto del Miocardio/complicaciones , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismoRESUMEN
Cardiac hypertrophy is a well-known major risk factor for poor prognosis in patients with cardiovascular diseases. Dysregulation of intracellular Ca2+ is involved in the pathogenesis of cardiac hypertrophy. However, the precise mechanism underlying cardiac hypertrophy remains elusive. Here, we investigate whether pressure-overload induced hypertrophy can be induced by destabilization of cardiac ryanodine receptor (RyR2) through calmodulin (CaM) dissociation and subsequent Ca2+ leakage, and whether it can be genetically rescued by enhancing the binding affinity of CaM to RyR2. In the very initial phase of pressure-overload induced cardiac hypertrophy, when cardiac contractile function is preserved, reactive oxygen species (ROS)-mediated RyR2 destabilization already occurs in association with relaxation dysfunction. Further, stabilizing RyR2 by enhancing the binding affinity of CaM to RyR2 completely inhibits hypertrophic signaling and improves survival. Our study uncovers a critical missing link between RyR2 destabilization and cardiac hypertrophy.
Asunto(s)
Señalización del Calcio , Calmodulina/metabolismo , Cardiomegalia , Canal Liberador de Calcio Receptor de Rianodina , Animales , Calcio/metabolismo , Señalización del Calcio/genética , Señalización del Calcio/fisiología , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Femenino , Corazón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/citología , Miocardio/metabolismo , Miocardio/patología , Presión , Unión Proteica , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Aberrant Ca2+ release from cardiac ryanodine receptors (RyR2) has been shown to be one of the most important causes of lethal arrhythmia in various types of failing hearts. We previously showed that dantrolene, a specific agent for the treatment of malignant hyperthermia, inhibits Ca2+ leakage from the RyR2 by correcting the defective inter-domain interaction between the N-terminal (1-619 amino acids) and central (2000-2500 amino acids) domains of the RyR2 and allosterically enhancing the binding affinity of calmodulin to the RyR2 in diseased hearts. In this study, we examined whether dantrolene inhibits this Ca2+ leakage, thereby preventing the pharmacologically inducible ventricular tachycardia in ventricular pressure-overloaded failing hearts. Ventricular tachycardia (VT) was easily induced after an injection of epinephrine in mice after 8 weeks of transverse aortic constriction-induced pressure-overload. Pretreatment with dantrolene almost completely inhibited the pharmacologically inducible VT. In the presence of dantrolene, the occurrence of both Ca2+ sparks and spontaneous Ca2+ transients was inhibited, which was associated with enhanced calmodulin binding affinity to the RyR2. These results suggest that dantrolene could be a new potent agent in the treatment of lethal arrhythmia in cases of acquired heart failure.
Asunto(s)
Dantroleno/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Relajantes Musculares Centrales/farmacología , Sustancias Protectoras/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/tratamiento farmacológico , Animales , Insuficiencia Cardíaca/patología , Ratones , Presión , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologíaRESUMEN
Proteome profiling based on two-dimensional (2D)-DIGE might be a useful tool for investigating drug-like compounds and the mode of action of drugs. However, obtaining data for profiling requires high labor costs, and it is difficult to control the reproducibility of spot positions because 2D-DIGE usually requires large-size glass plates and spot alignments are greatly affected by the quality of DryStrips and polyacrylamide gels (PAGs). Therefore, we have developed a novel platform by employing small size DryStrips and PAGs, and an image analysis strategy based on dual correction of spot alignment and volume. Our system can automatically detect a large number of consistent spots through all images. Cytosol fractions of HeLa cells treated with dimethyl sulfoxide (DMSO) or bortezomib were analyzed, 1697 consistent spots were detected, and 775 of them were significantly changed with the treatment. Deviations between different days and lot sets of DryStrips and PAGs were investigated by calculating the correlation coefficients. The mean values of the correlation between days and lot sets were 0.96 and 0.94, respectively. Clustering analysis of all the treatment data clearly separated the DMSO or bortezomib treated groups beyond day deviations. Thus, we have succeeded in developing an easy-to-handle 2D-DIGE system that can be a novel proteome profiling platform.
