RESUMEN
BACKGROUND/AIMS: We measured aspartyl (asparaginyl)-beta-hydroxylase (AAH) gene expression in human hepatocelluar carcinoma and surrounding uninvolved liver at both the mRNA and protein level and examined the regulation and function of this enzyme. METHODS: Since growth of HCC is mediated by signaling through the insulin-receptor substrate, type 1 (IRS-1), we examined-if AAH is a downstream gene regulated by insulin and IGF-1 in HCC cells. In addition, IRS-1 regulation of AAH was examined in a transgenic (Tg) mouse model in which the human (h) IRS-1 gene was over-expressed in the liver, and an in vitro model in which a C-terminus truncated dominant-negative hIRS-1 cDNA (hIRS-DeltaC) was over-expressed in FOCUS HCC cells. The direct effects of AAH on motility and invasiveness were examined in AAH-transfected HepG2 cells. RESULTS: Insulin and IGF-1 stimulation increased AAH mRNA and protein expression and motility in FOCUS and Hep-G2 cells. These effects were mediated by signaling through the Erk MAPK and PI3 kinase-Akt pathways. Over-expression of hIRS-1 resulted in high levels of AAH in Tg mouse livers, while over-expression of hIRS-DeltaC reduced AAH expression, motility, and invasiveness in FOCUS cells. Finally, over-expression of AAH significantly increased motility and invasiveness in HepG2 cells, whereas siRNA inhibition of AAH expression significantly reduced directional motility in FOCUS cells. CONCLUSIONS: The results suggest that enhanced AAH gene activity is a common feature of human HCC and growth factor signaling through IRS-1 regulates AAH expression and increases motility and invasion of HCC cells. Therefore, AAH may represent an important target for regulating tumor growth in vivo.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundario , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Oxigenasas de Función Mixta/metabolismo , Animales , Biopsia , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Factor de Crecimiento Epidérmico/farmacología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoglucemiantes/farmacología , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Ratones Transgénicos , Oxigenasas de Función Mixta/genética , Invasividad Neoplásica , Fosfoproteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaAsunto(s)
Absceso/etiología , Diverticulitis del Colon/complicaciones , Fondo de Saco Recto-Uterino , Infecciones por Escherichia coli/etiología , Absceso Hepático/complicaciones , Infecciones por Proteus/etiología , Enfermedades del Sigmoide/complicaciones , Absceso/terapia , Antibacterianos/uso terapéutico , Colon Sigmoide/cirugía , Diverticulitis del Colon/diagnóstico , Diverticulitis del Colon/cirugía , Drenaje/métodos , Infecciones por Escherichia coli/terapia , Humanos , Absceso Hepático/terapia , Masculino , Persona de Mediana Edad , Enfermedades Peritoneales/diagnóstico , Enfermedades Peritoneales/etiología , Enfermedades Peritoneales/terapia , Infecciones por Proteus/terapia , Enfermedades del Sigmoide/diagnóstico , Enfermedades del Sigmoide/cirugía , Ultrasonografía IntervencionalRESUMEN
BACKGROUND/AIMS: Insulin- and insulin growth factor-1 stimulated signaling through the insulin receptor substrate-1 (IRS-1) promotes hepatocellular proliferation and survival. IRS-1 over-expression in transgenic (Tg) mouse livers caused constitutive activation of Erk mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) resulting in significantly increased levels of DNA synthesis and larger hepatic masses relative to non-transgenic (non-Tg) littermates. However, the livers eventually ceased to grow but remained approximately 25% larger than non-Tg livers. We hypothesized that this growth homeostasis was achieved by parallel activation of pro-apoptosis pathways. METHODS: Since Fas-mediated apoptosis is a common mechanism of hepatocyte destruction, we investigated the potential role of Fas receptor as a regulator of hepatic mass in IRS-1 transgenic mice. RESULTS: Significantly increased Fas-receptor levels were detected in the livers of IRS-1 Tg compared to non-Tg mice by Western blot analysis. Functional activation of Fas-receptor in IRS-1 Tg livers was demonstrated by increased hepatocellular apoptosis caused by intravenous injection of anti-Fas (Jo-2). CONCLUSIONS: These findings suggest that the increased growth caused by IRS-1 over-expression is balanced by constitutive activation of pro-death mechanisms. Failure of the IRS-1 Tg mice to develop liver cancer may be due to preservation of pro-growth, pro-death homeostasis mechanisms.
Asunto(s)
Fosfoproteínas/fisiología , Receptor fas/metabolismo , Animales , Apoptosis/genética , Expresión Génica , Humanos , Etiquetado Corte-Fin in Situ , Proteínas Sustrato del Receptor de Insulina , Hígado/patología , Hígado/fisiopatología , Ratones , Ratones Transgénicos , Fosfoproteínas/genética , Regulación hacia ArribaRESUMEN
BACKGROUND: Aspartyl (asparaginyl) beta-hydroxylase (AAH) is an alpha-ketoglutarate-dependent dioxygenase that hydroxylates aspartate and asparagine residues in EGF-like domains of proteins. The consensus sequence for AAH beta-hydroxylation occurs in signaling molecules such as Notch and Notch homologs, which have roles in cell migration. AIM: This study evaluated the potential role of AAH in cell migration using cholangiocarcinoma cell lines as models due to their tendency to widely infiltrate the liver. METHODS: Five human cholangiocarcinoma cell lines established from human tumors were examined for AAH expression and motility. The effect of antisense oligodeoxynucleotide inhibition of AAH on cholangiocarcinoma cell migration was investigated. RESULTS: Western blot analysis detected the approximately 86 kDa AAH protein in all five cholangiocarcinoma cell lines, and higher levels of AAH in cell lines derived from moderately or poorly differentiated compared with well-differentiated tumors. Immunocytochemical staining and fluorescence activated cell sorting analysis revealed both surface and intracellular AAH immunoreactivity. Using the phagokinetic non-directional migration assay and a novel ATPLite luminescence-based directional migration assay, we correlated AAH expression with motility. Correspondingly, antisense and not sense or mutated antisense AAH oligodeoxynucleotides significantly inhibited AAH expression and motility in cholangiocarcinoma cells. CONCLUSIONS: AAH over-expression may contribute to the infiltrative growth pattern of cholangiocarcinoma cells by promoting motility.