RESUMEN

Elevated circulating estrogen levels are associated with increased risk of breast cancer in obese postmenopausal women. Following menopause, the biosynthesis of estrogens through CYP19 (aromatase)-mediated metabolism of androgen precursors occurs primarily in adipose tissue, and the resulting estrogens are then secreted into the systemic circulation. The potential links between obesity, inflammation, and aromatase expression are unknown. In both dietary and genetic models of obesity, we observed necrotic adipocytes surrounded by macrophages forming crown-like structures (CLS) in the mammary glands and visceral fat. The presence of CLS was associated with activation of NF-κB and increased levels of proinflammatory mediators (TNF-α, IL-1ß, Cox-2), which were paralleled by elevated levels of aromatase expression and activity in the mammary gland and visceral fat of obese mice. Analyses of the stromal-vascular and adipocyte fractions of the mammary gland suggested that macrophage-derived proinflammatory mediators induced aromatase and estrogen-dependent gene expression (PR, pS2) in adipocytes. Saturated fatty acids, which have been linked to obesity-related inflammation, stimulated NF-κB activity in macrophages leading to increased levels of TNF-α, IL-1ß, and Cox-2, each of which contributed to the induction of aromatase in preadipocytes. The discovery of the obesity → inflammation → aromatase axis in the mammary gland and visceral fat and its association with CLS may provide insight into mechanisms underlying the increased risk of hormone receptor-positive breast cancer in obese postmenopausal women, the reduced efficacy of aromatase inhibitors in the treatment of breast cancer in these women, and their generally worse outcomes. The presence of CLS may be a biomarker of increased breast cancer risk or poor prognosis.

Asunto(s)

Aromatasa/biosíntesis , Regulación Neoplásica de la Expresión Génica , Inflamación , Glándulas Mamarias Animales/metabolismo , Obesidad/metabolismo , Adipocitos/citología , Animales , Biomarcadores de Tumor , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Femenino , Humanos , Neoplasias Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos C57BL

RESUMEN

There is great interest in the role of polyunsaturated fatty acids (PUFAs) in promoting (n-6 class) or inhibiting (n-3 class) inflammation. Mammalian cells are devoid of desaturase that converts n-6 to n-3 PUFAs. Consequently, essential n-3 fatty acids must be supplied with the diet. We have studied the effect of endogenously produced n-3 PUFAs on colitis development in fat-1 transgenic mice carrying the Caenorhabditis elegans fat-1 gene encoding n-3 desaturase. Colonic cell lipid profile was measured by capillary gas chromatography in fat-1 and wild-type (WT) littermates fed standard diet supplemented with 10% (w/w) safflower oil rich (76%) in n-6 polyunsaturated linoleic acid (LA). Experimental colitis was induced by administrating 3% dextran sodium sulphate (DSS). Colitis was scored by histopatological analysis. Cyclooxygenase-2 (Cox-2) expression was evaluated by real time polymerase chain reaction. Prostaglandin E(2) (PGE(2)) levels and cytokine production were determined by enzyme and microsphere-based immunoassays, respectively. The n-6/n-3 PUFA ratios in colonic cells of fat-1 mice were markedly lower (9.83±2.62) compared to WT (54.5±9.24, P<.001). Results also showed an attenuation of colonic acute and chronic inflammation in fat-1 mice with significant decreases in PGE(2) production (P<.01) and Cox-2 expression (P<.01). High levels of colitis-induced proinflammatory cytokines, interleukin (IL)-18, IL-1α, IL-1ß, IL-6, monocytes chemotactic proteins 1, 2 and 3 (MCP 1,2,3), matrix metalloproteinase 9 and tumor necrosis factor α (TNF-α) were down-regulated in DSS acutely and chronically treated fat-1 mice. The expression of fat-1 gene in the colon was associated with endogenous n-3 PUFAs production, decreased Cox-2 expression, increased PGE(2) and cytokine production.