Asunto(s)
Proteoma , Proteómica/métodos , Electroforesis Bidimensional Diferencial en Gel , Animales , Bortezomib/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Dimetilsulfóxido/farmacología , Células HeLa , Humanos , Inhibidores de Proteasoma/farmacología , Proteoma/efectos de los fármacos , Células Sf9RESUMEN
BACKGROUND: Little is known about the pattern of isotope accumulation in the heart on 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography in patients with cardiac sarcoidosis (CS) complicated by ventricular aneurysm (VA).MethodsâandâResults:We prospectively enrolled 82 consecutive patients with CS; 54 patients with active CS (presence of abnormal 18F-FDG accumulation in the heart) were subdivided into VA (n=17) and non-VA groups (n=37). Strong 18F-FDG accumulation surrounding the VA and its disappearance in the VA center was observed in all patients with VA, probably because of scar formation at the VA. Peak standardized uptake value was higher around the VA than in the VA center (5.1±2.1 vs. 2.2±0.6, P=0.0003) and the VA center had no 18F-FDG accumulation (VA center: 2.2±0.6 vs. control area: 2.1±0.6, P=0.37). On the other hand, in non-VA patients with LV wall thinning (n=28), 18F-FDG accumulation was significantly high, even in the area of LV wall thinning (LV wall thinning area: 3.1±0.8 vs. control area: 2.0±0.6, P=0.00002). CONCLUSIONS: A pattern of strong 18F-FDG accumulation surrounding the VA and its disappearance in the VA center might be characteristic in patients with CS complicated by VA. Careful attention to FDG uptake would further elucidate CS pathophysiology and aid in the early treatment of VA.
Asunto(s)
Cardiomiopatías/diagnóstico por imagen , Fluorodesoxiglucosa F18/administración & dosificación , Aneurisma Cardíaco/diagnóstico por imagen , Miocarditis/diagnóstico por imagen , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radiofármacos/administración & dosificación , Sarcoidosis/diagnóstico por imagen , Corticoesteroides/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Cardiomiopatías/tratamiento farmacológico , Femenino , Aneurisma Cardíaco/tratamiento farmacológico , Humanos , Masculino , Persona de Mediana Edad , Miocarditis/tratamiento farmacológico , Valor Predictivo de las Pruebas , Estudios Prospectivos , Sarcoidosis/tratamiento farmacológico , Resultado del TratamientoRESUMEN
BACKGROUND: Tachycardia worsens cardiac performance in acute decompensated heart failure (ADHF). We investigated whether heart rate (HR) optimization by landiolol, an ultra-short-acting ß1-selective blocker, in combination with milrinone improved cardiac function in patients with ADHF and rapid atrial fibrillation (AF). METHODS AND RESULTS: We enrolled9 ADHF patients (New York Heart Association classification IV; HR, 138 ± 18 bpm; left ventricular [LV] ejection fraction, 28 ± 8%; cardiac index [CI], 2.1 ± 0.3 L/min-1/m-2; pulmonary capillary wedge pressure [PCWP], 24 ± 3 mm Hg), whose HRs could not be reduced using standard treatments, including diuretics, vasodilators, and milrinone. Landiolol (1.5-6.0 µg/kg-1/min-1, intravenous) was added to milrinone treatment to study its effect on hemodynamics. The addition of landiolol (1.5 µg/kg-1/min-1) significantly reduced HR by 11% without changing systolic blood pressure (BP) and resulted in a significant decrease in PCWP and a significant increase in stroke volume index (SVI), suggesting that HR reduction restores incomplete LV relaxation. Administration of more than 3.0 µg/kg-1/min-1 of landiolol decreased BP, CI, and SVI. CONCLUSION: The addition of landiolol at doses of <3.0 µg/kg/min to milrinone improved cardiac function in decompensated chronic heart failure with rapid atrial fibrillation by selectively reducing HR.
