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BACKGROUND AND AIMS: Endoscopic resection is accepted as standard treatment for intramucosal esophageal adenocarcinoma (EAC) that is well or moderately differentiated. Poor differentiation (PD) is judged as a risk factor for lymph node metastasis (LNM), and surgery is recommended. However, the evidence for this recommendation is weak. The aim of this study was to analyze the clinical course of patients after endoscopic resection of EAC with PD. METHODS: Patients undergoing endoscopic submucosal dissection for EAC were included from 16 German centers. Inclusion criteria were PD in the resection specimen, R0 resection, and endoscopic follow-up. Primary outcome was the metastasis rate during follow-up. Analysis was performed retrospectively in a prospectively collected database. RESULTS: Twenty-five patients with PD as single risk factor (group A) and 15 patients with PD and additional risk factors (submucosal invasion and/or lymphovascular invasion) (group B) were included. The metastasis rate was was 1 of 25 (4.0%; 95% CI, .4%-17.2%) in group A and 3 of 15 (20.0%; 95% CI, 6.0%-44.4%) in group B, respectively (P = .293). The rate of EAC-associated deaths was 1 of 25 (4%; 95% CI, .4%-17.2%) versus 3 of 15 (20%; 95% CI, 6.0%-44.4%) in group B (P = .293). The overall death rate was 7 of 25 (28.0%; 95% CI, 13.5%-47.3%) versus 3 of 15 (20%; 95% CI, 6.0%-44.4%) (P = .715). Median follow-up was 30 months (interquartile range, 15-53 months). CONCLUSIONS: During long-term follow-up, the risk of metastasis is low after endoscopic resection of mucosal EAC with PD as a single risk factor. A conservative approach seems justified in this small patient group. However, the treatment strategy must be determined on an individualized basis until further prospective data are available.
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HISTORY AND ADMISSION FINDINGS: We report on the case of an elderly patient with persisting diarrhea. Few weeks previous of admission the patient had received antibiotic therapy because of respiratory infection. On admission he seemed exsiccated and feeble. EXAMINATIONS: Macroscopic findings in colonoscopy showed proctosigmoiditis and membranous exsudations. Stool culture provided the evidence for an antibiotic-associated infection with pseudomonas aeruginosa. TREATMENT AND COURSE: The recommended oral therapy with ciprofloxacin proved to be effective. CONCLUSION: Complications with elderly patients are multimorbidity and diarrhea-induced prerenal failure. Frail patients can react strongly to antibiotic therapy with enteritis and dysbacteriosis.
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Antibacterianos/efectos adversos , Proctocolitis , Infecciones por Pseudomonas , Pseudomonas aeruginosa/aislamiento & purificación , Anciano , Antibacterianos/uso terapéutico , Humanos , Infecciones por Pseudomonas/microbiología , Infecciones del Sistema Respiratorio/tratamiento farmacológicoRESUMEN
The calcium-activated potassium channel KCa3.1 controls different cellular processes such as proliferation and volume homeostasis. We investigated the role of KCa3.1 in experimental and human liver fibrosis. KCa3.1 gene expression was investigated in healthy and injured human and rodent liver. Effect of genetic depletion and pharmacological inhibition of KCa3.1 was evaluated in mice during carbon tetrachloride induced hepatic fibrogenesis. Transcription, protein expression and localisation of KCa3.1 was analysed by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry. Hemodynamic effects of KCa3.1 inhibition were investigated in bile duct-ligated and carbon tetrachloride intoxicated rats. In vitro experiments were performed in rat hepatic stellate cells and hepatocytes. KCa3.1 expression was increased in rodent and human liver fibrosis and was predominantly observed in the hepatocytes. Inhibition of KCa3.1 aggravated liver fibrosis during carbon tetrachloride challenge but did not change hemodynamic parameters in portal hypertensive rats. In vitro, KCa3.1 inhibition leads to increased hepatocyte apoptosis and DNA damage, whereas proliferation of hepatic stellate cells was stimulated by KCa3.1 inhibition. Our data identifies KCa3.1 channels as important modulators in hepatocellular homeostasis. In contrast to previous studies in vitro and other tissues this channel appears to be anti-fibrotic and protective during liver injury.
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Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Adulto , Anciano , Animales , Apoptosis , Células Cultivadas , Femenino , Células Estrelladas Hepáticas/fisiología , Hepatocitos/fisiología , Humanos , Hígado/patología , Cirrosis Hepática/patología , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
BACKGROUND/AIMS: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC) can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. METHODS: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. RESULTS: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC)-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced ß1-integrin-dependent cyclic AMP (cAMP) signal with induction of the dual specificity mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1), which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4) and c-jun-NH2-terminal kinase (JNK) activation. Furthermore, TUDC induced a protein kinase A (PKA)-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a ß1-integrin-and PKA-dependent manner. In line with this, the ß1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp)-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. CONCLUSION: TUDC exerts its anti-apoptotic effect via a ß1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.