Asunto(s)

Colitis/metabolismo , Ciclooxigenasa 2/biosíntesis , Citocinas/biosíntesis , Dinoprostona/biosíntesis , Ácidos Grasos Omega-3/biosíntesis , Animales , Proteínas de Caenorhabditis elegans/genética , Colitis/inducido químicamente , Colitis/patología , Colon/metabolismo , Colon/patología , Sulfato de Dextran , Ácido Graso Desaturasas/genética , Ácidos Grasos Insaturados/farmacología , Ratones , Ratones Transgénicos

RESUMEN

Caseinphosphopeptides (CPPs) are considered as mineral carriers because of their ability to bind and solubilize calcium ions, with the possible role, yet to be definitely assessed, of improving calcium absorption at the intestinal level. Previous works demonstrated that CPPs improve calcium uptake, with increasing intracellular calcium concentration, by human differentiated tumor HT-29 cells, and that this effect correlates with the supramolecular structure of CPPs in the presence of calcium ions. The aim of the present study was to establish whether the CPP effect on calcium uptake is specific for HT-29 cells and depends on the differentiated state of the cells. To this purpose, HT-29 and Caco2 cells, two models of intestinal cells, were differentiated following appropriate protocols, including treatment with 1,25-(OH)2 vitamin D3. The CPP-dependent intracellular calcium rises were monitored at the single-cell level through fura2-fluorescence assays, and cell differentiation was assessed by biochemical and morphological methods. Results clearly showed that the ability to take up extracellular calcium ions under CPP stimulation is exhibited by both HT-29 and Caco2 cells, but only upon cell differentiation. This evidence adds novel support to the notion that CPPs favour calcium absorption, thus possibly acting as cellular bio-modulators and carrying a nutraceutical potential.

Asunto(s)

Calcio/farmacocinética , Caseínas/farmacología , Diferenciación Celular/fisiología , Quelantes/farmacología , Mucosa Intestinal/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Fosfopéptidos/farmacología , Fosfatasa Alcalina/metabolismo , Células CACO-2 , Calcitriol/farmacología , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Suplementos Dietéticos , Células HT29 , Humanos , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestructura , Cinética , Microvellosidades/enzimología , Complejo Sacarasa-Isomaltasa/metabolismo

RESUMEN

We have previously reported that sulindac, a non-steroidal anti-inflammatory drug, inhibited tumor formation in the small intestine but increased tumors in the colon of Apc(Min/+) mice, a model of human familial adenomatous polyposis. To further explore intestinal regional responses, we studied effects of sulindac on additional gene-targeted mouse models of human intestinal tumorigenesis; these were (i) Apc(1638N/+) mouse (chain termination mutation in exon 15 of the Apc gene); (ii) Mlh1(+/-) mouse (DNA mismatch repair deficiency, a mouse model of human hereditary non-polyposis colorectal cancer) and (iii) double-heterozygous Mlh1(+/-)Apc(1638N/+) mutant mouse. Mice were fed AIN-76A control diet with or without 0.02% sulindac for 6 months. Intestinal regional tumor incidence, multiplicity, volume and degree of inflammation were used as end points. The results showed the following: (i) sulindac inhibited tumor development in the small intestine of Apc(1638N/+) mice; (ii) in contrast, sulindac increased tumors in the small intestine of Mlh1 mutant mice, a neoplastic effect which persisted in heterozygous compound Mlh1(+/-)Apc(1638N/+) mutant mice; (iii) sulindac increased tumors in the cecum of all mice regardless of genetic background; (iv) sulindac decreased inflammation in the small intestine of Apc(1638N/+) mice, but it increased inflammation in the small intestine of Mlh1(+/-) mice and Mlh1(+/-)Apc(1638N/+) mice and (v) sulindac enhanced inflammation in the cecum of all mutant mice. Findings indicate that the effects of sulindac in the intestine of these mutant mouse models are probably related to genetic background and appear to be associated with its inflammatory-inducing response.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/genética , Proteína de la Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Neoplasias Colorrectales Hereditarias sin Poliposis , Mutación , Proteínas Nucleares/genética , Sulindac/efectos adversos , Sulindac/farmacología , Poliposis Adenomatosa del Colon/inducido químicamente , Poliposis Adenomatosa del Colon/tratamiento farmacológico , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Antineoplásicos/uso terapéutico , Ciego/efectos de los fármacos , Ciego/patología , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/inducido químicamente , Neoplasias Colorrectales Hereditarias sin Poliposis/tratamiento farmacológico , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Modelos Animales de Enfermedad , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Intestino Delgado/efectos de los fármacos , Intestino Delgado/patología , Ratones , Homólogo 1 de la Proteína MutL , Sulindac/uso terapéutico