Asunto(s)
Antagonistas Adrenérgicos beta/uso terapéutico , Fibrilación Atrial/tratamiento farmacológico , Cardiotónicos/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Milrinona/uso terapéutico , Morfolinas/uso terapéutico , Urea/análogos & derivados , Anciano , Anciano de 80 o más Años , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Hemodinámica/efectos de los fármacos , Humanos , Japón , Masculino , Estudios Prospectivos , Taquicardia , Resultado del Tratamiento , Urea/uso terapéuticoRESUMEN
BACKGROUND: Recently, new devices using oscillometric cuff have been developed to derive central blood pressure (BP) waveform from brachial BP waveform. Late systolic pressure augmentation of central BP waveform is a marker of central hemodynamics. Mobil-O-Graph, a cuff-based oscillometric device, can assess central augmentation pressure (AP) together with central BP. Central AP measurement using the Mobil-O-Graph in the European population was reported to be associated with age and sex. However, factors influencing central AP in the Asian population have not been shown. OBJECTIVES AND METHODS: We enrolled 110 normotensive volunteers (50 men; age range, 21â76 years). Central BP and AP were measured using the Mobil-O-Graph on the left arm with the subjects in the seated position after resting for at least 5 min. We compared central hemodynamics between the sexes. We investigated factors influencing central AP in uni- and multivariate linear regression analyses and age-related change in central AP using the Mobil-O-Graph in healthy Japanese individuals. RESULTS: Central AP were lower in men than in women (5.5 ± 2.8 vs. 11.0 ± 4.7 mm Hg, p < 0.001). The central AP in the total cohort was positively correlated with age (r = 0.325, p < 0.001) and inversely correlated with height (r = -0.601, p < 0.001) in the Pearson correlations. In multivariate regression analysis, the parameters influencing central AP (R>sup<2>/sup< = 0.467) were age (ß = 0.097, p < 0.001), sex (ß = -2.890, p = 0.010), and height (ß = -0.153, p = 0.031). The central AP (10.0 ± 4.8 mm Hg) in the ≥50-year-old group significantly increased compared with those in the 20- to 39-year-old group (6.7 ± 4.2 mm Hg, p < 0.05). CONCLUSIONS: Age, sex, and height influenced central AP, as assessed using the Mobil-O-Graph. Age-related increase in central AP was observed in normotensive Japanese individuals. Brachial cuff-based waveform recordings using the Mobil-O-Graph are feasible for the estimation of central AP in the Asian population.
RESUMEN
BACKGROUND: Ryanodine receptor (RyR2) is known to be a causal gene of catecholaminergic polymorphic ventricular tachycardia (CPVT), an important inherited disease. Some of the human CPVT-associated mutations have been found in a domain (4026-4172) that has EF hand motifs, the so-called calmodulin (CaM)-like domain (CaMLD). OBJECTIVE: The purpose of this study was to investigate the underlying mechanism by which CPVT is induced by a mutation at CaMLD. METHODS: A new N4103K/+ knock-in (KI) mice model was generated. RESULTS: Sustained ventricular tachycardia was frequently observed after infusion of caffeine plus epinephrine in KI mice. Endogenous CaM bound to RyR2 decreased even at baseline in isolated KI cardiomyocytes. Ca2+ spark frequency (CaSpF) was much higher in KI cells than in wild-type cells. Addition of GSH-CaM (higher affinity CaM to RyR2) significantly decreased CaSpF. In response to isoproterenol, spontaneous Ca2+ transient (SCaT) was frequently observed in intact KI cells. Incorporation of GSH-CaM into intact KI cells using a protein delivery kit decreased SCaT significantly. An assay using a quartz crystal microbalance technique revealed that mutated CaMLD peptide showed higher binding affinity to CaM binding domain (CaMBD) peptide. CONCLUSION: In the N4103K mutant, CaM binding affinity to RyR2 was significantly reduced regardless of beta-adrenergic stimulation. We found that this was caused by an abnormally tight interaction between CaMBD and mutated CaM-like domain (N4103K-CaMBD). Thus, CaMBD-CaMLD interaction may be a novel therapeutic target for treatment of lethal arrhythmia.