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Apoptosis/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Ácido Glicoquenodesoxicólico/antagonistas & inhibidores , Hepatocitos/efectos de los fármacos , Integrina beta1/genética , Ácido Tauroquenodesoxicólico/farmacología , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Ácido Glicoquenodesoxicólico/toxicidad , Hepatocitos/citología , Hepatocitos/metabolismo , Integrina beta1/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Masculino , Técnicas de Cultivo de Órganos , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Fosforilación , Cultivo Primario de Células , Flujo Pulsátil , Ratas , Ratas Wistar , Transducción de Señal , Simportadores/genética , Simportadores/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Insulin is known to induce hepatocyte swelling, which triggers via integrins and c-Src kinase an activation of the epidermal growth factor receptor (EGFR) and subsequent cell proliferation (1). Free fatty acids (FFAs) are known to induce lipoapoptosis in liver cells in a c-Jun-NH2-terminal kinase (JNK)-dependent, but death receptor-independent way (2). As non-alcoholic steatohepatitis (NASH) is associated with hyperinsulinemia and increased FFA-blood levels, the interplay between insulin and FFA was studied with regard to hepatocyte proliferation and apoptosis in isolated rat and mouse hepatocytes. Saturated long chain FFAs induced apoptosis and JNK activation in primary rat hepatocytes, but did not activate the CD95 (Fas, APO-1) system, whereas insulin triggered EGFR activation and hepatocyte proliferation. Coadministration of insulin and FFAs, however, abolished hepatocyte proliferation and triggered CD95-dependent apoptosis due to a JNK-dependent association of the activated EGFR with CD95, subsequent CD95 tyrosine phosphorylation and formation of the death-inducing signaling complex (DISC). JNK inhibition restored the proliferative insulin effect in presence of FFAs and prevented EGFR/CD95 association, CD95 tyrosine phosphorylation and DISC formation. Likewise, in presence of FFAs insulin increased apoptosis in hepatocytes from wild type but not from Alb-Cre-FAS(fl/fl) mice, which lack functional CD95. It is concluded that FFAs can shift insulin-induced hepatocyte proliferation toward hepatocyte apoptosis by triggering a JNK signal, which allows activated EGFR to associate with CD95 and to trigger CD95-dependent apoptosis. Such phenomena may contribute to the pathogenesis of NASH.
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Apoptosis/efectos de los fármacos , Ácidos Grasos no Esterificados/farmacología , Hepatocitos/patología , Hipoglucemiantes/farmacología , Insulina/farmacología , Receptor fas/metabolismo , Animales , Western Blotting , Membrana Celular/metabolismo , Proliferación Celular , Células Cultivadas , Receptores ErbB/metabolismo , Técnica del Anticuerpo Fluorescente , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Inmunoprecipitación , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The Src family kinases Yes, Fyn, and c-Src play a pivotal role in regulating diverse liver functions such as bile flow, proteolysis, apoptosis, and proliferation and are regulated by anisoosmotic cell volume changes, death receptor ligands, and bile acids. For example, cell swelling leads to an integrin-sensed and focal adhesion kinase-mediated activation of c-Src-triggering choleresis, proteolysis inhibition, regulatory volume decrease via p38MAPK and proliferation via the activation of the epidermal growth factor receptor and extracellular regulated kinases 1 and 2. In contrast, hepatocyte shrinkage generates an almost instantaneous oxidative stress response that triggers the activation of c-Jun N-terminal kinase and the Src family kinases Fyn and Yes. Whereas Fyn activation mediates cholestasis, Yes triggers CD95 activation and apoptosis. This review will discuss the role of Src family kinases in the regulation of liver function with emphasis on their role in osmo-signaling and bile acid signaling.
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Hígado/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Proteínas Proto-Oncogénicas c-yes/metabolismo , Familia-src Quinasas/metabolismo , Animales , Apoptosis , Ácidos y Sales Biliares/metabolismo , Proteína Tirosina Quinasa CSK , Proliferación Celular , Tamaño de la Célula , Humanos , Hígado/citología , Osmorregulación , Transducción de SeñalRESUMEN
We studied the downregulation of hepatobiliary transport systems and the effect of pharmacological heme oxygenase-1 (HO-1) preinduction by Hemoglobin-Glutamer 200 (HbG200) in cold ischemia-reperfused rat liver (I/R). Cold I/R reduced bile flow in the reperfusion period from 3.10±0.10 ml/3 h to 0.54±0.20 ml/3 h (p<0.05) and biliary taurocholate excretion from 45.9±13.81 µmol/3 h to 1.87±0.46 µmol/3 h (p<0.05). Mrp2, Bsep and Ntcp peak immunofluorescence in pericentral hepatocytes decreased to 79.0±2.6% (p<0.001), 80.6±8.4% (p<0.05) and 65.8±5.0% (p<0.01), respectively. Pre-induction of HO-1 by HbG200 was largely confined to pericentral hepatocytes. HO-1 induction attenuated the decreased bile flow (0.91±0.16 ml/3 h, p<0.05) and canalicular taurocholate secretion (4.33±1.71 µmol/3 h, p<0.05). Bsep and Mrp2 peak immunofluorescence in pericentral hepatocytes was largely restored. Activation of JNK and Fyn by cold I/R was significantly attenuated by HO-1. Inhibiting HO activity by tin protoporphyrin IX after HbG200 administration reversed the effect on bile flow and canalicular transporter expression. In conclusion, pericentral downregulation of Bsep and Mrp2 following cold I/R is ameliorated by inducing HO-1 and was associated with diminished hepatocellular JNK and Fyn signaling. HO-1 may serve as a therapeutic target to attenuate hepatocellular cholestasis following I/R injury.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Bilis/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemoglobinas/metabolismo , Isquemia/metabolismo , Daño por Reperfusión/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Isquemia/enzimología , Masculino , Ratas , Ratas Wistar , Daño por Reperfusión/enzimologíaRESUMEN