RESUMEN

Casein phosphopeptides (CPPs), originating by in vitro and/or in vivo casein digestion, are characterized by the ability to complex and solubilize calcium ions preventing their precipitation. Previous works demonstrated that CPPs improve calcium uptake by human differentiated intestinal tumor cell lines, are able to re-mineralize carious lesions in a dental enamel, and, as components of a diet, affect bone weight and calcium content in rats. The aim of the present study was to evaluate if CPPs can directly modulate bone cells activity and mineralization. Primary human osteoblast-like cells were established in culture from trabecular bone samples obtained from waste materials during orthopedic surgery. Commercial mixtures of bovine casein phosphopeptides were used. The CPP dependent intracellular calcium rises were monitored at the single cell level through fura2-fluorescence assays. Results show that CPPs: (i) stimulate calcium uptake by primary human osteoblast-like cells; (ii) increase the expression and activity of alkaline phosphatase, a marker of human osteoblast differentiation; (iii) affect the cell proliferation rate and the apoptotic level; (iv) enhance nodule formation by human SaOS-2. Taken together these results confirm the possibility that CPPs play a role as modulator of bone cell activity, probably sustained by their ability as calcium carriers. Although the exact mechanism by which CPPs act remains not completely clarified, they can be considered as potential anabolic factors for bone tissue engineering.

Asunto(s)

Calcio/metabolismo , Caseínas/metabolismo , Diferenciación Celular/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Fosfopéptidos/metabolismo , Fosfopéptidos/farmacología , Fosfatasa Alcalina/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , L-Lactato Deshidrogenasa/metabolismo , Osteoblastos/citología , Fosfopéptidos/química

RESUMEN

Both epidemiological and experimental findings have indicated that components of Western diets influence colonic tumorigenesis. Among dietary constituents, calcium and cholecalciferol have emerged as promising chemopreventive agents. We have demonstrated that a Western-style diet (WD) with low levels of calcium and cholecalciferol and high levels of (n-6) PUFA, increased the incidence of neoplasia in mouse intestine compared with a standard AIN-76A diet; models included wild-type mice and mice with targeted mutations. In the present study, adenomatous polyposis coli (Apc)(1638N/+) mice carrying a heterozygous Apc mutation were fed either an AIN-76A diet, a WD, or a WD supplemented with calcium and cholecalciferol (WD/Ca/VitD3). Diets were fed for 24 wk and effects on cellular and molecular events were assessed by performing immunohistochemistry in colonic epithelium along the crypt-to-surface continuum. Feeding WD to Apc(1638N/+) mice not only enhanced cyclin D1 expression in colonic epithelium compared with AIN-76A treatment as previously reported but also significantly increased the expression of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) concomitantly with a decrease in the proapoptotic Bcl2-associated X protein and the number of apoptotic epithelial cells. WD treatment enhanced mutant Apc-driven small intestinal carcinogenesis and also resulted in the formation of a small number of colonic adenomas (0.16 +/- 0.09; P < 0.05). By contrast, the WD/Ca/VitD3 diet reversed WD-induced growth, promoting changes in colonic epithelium. Importantly, Apc(1638N/+) mice fed the WD/Ca/VitD3 diet did not develop colonic tumors, further indicating that dietary calcium and cholecalciferol have a key role in the chemoprevention of colorectal neoplasia in this mouse model of human colon cancer.