Asunto(s)
Calcio/metabolismo , ADN/genética , Mutación , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Animales , Señalización del Calcio , Células Cultivadas , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Ratones , Miocitos Cardíacos/patología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologíaRESUMEN
BACKGROUND: Recently, we reported that urinary 8-hydroxy-2'-deoxyguanosine (U-8-OHdG), an oxidative stress marker, reflected inflammatory activity in cardiac sarcoidosis (CS). Here, we investigated whether U-8-OHdG levels were associated with ventricular tachycardia (VT) in patients with CS. METHODS AND RESULTS: This prospective cohort study enrolled 62 consecutive patients with CS, of whom 36 were diagnosed as having active CS based on abnormal 18F-flurodeoxyglucose accumulation in the heart on positron-emission tomography/computed tomography. The 36 patients with active CS were subdivided as having CS with sustained VT (CS-VT group; n=18) or CS without sustained VT (CS-nVT group; n=18). Twenty-seven patients diagnosed with idiopathic dilated cardiomyopathy served as heart failure controls. U-8-OHdG, brain natriuretic peptide, cardiac function indices, and immunohistological data from subendomyocardial biopsy samples were compared across groups. Immunohistochemical examination of ventricle biopsy samples revealed that the anti-8-OHdG antibody-positive area of cardiac tissue was significantly greater in CS-VT than in CS-nVT or dilated cardiomyopathy and significantly correlated with U-8-OHdG levels (n=58; R=0.61; P<0.00001), which were significantly higher in CS-VT than in CS-nVT (24.6±7.1 versus 15.2±3.8 ng/mg·Cr; P<0.0001). Other baseline characteristics did not differ between the groups. Multivariate analysis indicated that U-8-OHdG was an independent determinant factor for VT. Receiver operating characteristic curve analysis to identify patients with VT resulted in a U-8-OHdG cutoff value of 17.5 ng/mg·Cr (sensitivity, 89%; specificity, 83%; area under the curve, 0.90). CONCLUSIONS: U-8-OHdG levels are associated with VT in patients with active CS diagnosed by 18F-flurodeoxyglucose positron-emission tomography, providing additive and relevant information about the arrhythmia substrate.
Asunto(s)
Biomarcadores/orina , Cardiomiopatías/diagnóstico por imagen , Cardiomiopatías/orina , Desoxiguanosina/análogos & derivados , Estrés Oxidativo , Sarcoidosis/diagnóstico por imagen , Sarcoidosis/orina , Taquicardia Ventricular/diagnóstico por imagen , Taquicardia Ventricular/orina , 8-Hidroxi-2'-Desoxicoguanosina , Anciano , Cardiomiopatías/fisiopatología , Desoxiguanosina/orina , Ecocardiografía , Electrocardiografía , Femenino , Fluorodesoxiglucosa F18 , Humanos , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Tomografía de Emisión de Positrones , Estudios Prospectivos , Radiofármacos , Sarcoidosis/fisiopatología , Taquicardia Ventricular/fisiopatologíaRESUMEN
Signaling by extracellular signal-regulated kinase (ERK) plays an essential role in the induction of cell motility, but the precise mechanism underlying such regulation has remained elusive. We recently identified SH3P2 as a negative regulator of cell motility whose function is inhibited by p90 ribosomal S6 kinase (RSK)-mediated phosphorylation downstream of ERK. We here show that myosin 1E (Myo1E) is a binding partner of SH3P2 and that the interaction of the two proteins in the cytosol prevents the localization of Myo1E to the plasma membrane. Serum-induced phosphorylation of SH3P2 at Ser(202) by RSK results in dissociation of Myo1E from SH3P2 in the cytosol and the subsequent localization of Myo1E to the tips of lamellipodia mediated by binding of its TH2 domain to F-actin. This translocation of Myo1E is essential for lamellipodium extension and consequent cell migration. The ERK signaling pathway thus promotes cell motility through regulation of the subcellular localization of Myo1E.
