RESUMEN
The caveolin-1 scaffolding domain (CSD, amino acids 82-101 of caveolin-1) has been shown to suppress bleomycin-induced lung and skin fibrosis and angiotensin II (AngII)-induced myocardial fibrosis. To identify active subregions within CSD, we split its sequence into three slightly overlapping 8-amino acid subregions (82-89, 88-95, and 94-101). Interestingly, all three peptides showed activity. In bleomycin-treated mice, all three subregions suppressed the pathological effects on lung and skin tissue morphology. In addition, while bone marrow monocytes isolated from bleomycin-treated mice showed greatly enhanced migration in vitro toward CXCL12, treatment in vivo with CSD and its subregions almost completely suppressed this enhanced migration. In AngII-induced heart failure, both 82-89 and 88-95 significantly suppressed fibrosis (both Col I and HSP47 levels), microvascular leakage, and heart weight/ body weight ratio (HW/BW) while improving ventricular function. In contrast, while 94-101 suppressed the increase in Col I, it did not improve the other parameters. The idea that all three subregions can be active depending on the assay was further supported by experiments studying the in vitro migration of human monocytes in which all three subregions were extremely active. These studies are very novel in that it has been suggested that there is only one active region within CSD that is centered on amino acids 90-92. In contrast, we demonstrate here the presence of other active regions within CSD.
Asunto(s)
Caveolina 1/metabolismo , Movimiento Celular , Monocitos/metabolismo , Fibrosis Pulmonar/metabolismo , Enfermedades de la Piel/metabolismo , Animales , Bleomicina/efectos adversos , Bleomicina/farmacología , Ratones , Fibrosis Pulmonar/inducido químicamente , Enfermedades de la Piel/inducido químicamenteRESUMEN
Aging is a progressive, multifactorial, degenerative process in which deleterious changes occur in the biochemistry and function of organs. We showed that angiotensin II (AngII)-induced pathologies in the heart and kidney of young (3-month-old) mice are suppressed by the caveolin-1 scaffolding domain (CSD) peptide. Because AngII mediates many aging-associated changes, we explored whether CSD could reverse pre-existing pathologies and improve organ function in aged mice. Using 18-month-old mice (similar to 60-year-old humans), we found that >5-fold increases in leakage of serum proteins and >2-fold increases in fibrosis are associated with aging in the heart, kidney, and brain. Because tyrosine phosphorylation of cell junction proteins leads to the loss of microvascular barrier function, we analyzed the activation of the receptor tyrosine kinase PDGFR and the nonreceptor tyrosine kinases c-Src and Pyk2. We observed 5-fold activation of PDGFR and 2- to 3-fold activation of c-Src and Pyk2 in aged mice. Treatment with CSD for 4 weeks reversed these pathologic changes (microvascular leakage, fibrosis, kinase activation) in all organs almost down to the levels in healthy, young mice. In studies of heart function, CSD reduced the aging-associated increase in cardiomyocyte cross-sectional area and enhanced ventricular compliance in that echocardiographic studies demonstrated improved ejection fraction and fractional shortening and reduced isovolumic relation time. These results suggest that versions of CSD may be developed as treatments for aging-associated diseases in human patients based on the concept that CSD inhibits tyrosine kinases, leading to the inhibition of microvascular leakage and associated fibrosis, thereby improving organ function. SIGNIFICANCE STATEMENT: The caveolin-1 scaffolding domain (CSD) peptide reverses aging-associated fibrosis, microvascular leakage, and organ dysfunction in the heart, kidneys, and brain via a mechanism that involves the suppression of the activity of multiple tyrosine kinases, suggesting that CSD can be developed as a treatment for a wide range of diseases found primarily in the aged.
Asunto(s)
Envejecimiento/efectos de los fármacos , Envejecimiento/metabolismo , Caveolina 1/farmacología , Corazón/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/metabolismo , Envejecimiento/patología , Secuencia de Aminoácidos , Animales , Caveolina 1/química , Caveolina 1/genética , Femenino , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Tirosina QuinasasRESUMEN
Dysregulation of the renin-angiotensin system leads to systemic hypertension and maladaptive fibrosis in various organs. We showed recently that myocardial fibrosis and the loss of cardiac function in mice with transverse aortic constriction (TAC) could be averted by treatment with the caveolin-1 scaffolding domain (CSD) peptide. Here, we used angiotensin II (AngII) infusion (2.1 mg/kg/day for 2 wk) in mice as a second model to confirm and extend our observations on the beneficial effects of CSD on heart and kidney disease. AngII caused cardiac hypertrophy (increased heart weight to body weight ratio (HW/BW) and cardiomyocyte cross-sectional area); fibrosis in heart and kidney (increased levels of collagen I and heat shock protein-47 (HSP47)); and vascular leakage (increased levels of IgG in heart and kidney). Echocardiograms of AngII-infused mice showed increased left ventricular posterior wall thickness (pWTh) and isovolumic relaxation time (IVRT), and decreased ejection fraction (EF), stroke volume (SV), and cardiac output (CO). CSD treatment (i.p. injections, 50 µg/mouse/day) of AngII-infused mice significantly suppressed all of these pathological changes in fibrosis, hypertrophy, vascular leakage, and ventricular function. AngII infusion increased ß1 and ß3 integrin levels and activated Pyk2 in both heart and kidney. These changes were also suppressed by CSD. Finally, bone marrow cell (BMC) isolated from AngII-infused mice showed hyper-migration toward SDF1. When AngII-infused mice were treated with CSD, BMC migration was reduced to the basal level observed in cells from control mice. Importantly, CSD did not affect the AngII-induced increase in blood pressure (BP), indicating that the beneficial effects of CSD were not mediated via normalization of BP. These results strongly indicate that CSD suppresses AngII-induced pathological changes in mice, suggesting that CSD can be developed as a treatment for patients with hypertension and pressure overload-induced heart failure.
