RESUMEN
The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Neoplasias Mamarias Animales/patología , Péptidos Cíclicos/farmacología , alfa-Fetoproteínas/farmacología , Animales , Apoptosis/efectos de los fármacos , Perros , Factor de Crecimiento Epidérmico/metabolismo , Estradiol/metabolismo , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/efectos de los fármacos , Receptor beta de Estrógeno/efectos de los fármacos , Femenino , Factor de Crecimiento Similar a EGF de Unión a Heparina , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias Mamarias Animales/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Estadificación de Neoplasias , Fosforilación , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Tamoxifeno/farmacología , Células Tumorales CultivadasRESUMEN
A stable cyclized 9-mer peptide (cP) containing the active site of alpha-alpha fetoprotein (alphaFP) has been shown to be effective for prevention of estrogen-stimulated tumor cell proliferation in culture or of xenographt growth in immunodeficient mice. cP does not block 17beta-estradiol (E2) binding to its receptors, but rather appears to interfere with intracellular processing of the signal that supports growth. To obtain insight on that mechanism we studied the effect of cP on the proliferation of MCF-7 cells in culture. Proliferation in the presence of 2 microM E2 is decreased up to 40% upon addition of 2 microg ml(-1) cP to the medium; the presence of cP did not increase cell death, cP reduced also the proliferation of estrogen-dependent ZR75-1 cells but had no effect on autonomous MDA-MB-231 cells, cP did not modify the number of binding sites for labeled E2 or affected cell death. We detected increased nuclear p21Cip1 immunoreactivity after cP treatment. Our results suggest that cP acts via p21Cip1 to slow the process of MCF-7 cells through the cycle.
Asunto(s)
Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Estradiol/farmacología , Péptidos Cíclicos/farmacología , alfa-Fetoproteínas/farmacología , Animales , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Ratones , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This study was aimed to obtain additional information on the activity of a cyclized 9-amino acid peptide (cP) containing the active site of alpha fetoprotein, which inhibits the estrogen-stimulated proliferation of tumor cells in culture and of xenografts in immunodeficient mice. Breast cancer cells cultured in the presence of 2 nM estradiol were exposed to cP for different periods and their proliferation, estradiol binding parameters, clustering tendency and expression of E-cadherin and p21Cip1 were analyzed by biochemical and cell biology methods. The proliferation of MCF7 cells was significantly decreased by the addition of 2 microg/ml cP to the medium. cP did not increase cell death rate nor alter the number of binding sites for estradiol nor the endogenous aromatase activity of MCF7 cells. cP also decreased the proliferation of estrogen-dependent ZR75-1 cells but had no effect on estrogen-independent MDA-MB-231 cells. An increased nuclear p21Cip1 expression detected after cP treatment suggests that cP slows MCF7 cell proliferation via this regulator. We propose that cP could represent a novel breast cancer therapeutic agent whose mechanism of action is different from that of tamoxifen or of inhibitors of aromatase.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Estradiol/farmacología , Glándulas Mamarias Humanas/efectos de los fármacos , alfa-Fetoproteínas/farmacología , Animales , Antineoplásicos/química , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Ratones Desnudos , Péptidos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , alfa-Fetoproteínas/químicaRESUMEN
Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment I, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P<0.05) were formed when FCS was used compared to PVA and 0.25mg/ml of fetuin. In Experiment II, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P<0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P<0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P<0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes.
Asunto(s)
Bovinos/embriología , Desarrollo Embrionario y Fetal/efectos de los fármacos , Fertilización In Vitro/efectos de los fármacos , Zona Pelúcida/efectos de los fármacos , alfa-Fetoproteínas/farmacología , Animales , Blastocisto/fisiología , Fase de Segmentación del Huevo , Medios de Cultivo , Técnicas de Cultivo , Femenino , Sangre Fetal , Oocitos/citología , Oocitos/fisiología , Folículo Ovárico/citología , Alcohol Polivinílico , Albúmina Sérica Bovina , Zona Pelúcida/fisiologíaRESUMEN
Animal lectins are classified on the basis of structural and functional studies in two types: the C-type, characterized by their dependence on calcium ions and the S-type which are not calcium-dependent, but thiol-dependent. In this late one, a group has been extensively studied as the S-Lac type. They are extracted with saline buffers added with lactose in presence of thiol agents, and constitute a family of structurally related protein which contain a series of conserved amino acids. They specifically bind to complementary glicoconjugates, and their biosynthesis and localization are developmentally regulated. Their role could be related to several biological activities in different organs.