Asunto(s)
Movimiento Celular , Sistema de Señalización de MAP Quinasas , Miosina Tipo I/metabolismo , Seudópodos/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Péptidos y Proteínas de Señalización Intracelular , Modelos Biológicos , Miosina Tipo I/química , Fosforilación , Fosfoserina/metabolismo , Prolina/metabolismo , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Proteínas/química , Proteínas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDaRESUMEN
Extracellular signal-regulated kinase (ERK) has been implicated in the development of insulin resistance associated with obesity and type 2 diabetes mellitus. We have now examined the potential of pharmacological targeting of the ERK pathway with MEK (ERK kinase) inhibitors (PD184352 and PD0325901) for the treatment of obesity-associated insulin resistance. The effects of PD184352 and PD0325901 on the expression of adipocytokines and lipolysis activity were thus examined in 3T3-L1 adipocytes maintained in long-term culture as a model of adipocyte hypertrophy. Leptin receptor-deficient (db/db) mice and high-fat diet-fed KKAy mice, both of which are models of type 2 diabetes, were also treated orally with PD184352 to examine its effects on the diabetic condition. ERK activity was increased in hypertrophic 3T3-L1 adipocytes as well as in adipose tissue of db/db mice and high-fat diet-fed KKAy mice, and this enhanced ERK signaling was associated with dysregulation of adipocytokine expression and increased lipolysis activity. Specific blockade of the ERK pathway in hypertrophic 3T3-L1 adipocytes by MEK inhibitors ameliorated the dysregulation of adipocytokine expression and suppressed the enhanced lipolysis activity. Furthermore, repeated oral administration of PD184352 normalized hyperglycemia and hyperlipidemia and improved insulin sensitivity and glucose tolerance in the diabetic mice. These results suggest that sustained activation of the ERK pathway in adipocytes is associated with the pathogenesis of type 2 diabetes and that selective blockade of this pathway with MEK inhibitors warrants further study as a promising approach to the treatment of insulin resistance and type 2 diabetes.
Asunto(s)
Adipocitos/efectos de los fármacos , Benzamidas/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Difenilamina/análogos & derivados , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Resistencia a la Insulina , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Células 3T3-L1 , Adipocitos/metabolismo , Adipoquinas/metabolismo , Adiponectina/metabolismo , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Quimiocina CCL2/efectos de los fármacos , Quimiocina CCL2/metabolismo , Dieta Alta en Grasa , Difenilamina/farmacología , Modelos Animales de Enfermedad , Ácidos Grasos no Esterificados/metabolismo , Prueba de Tolerancia a la Glucosa , Hiperlipidemias/metabolismo , Immunoblotting , Técnicas In Vitro , Insulina/metabolismo , Interleucina-6/metabolismo , Lipólisis/efectos de los fármacos , Masculino , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Leptina/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Triglicéridos/metabolismo , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
CONCLUSION: We demonstrated that repression of keratinocyte growth factor (KGF) receptor (KGFR) could be a potentially useful strategy in the conservative treatment of middle ear cholesteatoma. OBJECTIVES: Recently, the use of a selective inhibitor of the KGFR, SU5402, in an in vitro experiment resulted in the inhibition of the differentiation and proliferation of epithelial cells through KGF secretion by fibroblasts isolated from the cholesteatoma. In this study, we investigated the effects of the KGFR inhibitor during middle ear cholesteatoma formation in vivo. METHODS: Based on the role of KGF in the development of cholesteatoma, Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal of rats five times on every fourth day. Ears transfected with empty vector were used as controls. KGFR selective inhibitor (SU5402) or MEK inhibitor (PD0325901) was administered in the right ear of five rats after vector transfection. In the control, 2% DMSO in PBS was administered in the other ears after vector transfection. RESULTS: The use of a selective KGFR inhibitor, SU5402, completely prevented middle ear cholesteatoma formation in the rats.
Asunto(s)
Colesteatoma del Oído Medio/prevención & control , Pirroles/uso terapéutico , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Animales , Benzamidas , Colesteatoma del Oído Medio/metabolismo , Difenilamina/análogos & derivados , Evaluación Preclínica de Medicamentos , Conducto Auditivo Externo/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Vectores Genéticos , Masculino , Ratas Sprague-Dawley , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , TransfecciónRESUMEN
The ERK pathway is up-regulated in various human cancers and represents a prime target for mechanism-based approaches to cancer treatment. Specific blockade of the ERK pathway alone induces mostly cytostatic rather than pro-apoptotic effects, however, resulting in a limited therapeutic efficacy of the ERK kinase (MEK) inhibitors. We previously showed that MEK inhibitors markedly enhance the ability of histone deacetylase (HDAC) inhibitors to induce apoptosis in tumor cells with constitutive ERK pathway activation in vitro. To evaluate the therapeutic efficacy of such drug combinations, we administered the MEK inhibitor PD184352 or AZD6244 together with the HDAC inhibitor MS-275 in nude mice harboring HT-29 or H1650 xenografts. Co-administration of the MEK inhibitor markedly sensitized the human xenografts to MS-275 cytotoxicity. A dose of MS-275 that alone showed only moderate cytotoxicity thus suppressed the growth of tumor xenografts almost completely as well as induced a marked reduction in tumor cellularity when administered with PD184352 or AZD6244. The combination of the two types of inhibitor also induced marked oxidative stress, which appeared to result in DNA damage and massive cell death, specifically in the tumor xenografts. The enhanced therapeutic efficacy of the drug combination was achieved by a relatively transient blockade of the ERK pathway. Administration of both MEK and HDAC inhibitors represents a promising chemotherapeutic strategy with improved safety for cancer patients.