Asunto(s)

Poliposis Adenomatosa del Colon/prevención & control , Apoptosis/efectos de los fármacos , Calcio de la Dieta/farmacología , Colecalciferol/farmacología , Ciclina D1/metabolismo , Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/patología , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Peso Corporal , Calcio de la Dieta/uso terapéutico , Pruebas de Carcinogenicidad , Colecalciferol/uso terapéutico , Colon/patología , Ciclina D1/genética , Dieta , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Mutación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Distribución Aleatoria , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

Casein phosphopeptides (CPPs) form aggregated complexes with calcium phosphate and induce Ca2+ influx into HT-29 cells that have been shown to be differentiated in culture. The relationship between the aggregation of CPPs assessed by laser light scattering and their biological effect was studied using the CPPs beta-CN(1-25)4P and alpha(s1)-CN(59-79)5P, the commercial mixture CPP DMV, the 'cluster sequence' pentapeptide, typical of CPPs, and dephosphorylated beta-CN(1-25)4P, [beta-CN(1-25)0P]. The biological effect was found to be: (a) maximal with beta-CN(1-25)4P and null with the 'cluster sequence'; (b) independent of the presence of inorganic phosphate; and (c) maximal at 4 mmol.L(-1) Ca2+. The aggregation of CPP had the following features: (a) rapid occurrence; (b) maximal aggregation by beta-CN(1-25)4P with aggregates of 60 nm hydrodynamic radius; (c) need for the concomitant presence of Ca2+ and CPP for optimal aggregation; (d) lower aggregation in Ca2+-free Krebs/Ringer/Hepes; (e) formation of bigger aggregates (150 nm radius) with beta-CN(1-25)0P. With both beta-CN(1-25)4P and CPP DMV, the maximum biological activity and degree of aggregation were reached at 4 mmol.L(-1) Ca2+.

Asunto(s)

Calcio/metabolismo , Caseínas/metabolismo , Fosfopéptidos/fisiología , Secuencia de Aminoácidos , Células HT29 , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Conformación Proteica , Relación Estructura-Actividad

RESUMEN

Niemann-Pick type A is a disease characterized by the absence of a functional SMPD1 (acidic sphingomyelinase) gene and the abnormal accumulation of sphingomyelin. Under these conditions, also sphingosylphosphocholine (SPC, a sphingomyelin metabolite) accumulates in various tissues, including the brain, where it might act as a toxic stimulus, contributing to the appearance of the neurological symptoms. We studied the effects of SPC on astrocytic and neuronal cultures from rat. In particular, we investigated the possibility that SPC acts on astrocytes and that this represents the first step leading to neurodegeneration. Our results show that acute administration of SPC to astrocytes in culture promotes Ca2+ responses and a release of glutamate that, in turn, leads to cytosolic [Ca2+] elevation in neurons. We also show that chronic stimulation by SPC leads astrocytes to proliferate, but can also change their phenotype towards an activated state that might contribute to the inflammatory responses. Interestingly, upon acute SPC stimulation, activated astrocytes release more glutamate. In conclusion, we show that both chronic and acute exposure to SPC can constitute harmful signals that may have a role in the sequence of events leading to neurodegeneration.

