RESUMEN
Breast cancer (BC) has a poor prognosis due to its strong metastatic ability. Accumulating data present ether à go-go (hEag1) K(+) channels as relevant player in controlling cell cycle and proliferation of non-invasive BC cells. However, the role of hEag1 in invasive BC cells migration is still unknown. In this study, we studied both the functional expression and the involvement in cell migration of hEag1 in the highly metastatic MDA-MB-231 human BC cells. We showed that hEag1 mRNA and proteins were expressed in human invasive ductal carcinoma tissues and BC cell lines. Functional activity of hEag1 channels in MDA-MB-231 cells was confirmed using astemizole, a hEag1 blocker, or siRNA. Blocking or silencing hEag1 depolarized the membrane potential and reduced both Ca(2+) entry and MDA-MB-231 cell migration without affecting cell proliferation. Recent studies have reported that Ca(2+) entry through Orai1 channels is required for MDA-MB-231 cell migration. Down-regulation of hEag1 or Orai1 reduced Ca(2+) influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca(2+) influx or cell migration were observed in cells co-transfected with sihEag1 and siOrai1. Finally, both Orai1 and hEag1 are expressed in invasive breast adenocarcinoma tissues and invaded metastatic lymph node samples (LNM(+)). In conclusion, this study is the first to demonstrate that hEag1 channels are involved in the serum-induced migration of BC cells by controlling the Ca(2+) entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Movimiento Celular/fisiología , Canales de Potasio Éter-A-Go-Go/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Canales de Calcio/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Línea Celular Tumoral , Supervivencia Celular , Canales de Potasio Éter-A-Go-Go/genética , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Ganglios Linfáticos/patología , Manganeso , Invasividad Neoplásica , Proteína ORAI1 , Técnicas de Placa-Clamp , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Vascular calcification is frequent in patients with chronic kidney disease. Osteoprotegerin (OPG, a soluble factor which blocks osteoclast differentiation) has recently been implicated in the genesis of vascular calcification. Given that OPG can bind the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we hypothesized that the TRAIL protein is involved in the formation of vascular calcification both in vitro and in vivo. Using an immunohistochemical approach, we evaluated TRAIL and OPG expression on aortic valves slides from non-uremic and uremic wild type and apolipoprotein knockout (Apo E(-/-) ) mice. We also tested the in vitro effects of TRAIL on cultured primary human vascular smooth muscle cells (hVSMC). We further assayed serum soluble TRAIL (sTRAIL) levels in hemodialysis patients and correlated them with vascular calcification scores. Our results demonstrated that: (i) TRAIL and OPG were expressed inside the atheroma plaque in non-uremic ApoE(-/-) mice, but not in wild type mice; and (ii) uremia enhanced the expression levels. TRAIL enhanced the phosphate-induced mineralization of hVSMCs in a dose-dependent manner. In clinical terms, we demonstrated that sTRAIL is depressed in the sera of hemodialysis patients, but was not correlated with vascular calcification. Our results suggest that TRAIL may be involved in the formation of vascular calcification in certain experimental settings; however, its role in chronic kidney disease patients requires further evaluation.
Asunto(s)
Calcinosis/fisiopatología , Enfermedades Renales/fisiopatología , Osteoprotegerina/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Anciano , Animales , Apolipoproteínas E/genética , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedades Renales/terapia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Osteoprotegerina/sangre , Osteoprotegerina/genética , Placa Aterosclerótica/patología , Diálisis Renal/métodos , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Uremia/fisiopatologíaRESUMEN
sp(2)-Iminosugar-type castanospermine analogues bearing an alpha-configured N-, S-, or C-linked pseudoanomeric group have been designed as selective inhibitors of the neutral alpha-glucosidases involved in N-glycoprotein processing; evaluation in breast cancer cell growth indicated a significant antiproliferative potential that was dependent on the nature of the pseudoanomeric group.
Asunto(s)
Antineoplásicos/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores de Glicósido Hidrolasas , Glicósidos/química , Indolizinas/química , Monosacáridos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Indolizinas/farmacología , Estructura MolecularRESUMEN
BACKGROUND: Chronic kidney disease (CKD) is associated with accelerated atherosclerosis and an inadequate inflammatory response which may account for the high morbidity and mortality observed in this population. In vitro and preclinical evidence suggests that the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) might be involved in both the atherosclerosis pathway and modulation of the inflammatory response. The aim of the present study was thus to (i) determine serum levels of soluble TRAIL (sTRAIL) in a cohort of CKD patients, (ii) assess the relationship between sTRAIL and other inflammatory biomarkers (C-reactive protein and albumin) and (iii) evaluate the association between serum sTRAIL levels and the mortality risk. METHODS: One hundred and thirty patients (mean +/- SD age: 67 +/- 12; 62% males; 8% at CKD stage 2, 26% at stage 3, 27% at stage 4, 8% at stage 5 and 31% at stage 5D) were assayed for sTRAIL and the selected biochemical parameters and then prospectively monitored for mortality. RESULTS: CKD stage 5D patients had significantly lower serum sTRAIL levels (median: 46 pg/ml) than patients at CKD stages 2 and 3 (median: 62 pg/ml) or stages 4 and 5 (median: 71 pg/ml). There was no correlation between serum sTRAIL and the estimated glomerular filtration rate (GFR) (r(2) = 0.017, P = 0.22) in pre-dialysis patients. In a multivariate regression analysis, the body mass index (beta = 1.48, P = 0.001) and the serum C-reactive protein (CRP) level (beta = -8.841, P < 0.0001) were independently associated with serum sTRAIL. During follow-up (mean: 772 +/- 286 days), 36 patients died (19 from cardiovascular events, 8 from infectious events and 9 from other causes). The lowest sTRAIL levels (first tertile) were associated with the worst all-cause survival (P = 0.010). Cox regression analyses (with non-cumulative models including age, albumin and CRP as covariates) confirmed the low serum sTRAIL level (first tertile) as an independent predictor of all-cause mortality. CONCLUSIONS: Circulating sTRAIL is a negative marker for inflammation and is inversely associated with the mortality risk in CKD patients. Further studies are needed to better understand the role of sTRAIL as an inflammatory marker and to confirm its protective role in the CKD population.