Asunto(s)
Benzamidas/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Sistema de Señalización de MAP Quinasas , Piridinas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis , Bencimidazoles/farmacología , Sinergismo Farmacológico , Femenino , Células HT29 , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Estrés Oxidativo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Blockade of the ERK signaling pathway by ERK kinase (MEK) inhibitors selectively enhances the induction of apoptosis by microtubule inhibitors in tumor cells in which this pathway is constitutively activated. We examined the mechanism by which such drug combinations induce enhanced cell death by applying time-lapse microscopy to track the fate of individual cells. MEK inhibitors did not affect the first mitosis after drug exposure, but most cells remained arrested in interphase without entering a second mitosis. Low concentrations of microtubule inhibitors induced prolonged mitotic arrest followed by exit of cells from mitosis without division, with most cells remaining viable. However, the combination of a MEK inhibitor and a microtubule inhibitor induced massive cell death during prolonged mitosis. Impairment of spindle assembly checkpoint function by RNAi-mediated depletion of Mad2 or BubR1 markedly suppressed such prolonged mitotic arrest and cell death. The cell death was accompanied by up-regulation of the pro-apoptotic protein Bim (to which MEK inhibitors contributed) and by down-regulation of the anti-apoptotic protein Mcl-1 (to which microtubule and MEK inhibitors contributed synergistically). Whereas RNAi-mediated knockdown of Bim suppressed cell death, stabilization of Mcl-1 by RNAi-mediated depletion of Mule slowed its onset. Depletion of Mcl-1 sensitized tumor cells to MEK inhibitor-induced cell death, an effect that was antagonized by knockdown of Bim. The combination of MEK and microtubule inhibitors thus targets Bim and Mcl-1 in a cooperative manner to induce massive cell death in tumor cells with aberrant ERK pathway activation.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Sistema de Señalización de MAP Quinasas/genética , Proteínas Mad2 , Proteínas de la Membrana/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Nephronophthisis (NPHP), the most frequent genetic cause of end-stage kidney disease in children and young adults, is characterized by a variable number of renal cysts associated with cortical tubular atrophy and interstitial fibrosis. The p38 mitogen-activated protein kinase (MAPK) pathway is an important intracellular signaling pathway involved in the production of profibrotic mediators. The relationship between p38 MAPK and renal fibrosis in NPHP2 is unknown. METHODS: We administered a selective p38 MAPK inhibitor, FR167653, in a NPHP2 mouse model (inv/inv, invΔC mice) from 3 to 6 weeks old, and the kidneys were examined at 6 weeks of age. Phosphorylation of p38 MAPK (p-p38 MAPK) protein levels, the degree of renal fibrosis, messenger RNA (mRNA) levels for extracellular matrix genes and mRNA levels for transforming growth factor in the kidneys were studied. Effect of an extracellular signal-regulated protein kinase (ERK) kinase (MEK) inhibitor on renal fibrosis was also evaluated. RESULTS: Expression of extracellular matrix genes and p-p38 MAPK were increased in the NPHP2 mouse model kidney. FR167653 successfully decreased p-p38 MAPK levels, the degree of fibrosis and extracellular matrix gene expressions. However, the FR167653 did not prevent cyst expansion, abnormal cell proliferation and acceleration of apoptosis and did not influence ERK activation. In contrast, MEK inhibition reduced both cyst expansion and fibrosis without affecting p38 MAPK activation. CONCLUSIONS: These results suggest that inhibition of p38 MAPK reduced renal fibrosis but not cyst expansion, cell proliferation and apoptosis in NPHP2 model mice. Our results suggest that p38 MAPK and ERK signaling pathways independently affect renal fibrosis in inv mutant mice.