Asunto(s)

Astrocitos/efectos de los fármacos , Síndromes de Neurotoxicidad/patología , Enfermedad de Niemann-Pick Tipo A/patología , Fosforilcolina/análogos & derivados , Esfingosina/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/patología , Colorantes Fluorescentes , Fura-2 , Ácido Glutámico/metabolismo , Hipocampo/patología , Microscopía por Video , Necrosis/patología , Degeneración Nerviosa/patología , Fenotipo , Fosforilcolina/farmacología , Ratas , Ratas Sprague-Dawley , Esfingosina/farmacología

RESUMEN

BACKGROUND: Various differentiation-inducing agents or harvesting of spontaneously late post-confluence cultures have been used to differentiate the human colon carcinoma Caco-2 cell line. We report a new procedure to generate pre-confluent subcultures of Caco-2 population at various stages of differentiation without altering culture conditions. MATERIALS AND METHODS: Ultrastructural analysis, cell proliferation activity and biochemical markers of differentiation were evaluated at different passages. RESULTS: Subcultures of Caco-2 cells at pre-confluence, exhibiting progressive acquisition of a more benign differentiation phenotype, were generated. Early passages of Caco-2 cells showed a well-developed brush border and incomplete junctional apparatus; subsequent subcultures yielded cell populations with well-developed junctions similar to those of small intestinal cells. CONCLUSION: These culture conditions represent a new versatile model not only to progressively induce the differentiation program in Caco-2 cells at pre-confluence without changes of culture media, but also to explore mechanistic modes of drug transport and tumor development.

Asunto(s)

Células CACO-2/patología , Diferenciación Celular/fisiología , Células CACO-2/enzimología , Células CACO-2/ultraestructura , Recuento de Células , Técnicas de Cultivo de Célula/métodos , Procesos de Crecimiento Celular/fisiología , Humanos , Fenotipo

RESUMEN

S1P is involved in the regulation of multiple biological processes (cell survival, growth, migration and differentiation) both in neurons and glial cells. The study was aimed at investigating the possible effects of S1P on calcium signaling in cerebellar astrocytes and differentiated granule cells. In cerebellar astrocytes S1P is able to mediate calcium signaling mainly through Gi protein coupled receptors, whereas in differentiated neurons it failed to evoke any calcium signaling, despite acting both extracellularly and intracellularly. The data indicate strict cell specificity in S1P-evoked calcium response, which could be relevant to communication between neurons and glial cells in the cerebellum.

Asunto(s)

Astrocitos/fisiología , Señalización del Calcio/efectos de los fármacos , Lisofosfolípidos/fisiología , Esfingosina/análogos & derivados , Animales , Señalización del Calcio/fisiología , Diferenciación Celular , Células Cultivadas , Cerebelo/citología , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Ratas , Esfingosina/farmacología , Esfingosina/fisiología

RESUMEN

Casein phosphopeptides beta-CN(1-25)4P and alpha(s1)-CN(59-79)5P, from beta- and alpha(s1)-casein, respectively, both carrying the characteristic 'acidic motif' Ser(P)-Ser(P)-Ser(P)-Glu-Glu, were chemically synthesized and administered to HT-29 cells differentiated in culture, which are a used model of intestinal epithelium for absorption studies. Both casein phosphopeptides caused an increase of [Ca(2+)](i) due to influx of extracellular Ca(2+). The response was quantitatively higher with beta-CN(1-25)4P than alpha(s1)-CN(59-79)5P. The synthetic peptide corresponding to the 'acidic motif' was ineffective and the dephosphorylated form of beta-CN(1-25)4P almost inactive. The lack of the N-terminally located five amino acids, or sequence modifications within the N-terminal segment of beta-CN(1-25)4P, caused a total loss of activity, whereas the lack of the C-terminal segment preserved activity. In conclusion, the influx of calcium into HT-29 cells caused by beta-CN(1-25)4P appears to depend on the phosphorylated 'acidic motif' and the preceding N-terminal region.

Asunto(s)

Calcio/metabolismo , Caseínas/química , Caseínas/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Transporte Iónico , Cinética , Datos de Secuencia Molecular , Fosfopéptidos/química , Fosfopéptidos/farmacología , Fosforilación