Asunto(s)
Angiotensina II/administración & dosificación , Caveolina 1/administración & dosificación , Corazón/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/patología , Miocardio/patología , Fragmentos de Péptidos/administración & dosificación , Angiotensina II/fisiología , Angiotensinas/antagonistas & inhibidores , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Permeabilidad Capilar/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Fibrosis/etiología , Fibrosis/patología , Fibrosis/prevención & control , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
Bromelain is a proteolytic enzyme extracted from the stems and the immature fruits of pineapple that was found to be antitumorigenic in different in vitro models. Bromelain has been reported to promote apoptosis, particularly in breast cancer cells, with the up-regulation of c-Jun N-terminal kinase and p38 kinase. Our study was designed to determine if bromelain could induce apoptosis in GI-101A breast cancer cells. GI-101A cells were treated with increasing concentrations of bromelain for 24 hours. The effect of bromelain for inducing cell death via activation of the apoptosis mechanism in GI-101A cells was further determined by using caspase-9 and caspase-3 assays along with the M30-Apoptosense assay to measure cytokeratin 18 (CK18) levels in the cytoplasm of the cultured cancer cells. A dose-dependent increase in the activities of caspase-9 and caspase-3 coinciding with elevation of CK18 levels was found in bromelain-treated cells compared with control cells. Furthermore, the apoptosis induction by bromelain was confirmed by DNA fragmentation analysis and 4,6'-diamino-2-phenylindole dihydrochloride fluorescence staining of the nucleus. Our results indicate an increase in apoptosis-related cell death in breast cancer cells with increasing concentrations of bromelain.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Bromelaínas/farmacología , Neoplasias de la Mama/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Fragmentación del ADN , Femenino , Humanos , Queratina-18/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac alpha- and beta-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 --> Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality.
Asunto(s)
Cardiomegalia/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animales , Gatos , ADN Complementario/metabolismo , Espectrometría de Masas/métodos , Microscopía Confocal/métodos , Mutación , Miocardio/metabolismo , Miocitos Cardíacos/citología , Fosforilación , Presión , Estructura Terciaria de Proteína , Serina/químicaRESUMEN
BACKGROUND: Oxidative stress is implicated in oral carcinogenesis and has been found to be aggravated during radiotherapy. A great deal of attention has been focused on the possible therapeutic implications of selenium as a potent antioxidant. We determined whether selenium supplementation to radiation treated oral cancer patients render improvement in the antioxidant status against oxidative stress. METHOD: Blood samples were collected from stage (III) oral cancer patients before initiating radiotherapy (Group B) (n=63) and this group is bifurcated into Group C-patients given radiotherapy alone (n=27) and Group D-patients given radiotherapy and supplemented with selenium (400 mug/day for 6 months) (n=36). Both Group C and D were followed up for 6 months. We evaluated the plasma selenium concentration, non-enzymatic system including GSH, vitamins E, C, A and ceruloplasmin and enzymatic antioxidant system including superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase. RESULTS: The concentrations of selenium, all non-enzymatic antioxidants and the activities of enzymatic antioxidants were found to be lowered in oral cancer patients (Group B), compared to normal (Group A) (p<0.05). Similar decrease in the concentration of selenium and antioxidants status was observed in radiotherapy group (Group C) (p<0.05). On the contrary, selenium group (Group D) showed marked increase in the concentrations of selenium and antioxidant status at 6 months compared to radiation group (Group C) (p<0.05). CONCLUSION: The observed result represents the antioxidant property of selenium through the improvement of antioxidant defense system. Selenium supplementation could be of great interest in protecting cells against oxidative stress.
Asunto(s)
Antioxidantes/metabolismo , Carcinoma de Células Escamosas/metabolismo , Enzimas/sangre , Neoplasias de la Boca/metabolismo , Selenio/sangre , Selenito de Sodio/administración & dosificación , Adulto , Anciano , Antioxidantes/análisis , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/radioterapia , Ceruloplasmina/análisis , Suplementos Dietéticos , Femenino , Estudios de Seguimiento , Glutatión/sangre , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/radioterapia , Estadificación de Neoplasias , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Resultado del TratamientoRESUMEN
A body of evidence has accumulated implicating free radical generation and reaction of arsenic with protein thiols in the biochemical and molecular mechanisms of arsenic toxicity. Brain readily undergoes oxidative damage, so it is important to determine whether arsenic-induced changes in rat brain may be associated with oxidative events. An increase in oxidative stress may contribute to the development of protein damage in rat brain. Present experiments were performed to study the effect of arsenic (sodium arsenite, 100 ppm mixed in drinking water) on protein oxidation and further to demonstrate the potential of dl-alpha-lipoic acid (70 mg/kg body weight) against arsenic-induced changes in different anatomic regions (cortex, striatum, cerebellum, hypothalamus and hippocampus) of the brain of male Wistar rats. We report here that arsenic treated rats had a significantly higher level of oxidised protein as assessed by increased carbonyl residues and decreased protein thiols (protein sulfhydryls) as compared to control rats in all five regions studied, with the most notable changes occurring in the cortex, striatum and hippocampus. Coadministration of lipoic acid along with arsenic resulted in reversal of the arsenic induced trends in carbonyl and sulfhydryl concentrations. The results of the study showed, lipoic acid treatment reduces oxidative protein damage in arsenic intoxicated rat brain regions, which is associated with its antioxidant activity that combines free radical scavenging and metal chelating properties.