Asunto(s)
Modelos Animales de Enfermedad , Fibrosis/prevención & control , Enfermedades Renales/prevención & control , Pirazoles/farmacología , Piridinas/farmacología , Factores de Transcripción/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Apoptosis , Western Blotting , Proliferación Celular , Quistes/tratamiento farmacológico , Quistes/enzimología , Quistes/prevención & control , Fibrosis/tratamiento farmacológico , Fibrosis/enzimología , Inhibidores de Crecimiento/farmacología , Humanos , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/enzimología , Ratones , Ratones Transgénicos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The extracellular signal-regulated kinase (ERK) pathway is a major determinant in the control of diverse cellular processes such as proliferation, survival, and motility. This pathway is often upregulated in human cancers and as such represents an attractive target for mechanism-based approaches to cancer treatment. However, specific blockade of the ERK pathway alone induces mostly cytostatic rather than proapoptotic effects, resulting in limited therapeutic efficacy. Blockade of the constitutively activated ERK pathway by an ERK kinase (MEK) inhibitor sensitizes tumor cells to apoptotic cell death induced by several cytotoxic anticancer agents including microtubule-destabilizing agents and histone deacetylase inhibitors, not only in vitro but also in tumor zenografts in vivo. Thus, low concentrations of these anticancer drugs that by themselves show little cytotoxicity effectively kill tumor cells in which the ERK pathway is constitutively activated when co-administrated with a MEK inhibitor. The combination of a cytostatic signaling pathway inhibitor (MEK inhibitors) and conventional anticancer drugs (microtubule-destabilizing agents or histone deacetylase inhibitors) provides an excellent basis for the development of safer anticancer chemotherapies with enhanced efficacy through lowering the required dose of the latter cytotoxic drugs.
Asunto(s)
Antineoplásicos/uso terapéutico , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/uso terapéutico , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Moduladores de Tubulina/uso terapéutico , Animales , Apoptosis , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Terapia Molecular Dirigida , Neoplasias/metabolismoRESUMEN
Although the extracellular signal-regulated kinase (ERK) pathway functions downstream of Ras in induction of the cell motility response, the detailed molecular mechanism by which this pathway regulates cell motility has remained elusive. The application of a functional expression cloning strategy to discover proteins that regulate cell motility has resulted in the identification of an SH3 domain-containing protein, SH3P2. Overexpression of SH3P2 in HeLa S3 cells inhibited cell motility, whereas RNA interference-mediated depletion of SH3P2 enhanced motility in various tumor cell lines, suggesting that SH3P2 functions as a negative regulator of cell motility. The expression level of SH3P2 alone did not correlate well with the motility of tumor cells, however. SH3P2 was phosphorylated on Ser(202) by ribosomal S6 kinase (RSK) in an ERK pathway-dependent manner, and such phosphorylation inhibited the ability of SH3P2 to suppress cell motility. The RSK inhibitor BI-D1870 suppressed SH3P2 phosphorylation and tumor cell motility as effectively as did the MEK inhibitor PD184352. Furthermore, expression of the unphosphorylatable SH3P2 mutant SH3P2(S202A) inhibited tumor cell motility, indicating that phosphorylation of SH3P2 at Ser(202) is a key determinant of such motility. These results suggest that SH3P2 is an essential molecule that functions downstream of the ERK pathway to modulate cell motility.