Asunto(s)
Inflamación/sangre , Enfermedades Renales/sangre , Enfermedades Renales/mortalidad , Ligando Inductor de Apoptosis Relacionado con TNF/sangre , Anciano , Biomarcadores/sangre , Proteína C-Reactiva/metabolismo , Enfermedad Crónica , Estudios de Cohortes , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Estudios Retrospectivos , Factores de Riesgo , Albúmina Sérica/metabolismo , Tasa de SupervivenciaRESUMEN
In this work, we investigated the effect of inorganic phosphate (Pi) on the differentiation of monocyte/macrophage precursors into an "osteoclastic" phenotype, and we delineated the molecular mechanisms which could be involved in this phenomenon. This was achieved by stimulating human peripheral blood monocytic cells and RAW 264.7 monocyte-macrophage precursor cells to differentiate into osteoclast-like cells in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). RANKL has been previously reported to stimulate the signaling kinases ERK 1/2, p38, Akt, JNK, and the DNA-binding activity of the transcription factors AP-1 and NF-kappaB. Increase in extracellular Pi concentration (1.5-4.5 mM) dose-dependently inhibits both osteoclastic differentiation and bone resorption activity induced by RANKL and M-CSF. Pi was found to specifically inhibit the RANKL-induced JNK and Akt activation, while RANKL-induced p38 and ERK 1/2 phosphorylation were not significantly affected. Moreover, we found that Pi significantly reduced the RANKL-stimulated DNA-binding activity of NF-kappaB. The effects of Pi on osteoclast differentiation and DNA-binding activity of NF-kappaB were prevented by Foscarnet, a sodium-phosphate cotransport inhibitor, suggesting that the effects of Pi occur subsequently to its intake. These results demonstrate that Pi downregulates the differentiation of osteoclasts via a negative feedback exerted on RANK-RANKL signaling.
Asunto(s)
Espacio Extracelular/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Fosfatos/farmacología , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Fosfatasa Ácida/metabolismo , Animales , Resorción Ósea , Diferenciación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Foscarnet/farmacología , Humanos , Isoenzimas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Osteoclastos/citología , Osteoclastos/enzimología , Osteogénesis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ligando RANK/farmacología , Proteínas Cotransportadoras de Sodio-Fosfato/antagonistas & inhibidores , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Fosfatasa Ácida Tartratorresistente , Factor de Transcripción AP-1/metabolismoRESUMEN
BACKGROUND: In chronic kidney disease (CKD) patients, the intake of calcium-based phosphate binders is associated with a marked progression of coronary artery and aortic calcification, in contrast to patients receiving calcium-free phosphate binders. The aim of this study was to reexamine the role of calcium carbonate in vascular calcification and to analyse its effect on aortic calcification-related gene expression in chronic renal failure (CRF). METHODS: Mice deficient in apolipoprotein E underwent either sham operation or subtotal nephrectomy to create CRF. They were then randomly assigned to one of the three following groups: a control non-CRF group and a CRF group fed on standard diet, and a CRF group fed on calcium carbonate enriched diet, for a period of 8 weeks. Aortic atherosclerotic plaque and calcification were evaluated using quantitative morphologic image processing. Aortic gene and protein expression was examined using immunohistochemistry and Q-PCR methods. RESULTS: Calcium carbonate supplementation was effective in decreasing serum phosphorus but was associated with a higher serum calcium concentration. Compared with standard diet, calcium carbonate enriched diet unexpectedly induced a significant decrease of both plaque (p<0.05) and non-plaque-associated calcification surface (p<0.05) in CRF mice. It also increased osteopontin (OPN) protein expression in atherosclerotic lesion areas of aortic root. There was also a numerical increase in OPN and osteoprotegerin gene expression in total thoracic aorta but the difference did not reach the level of significance. Finally, calcium carbonate did not change the severity of atherosclerotic lesions. CONCLUSION: In this experimental model of CRF, calcium carbonate supplementation did not accelerate but instead decreased vascular calcification. If our observation can be extrapolated to humans, it appears to question the contention that calcium carbonate supplementation, at least when given in moderate amounts, necessarily enhances vascular calcification. It is also compatible with the hypothesis of a preponderant role of phosphorus over that of calcium in promoting vascular calcification in CRF.