Asunto(s)
Galactósidos/metabolismo , Lectinas/metabolismo , Secuencia de Aminoácidos , Anfibios , Animales , Asialoglicoproteínas/farmacología , Lubina , Sitios de Unión , Bufo arenarum , Carbohidratos/farmacología , Bovinos , Embrión de Pollo , Pollos , Pez Eléctrico , Fetuínas , Galectinas , Hemaglutininas/farmacología , Humanos , Peces Killi , Lectinas/antagonistas & inhibidores , Lectinas/fisiología , Ratones , Datos de Secuencia Molecular , Peso Molecular , Conejos , Ratas , Solubilidad , Vertebrados , Xenopus laevis , alfa-Fetoproteínas/farmacologíaRESUMEN
Sobre la base de estudios estructurales y funcionales, las lectinas animales se han clasificado en dos tipos: el Tipo C, caracterizado por su dependencia de los iones de calcio y el Tipo S que no es calciodependiente, sino tioldependiente. Entre estas últimas, se ha estudiado ampliamente el grupo de lectinas S-Lac, que son extraídas con buffers salinos con lactosa, en presencia de tioles. Contituyen una familia de proteínas realcionadas estructuralmente, que contienen una serie de aminoácidos conservados. Se unen específicamente a glicoconjugados complementarios y su biosíntesis y localización son reguladas por el desarrolo. Su rol puede relacionarse con diversas actividades biológicas que poderían variar según el órgano. (AU)
Asunto(s)
Humanos , Animales , Bovinos , Ratas , Lectinas/metabolismo , Galactósidos/metabolismo , Lectinas/antagonistas & inhibidores , Lectinas/fisiología , Carbohidratos/farmacología , Hemaglutininas/farmacología , alfa-Fetoproteínas/farmacología , Datos de Secuencia Molecular , Secuencia de Aminoácidos , Solubilidad , Peso Molecular , Sitios de Unión , Pez Eléctrico , Fundulidae , Anfibios , Bufo arenarum , Xenopus laevisRESUMEN
Sobre la base de estudios estructurales y funcionales, las lectinas animales se han clasificado en dos tipos: el Tipo C, caracterizado por su dependencia de los iones de calcio y el Tipo S que no es calciodependiente, sino tioldependiente. Entre estas últimas, se ha estudiado ampliamente el grupo de lectinas S-Lac, que son extraídas con buffers salinos con lactosa, en presencia de tioles. Contituyen una familia de proteínas realcionadas estructuralmente, que contienen una serie de aminoácidos conservados. Se unen específicamente a glicoconjugados complementarios y su biosíntesis y localización son reguladas por el desarrolo. Su rol puede relacionarse con diversas actividades biológicas que poderían variar según el órgano.
Asunto(s)
Humanos , Animales , Bovinos , Ratas , Galactósidos/metabolismo , Lectinas/metabolismo , alfa-Fetoproteínas/farmacología , Secuencia de Aminoácidos , Anfibios , Sitios de Unión , Bufo arenarum , Carbohidratos/farmacología , Pez Eléctrico , Fundulidae , Hemaglutininas/farmacología , Lectinas/antagonistas & inhibidores , Lectinas/fisiología , Datos de Secuencia Molecular , Peso Molecular , Solubilidad , Xenopus laevisRESUMEN
Neuraminidase (NA) is an enzyme produced by several microorganisms, which is capable of liberating sialic acid from glycoproteins and modifying cellular adhesion mechanisms. NA is considered a virulence factor in some bacterial species and has been implicated in the pathogenesis of acute poststreptococcal glomerulonephritis, a disease in which glomerular leukocyte infiltration is a prominent feature. We examined the effect of NA on kidney infiltration by neutrophils (PMN), T lymphocytes (TL) and monocyte-macrophages (MM). Intravenous injection of NA resulted in an early increase in the number of PMN (1 hr, 3.42 +/- 0.19 cells/cgs, mean +/- SEM; 3 hr, 3.63 +/- 0.13; 6 hr, 2.9 +/- 0.24; controls, 1.53 +/- 0.18; P < 0.001) and MM (1 hr, 3.49 +/- 0.16; 3 hr, 4.02 +/- 0.2; 6 hr, 3.88 +/- 0.27; controls 1.43 +/- 0.14; P < 0.001) in the glomeruli, while TL increased later (24 hr, 2.29 +/- 0.14; 48 hr, 2.4 +/- 0.2; 72 hr, 2.16 +/- 0.15; controls 0.7 +/- 0.07; P < 0.001). PMN and TL were also increased in the interstitium (up to ninefold for PMN and up to threefold for TL). Following i.v. injection of 51Cr-labeled NA-treated leukocytes, renal radioactive uptake was significantly increased at all times tested (percent radioactivity/gram of tissue after PMN injection, 3 hr, 5.57 +/- 0.46, mean +/- SEM; 12 hr, 5.38 +/- 0.77; 60 hr, 6.51 +/- 1.1; controls, 1.26 +/- 0.17, 1.75 +/- 0.25, and 2.46 +/- 0.08, respectively; P < 0.001 in each case.