Asunto(s)
Movimiento Celular , Proteínas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Serina/metabolismo , Línea Celular Tumoral , Células HeLa , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosforilación , Proteínas/genética , Pteridinas/farmacología , Interferencia de ARN , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Serina/genéticaRESUMEN
PURPOSE: The extracellular signal-regulated kinase (ERK) pathway is upregulated in human cancers and represents a target for mechanism-based approaches to cancer treatment. However, specific blockade of the ERK pathway alone induces mostly cytostatic rather than proapoptotic effects, resulting in a limited therapeutic efficacy of inhibitors that target the mitogen-activated protein kinase/ERK kinase (MEK). Given the cytoprotective role of the ERK pathway, we examined whether its blockade by the MEK inhibitor PD184352 might enhance the therapeutic efficacy of anticancer drugs in human tumor xenograft models. EXPERIMENTAL DESIGN: We recently showed that blockade of the ERK pathway by MEK inhibitors enhances the induction of apoptosis by microtubule-destabilizing agents, including TZT-1027 and vinorelbine, in various tumor cells with aberrant activation of the ERK pathway in vitro. We here examined the therapeutic efficacy of the combination of PD184352 with TZT-1027 or vinorelbine in nude mice harboring HT-29 or HT1080 tumor xenografts, in which the ERK pathway is activated as a result of mutations of BRAF and NRAS, respectively. RESULTS: Coadministration of PD184352 markedly sensitized HT-29 or HT1080 tumor xenografts to TZT-1027-induced or vinorelbine-induced cytotoxicity. Low doses of TZT-1027 or vinorelbine that by themselves showed little or moderate cytotoxicity thus suppressed the growth of HT-29 xenografts almost completely and induced essentially complete regression of HT1080 xenografts when administered with PD184352. The enhanced therapeutic efficacy of the drug combinations was achieved by a relatively transient blockade of the ERK pathway. CONCLUSIONS: Administration of both a MEK inhibitor and a microtubule-destabilizing agent represents a promising chemotherapeutic strategy with improved safety for cancer patients.
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Moduladores de Tubulina/farmacología , Animales , Sinergismo Farmacológico , Femenino , Células HT29 , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Vinblastina/análogos & derivados , Vinblastina/farmacología , Vinorelbina , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Deregulated activation of protein tyrosine kinases, such as the epidermal growth factor receptor (EGFR) and Abl, is associated with human cancers including non-small cell lung cancer (NSCLC) and chronic myeloid leukemia (CML). Although inhibitors of such activated kinases have proved to be of therapeutic benefit in individuals with NSCLC or CML, some patients manifest intrinsic or acquired resistance to these drugs. We now show that, whereas blockade of either the extracellular signal-regulated kinase (ERK) pathway or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway alone induced only a low level of cell death, it markedly sensitized NSCLC or CML cells to the induction of apoptosis by histone deacetylase (HDAC) inhibitors. Such enhanced cell death induced by the respective drug combinations was apparent even in NSCLC or CML cells exhibiting resistance to EGFR or Abl tyrosine kinase inhibitors, respectively. Co-administration of a cytostatic signaling pathway inhibitor may contribute to the development of safer anticancer strategies by lowering the required dose of cytotoxic HDAC inhibitors for a variety of cancers.
Asunto(s)
Analgésicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Resistencia a Antineoplásicos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Neoplasias Pulmonares/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Benzamidas/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Gefitinib , Humanos , Mesilato de Imatinib , Fosfatidilinositol 3-Quinasas/metabolismo , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinas/farmacología , Quinazolinas/farmacologíaRESUMEN
Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage kidney disease in children and young adults. Inv mice are a model for human nephronophthisis type 2 (NPHP2) and characterized by multiple renal cysts and situs inversus. Renal epithelial cells in inv cystic kidneys show increased cell proliferation. We studied the ERK pathway to understand the mechanisms that induce cell proliferation and renal cyst progression in inv kidneys. We studied the effects of ERK suppression by administering PD184352, an oral mitogen-activated protein kinase kinase (MEK) inhibitor on renal cyst expansion, extracellular signal-regulated protein kinase (ERK) activity, bromo-deoxyuridine (BrdU) incorporation and expression of cell-cycle regulators in invDeltaC kidneys. Phosphorylated ERK (p-ERK) level increased along with renal cyst enlargement. Cell-cycle regulators showed a high level of expression in invDeltaC kidneys. PD184352 successfully decreased p-ERK level and inhibited renal cyst enlargement. The inhibitor also decreased expression of cell-cycle regulators and BrdU incorporation in renal epithelial cells. The present results showed that ERK regulated renal cell proliferation and cyst expansion in inv mutants.