RESUMEN
Chronic inflammation in the stomach induces metaplasia, the pre-cancerous lesion that precedes inflammation-driven neoplastic transformation. While Hedgehog signaling contributes to the initiation of some cancers, its role in gastric transformation remains poorly defined. We found that Helicobacter-infected C57BL/6 mice develop extensive mucous cell metaplasia at 6 month but not at 2 months post-infection. Gastric metaplasia coincided with the appearance of CD45(+)MHCII(+)CD11b(+)CD11c(+) myeloid cells that were normally not present in the chronic gastritis at 2 months. The myeloid regulatory gene Schlafen-4 was identified in a microarray analysis comparing infected WT versus Gli1 null mice and was expressed in the CD11b(+)CD11c(+) myeloid population. Moreover this same population expressed IL-1ß and TNFα pro-inflammatory cytokines. By 6 months, the mucous neck cell metaplasia (SPEM) expressed IL-6, phosphorylated STAT3 and the proliferative marker Ki67. Expression was not observed in Gli1 mutant mice consistent with the requirement of Gli1 to induce this pre-neoplastic phenotype. Ectopic Shh ligand expression alone was not sufficient to induce SPEM, but with Helicobacter infection synergistically increased the histologic severity observed with the inflammation. Therefore Hedgehog signaling is required, but is not sufficient to generate pre-neoplastic changes during chronic gastritis. Gli1-dependent myeloid cell differentiation plays a pivotal role in the appearance of myeloid cell subtypes ostensibly required for SPEM development. Moreover, it suggests that therapies capable of targeting this phenotypic switch might prevent progression to metaplasia, the pre-neoplastic change that develops prior to dysplasia and gastric cancer, which also occurs in other epithelial-derived neoplasias initiated by chronic inflammation.
Asunto(s)
Gastritis/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter felis , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Mieloides/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Enfermedad Crónica , Citocinas/biosíntesis , Citocinas/genética , Gastritis/genética , Gastritis/microbiología , Gastritis/patología , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/patología , Infecciones por Helicobacter/prevención & control , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Metaplasia , Ratones , Ratones Transgénicos , Células Mieloides/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Proteína con Dedos de Zinc GLI1RESUMEN
OBJECTIVES: Normally, sonic hedgehog (Shh) is expressed in the pancreas during fetal development and transiently after tissue injury. Although pancreatic cancers express Shh, it is not known if the protein is secreted into the blood and whether its plasma levels change with pancreatic transformation. The goal of this study was to develop an enzyme-linked immunosorbent assay to detect human Shh in blood and determine its levels in subjects with and without pancreatic cancer. METHODS: A human Shh enzyme-linked immunosorbent assay was developed, and plasma Shh levels were measured in blood samples from healthy subjects and patients with pancreatitis or pancreatic cancer. The biological activity of plasma Shh was tested using NIH-3T3 cells. RESULTS: The mean levels of Shh in human blood were lower in patients with pancreatitis and pancreatic cancer than in healthy subjects. Hematopoietic cells did not express Shh, suggesting that Shh is secreted into the bloodstream. Plasma fractions enriched with Shh did not induce Gli-1 messenger RNA, suggesting that the protein was not biologically active. CONCLUSIONS: Shh is secreted from tissues and organs into the circulation, but its activity is blocked by plasma proteins. Reduced plasma levels were found in pancreatic cancer patients, but alone were not sufficient to predict pancreatic cancer.
Asunto(s)
Proteínas Hedgehog/sangre , Neoplasias Pancreáticas/sangre , Adolescente , Adulto , Anciano , Animales , Proteínas Sanguíneas/farmacología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Fibrosis , Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/farmacología , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Persona de Mediana Edad , Células 3T3 NIH , Páncreas/metabolismo , Páncreas/patología , Pancreatitis/sangre , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Proteína con Dedos de Zinc GLI1RESUMEN
ZBP-89 can enhance tumor cells to death stimuli. However, the molecular mechanism leading to the inhibitory effect of ZBP-89 is unknown. In this study, 4 liver cell lines were used to screen for the target of ZBP-89 on cell death pathway. The identified Bak was further analyzed for its role in ZBP-89-mediated apoptosis. The result showed that ZBP-89 significantly and time-dependently induced apoptosis. It significantly upregulated the level of pro-apoptotic Bak. ZBP-89 targeted a region between -457 and -407 of human Bak promoter to stimulate Bak expression based on the findings of Bak promoter luciferase report gene assay and electrophoretic mobility shift assay. ZBP-89-induced Bak increase and ZBP-89-mediated apoptosis were markedly suppressed by Bak siRNA, confirming that Bak was specifically targeted by ZBP-89 to facilitate apoptosis. In conclusion, this study demonstrated that ZBP-89 significantly induced apoptosis of HCC cells via promoting Bak level.
Asunto(s)
Apoptosis , Carcinoma Hepatocelular/patología , Proteínas de Unión al ADN/metabolismo , Neoplasias Hepáticas/patología , Factores de Transcripción/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Luciferasas/metabolismo , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genética , Factores de Transcripción/genética , Células Tumorales Cultivadas , Proteína Destructora del Antagonista Homólogo bcl-2/antagonistas & inhibidores , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismoRESUMEN
BACKGROUND & AIMS: Hypochlorhydria during Helicobacter pylori infection inhibits gastric Sonic Hedgehog (Shh) expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through intracellular calcium (Ca2(+)(i))-dependent protein kinase C (PKC) or cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation. METHODS: We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1, a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) + 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), PKC-overexpressing adenoviruses, and PKC inhibitors were used to modulate Ca(2+)(i)-release, PKC activity, and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H(+)/K(+)-ß-cholera-toxin-overexpressing mice. RESULTS: Mice that expressed secreted hedgehog-interacting protein-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression also was repressed in the hyperchlorhydric H(+)/K(+)-ß-cholera-toxin model with increased cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca(2+)(i) release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin-, and carbachol-mediated release of Ca(2+)(i) induced Shh expression. Ca(2+)-chelation with BAPTA + EGTA reduced Shh expression. Overexpression of PKC-α, -ß, and -δ (but not PKC-ϵ) induced an Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene. CONCLUSIONS: Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca(2+)(i)-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin.
Asunto(s)
Aclorhidria/metabolismo , Calcio/metabolismo , Ácido Gástrico/metabolismo , Proteínas Hedgehog/genética , Proteína Quinasa C/metabolismo , Aclorhidria/fisiopatología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Gastrinas/genética , Gastrinas/metabolismo , Regulación de la Expresión Génica/fisiología , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Proteínas Hedgehog/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal/fisiologíaRESUMEN
ZBP-89 inhibits the some tumor cells but its role in HCC is unknown. We investigated effect of ZBP-89 on cell death of 5 HCC cell lines with different status of p53. We found that ZBP-89 significantly induced cell death of all HCC cells particularly those with wild-type p53. The inhibition was well correlated with the induction of caspase-6 activity. The inhibition of caspase-6 abolished the effect of ZBP-89. ZBP-89 reduced the cells in G2-M but increased them in S phase. With the changes in caspase-6 and cell cycle, ZBP-89 greatly enhanced the killing effectiveness of 5-fluorouracil or staurosporine in HCC cells.