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Riñón/metabolismo , Macrófagos/fisiología , Neuraminidasa/farmacología , Neutrófilos/fisiología , Linfocitos T/fisiología , Animales , Recuento de Células , Movimiento Celular/efectos de los fármacos , Ferritinas/farmacología , Técnicas para Inmunoenzimas , Inyecciones Intravenosas , Riñón/efectos de los fármacos , Macrófagos/efectos de los fármacos , Neuraminidasa/administración & dosificación , Neutrófilos/efectos de los fármacos , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Linfocitos T/efectos de los fármacos , alfa-Fetoproteínas/farmacologíaRESUMEN
We have examined the effect of human alpha-fetoprotein (h-alpha FP), at concentrations ranging from the physiological to pathological circulating levels, on the native and alpha IFN- and IL2-boosted NK activity of normal peripheral blood lymphocytes (PBL). The cytotoxic function of unstimulated PBL was unchanged after a 16 hr incubation with 200 to 8000 ng/ml h-alpha FP. By contrast, this treatment significantly reduced the responsiveness of PBL to the NK-enhancer factors IFN and Interleukin 2. Optimal inhibition was observed when cells were pre-incubated with h-alpha FP before being stimulated with IFN. The sensitivity of PBL to the h-alpha FP mediated inhibition was restricted to the very early times (30 min) of incubation with the stimulating agents.
Asunto(s)
Células Asesinas Naturales/efectos de los fármacos , alfa-Fetoproteínas/farmacología , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Técnicas In Vitro , Interferón-alfa/farmacología , Interleucina-2/farmacología , Células Asesinas Naturales/inmunología , Cinética , alfa-Fetoproteínas/inmunologíaRESUMEN
The presence in the 100,000 g supernatant of rat brain homogenate of an inhibitor of the sialyltransferase has been confirmed. It is also present in chicken and bovine brain and in other rat and bovine organs. The inhibitor has been purified, a preparation with a specific activity 130-fold higher than that of the original 100,000 g supernatant of brain being obtained. It runs as a single peak in polyacrylamide-gel electrophoresis; when run in the presence of SDS, two components appeared. The apparent Mr of the components were 14,800 and 22,400. The inhibitor has been characterized as a heat-stable protein of acidic nature. It has effect on the glycolipid and the glycoprotein sialyltransferase activities but has no effect on the galactosaminyltransferase activity.
Asunto(s)
Asialoglicoproteínas , Sialiltransferasas/antagonistas & inhibidores , Aminoácidos/análisis , Animales , Encéfalo/enzimología , Bovinos , Pollos , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Quimotripsina/farmacología , Fetuínas , Cinética , Peso Molecular , Ratas , Especificidad de la Especie , Temperatura , Distribución Tisular , Tripsina/farmacología , alfa-Fetoproteínas/farmacología , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Estudou-se o efeito de alfafetoproteína (AFP) sobre a proliferaçäo de linfócitos periféricos humanos estimulados por fitohemaglutinina (PHA), concanavalina A (Con A), pokeweed mitogênio (PWM) e por células alogênicas em culturas mistas de linfócitos, ao medir-se e comparar-se a incorporaçäo de timidina triciada (parâmetros de crescimento celular) em culturas contendo AFP humanas purificada e em culturas sem a proteína (controles). Nenhuma alteraçäo quantitativa na reatividade linfocitária foi verificada nas culturas com AFP