RESUMEN
BACKGROUND: The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts. METHODS: To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs. RESULTS: We show that tumour spheroids generate 'circular chemorepellent-induced defects' (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with â¼50% attenuation of CCID formation by treatment of cells with 10 µM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general. INTERPRETATION: These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell-cell contacts, migration, and the generation of CCID.
Asunto(s)
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Neoplasias de la Mama/patología , Carcinoma/patología , Transdiferenciación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , FN-kappa B/fisiología , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Técnicas de Cocultivo , Células Endoteliales/fisiología , Femenino , Humanos , Mesodermo/efectos de los fármacos , Mesodermo/fisiología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Invasividad Neoplásica , Nitrilos/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonas/farmacología , Células Tumorales CultivadasRESUMEN
Puromycin aminonucleoside nephrosis (PAN), a rat model of human minimal change nephropathy, is characterized by extensive flattening of glomerular epithelial cell (podocyte) foot processes and by severe proteinuria. For comparison of expression of glomerular membrane proteins of normal and PAN rats, a membrane protein fraction of isolated rat glomeruli was prepared and monoclonal antibodies were raised against it. An IgG-secreting clone designated LF3 was selected that specifically immunolabeled podocytes of normal but not of PAN rats. The antigen of LF3 IgG was identified as a 43-kd glycoprotein. Molecular cloning of its cDNA was performed in a delta gt11 expression library prepared from mRNA of isolated rat glomeruli. The predicted amino acid sequence indicated a 166-amino-acid integral membrane protein with a single membrane-spanning domain, two potential phosphorylation sites in its short cytoplasmic tail, and six potential O-glycosylation sites in the large ectodomain. High amino acid sequence identities were found to membrane glycoproteins of rat lung and bone and mouse thymus epithelial cells as well as to a phorbol-ester-induced protein in a mouse osteoblast cell line and to a canine influenza C virus receptor. In PAN, expression of this 43-kd protein was selectively reduced to < 30%, as determined by quantitative immunogold electron microscopy, immunoblotting, and Northern blotting. These data provide evidence that transcription of the 43-kd transmembrane podocyte glycoprotein is specifically down-regulated in PAN. To indicate that this protein could be associated with transformation of arborized foot processes to flat feet (Latin, pes planus) we have called it podoplanin.
Asunto(s)
Regulación hacia Abajo , Glomérulos Renales/metabolismo , Glicoproteínas de Membrana/metabolismo , Nefrosis/metabolismo , Secuencia de Aminoácidos , Animales , Perros , Epitelio/química , Epitelio/metabolismo , Epitelio/ultraestructura , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Glomérulos Renales/química , Glomérulos Renales/ultraestructura , Lectinas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Peso Molecular , Nefrosis/inducido químicamente , Nefrosis/patología , Inhibidores de la Síntesis de la Proteína , Puromicina , Conejos , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoAsunto(s)
Antígenos HLA/genética , Enfermedades Linfáticas/genética , Síndrome Mucocutáneo Linfonodular/genética , Preescolar , Femenino , Antígenos HLA-B , Humanos , Lactante , Israel , Masculino , Síndrome Mucocutáneo Linfonodular/epidemiología , Síndrome Mucocutáneo Linfonodular/inmunología , FenotipoRESUMEN
Treatment of mice with griseofulvin for 8 months induced hepatocellular nodules in the liver which persist after discontinuation of griseofulvin feeding. We investigated the porphyrogenic effect of griseofulvin on these nodules and surrounding nonneoplastic liver after renewed short-term exposure of tumor-bearing mice to this agent. Griseofulvin treatment for 4 days led to marked elevation of the activity of 5-aminolevulinate synthase in peritumoral (3.8-fold) and control (6-fold) liver. The increase in enzyme activity was much less pronounced in the nodules (1.5-fold). Ferrochelatase activity was markedly decreased under the same experimental conditions in both peritumoral and control livers (to 18 and 13.5%, respectively, of the pretreatment values), but the effect was considerably smaller in nodules (to 40% of the pretreatment value). These results may explain the lack of porphyrin accumulation in tumor tissue.