UNLABELLED: Ursodeoxycholic acid, which in vivo is converted to its taurine conjugate tauroursodeoxycholic acid (TUDC), is a mainstay for the treatment of cholestatic liver disease. Earlier work showed that TUDC exerts its choleretic properties in the perfused rat liver in an α5 ß1 integrin-mediated way. However, the molecular basis of TUDC-sensing in the liver is unknown. We herein show that TUDC (20 µmol/L) induces in perfused rat liver and human HepG2 cells the rapid appearance of the active conformation of the ß1 subunit of α5 ß1 integrins, followed by an activating phosphorylation of extracellular signal-regulated kinases. TUDC-induced kinase activation was no longer observed after ß1 integrin knockdown in isolated rat hepatocytes or in the presence of an integrin-antagonistic hexapeptide in perfused rat liver. TUDC-induced ß1 integrin activation occurred predominantly inside the hepatocyte and required TUDC uptake by way of the Na(+) /taurocholate cotransporting peptide. Molecular dynamics simulations of a 3D model of α5 ß1 integrin with TUDC bound revealed significant conformational changes within the head region that have been linked to integrin activation before. CONCLUSIONS: TUDC can directly activate intrahepatocytic ß1 integrins, which trigger signal transduction pathways toward choleresis. (HEPATOLOGY 2013).
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Hepatocitos/metabolismo , Integrina alfa5beta1/metabolismo , Ácido Tauroquenodesoxicólico/metabolismo , Regulación Alostérica/fisiología , Animales , Dimerización , Proteínas Fluorescentes Verdes/genética , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Integrina alfa5beta1/química , Integrina alfa5beta1/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Relación Estructura-Actividad , Simportadores/genética , Simportadores/metabolismo , Ácido Tauroquenodesoxicólico/farmacología , Ácido Taurocólico/metabolismo , Ácido Taurocólico/farmacología , Ácido Ursodesoxicólico/farmacocinéticaRESUMEN
Hepatocyte volume regulation has been shown to play an important role in cellular metabolism, proliferation, viability and especially in hepatic functions such as bile formation and proteolysis. Recent studies on liver explants led to the assumption that cell volume changes present a trigger for outside-in signaling via integrins, a protein family involved in mediating cellular response to binding to the extracellular matrix (ECM). However, it remains elusive how these volume change related signaling events are transducted on a single cell level and how these events are influenced and controlled by ECM interactions. One could speculate that an increase in cell volume leads to an increase in integrin/ECM contacts which causes activation of integrins, which act as mechano-sensors. In order to test this idea, it was an important issue to quantify the cell volume-dependence of the contact areas between the cell and the surrounding ECM. In this study we used two wavelength reflection interference contrast microscopy (DW-RICM) to directly observe the dynamics of cell-substrate contacts, mimicking cell-ECM interactions, in response to a controlled and well-defined volume change induced by hypoosmotic stimulation. This is the first time a non-invasive, label-free method is used to uncover a volume change related response of in vitro hepatocytes in real time. The cell cluster analysis we present here agrees well with previous studies on ex vivo whole liver explants. Moreover, we show that the increase in contact area after cell swelling is a reversible process, while the reorganisation of contacts depends on the type of ECM molecules presented to the cells. As our method complements common whole liver studies providing additional insight on a cell cluster level, we expect this technique to be particular suitable for further detailed studies of osmotic stimulation not only in hepatocytes, but also other cell types.
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Apoptosis/fisiología , Matriz Extracelular/metabolismo , Hepatocitos/efectos de los fármacos , Soluciones Hipotónicas/farmacología , Microscopía de Interferencia/métodos , Receptor fas/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Presión Osmótica , Ratas , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacosRESUMEN
Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation.
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Apoptosis , Receptores ErbB/metabolismo , Hígado/citología , Hígado/fisiología , Regeneración , Receptor fas/metabolismo , Animales , Proliferación Celular , Células Hep G2 , Humanos , Hígado/enzimología , Hígado/metabolismoRESUMEN
Changes in mammalian cell volume as induced by either anisoosmolarity, hormones, nutrients or oxidative stress critically contribute to the regulation of metabolism, membrane transport, gene expression and the susceptibility to cellular stress. Osmosensing, i.e. the registration of cell volume changes, triggers signal transduction pathways towards effector pathways (osmosignaling) which link alterations of cell volume to changes in cell function. This review summarizes our own work on the understanding of how osmosensing and osmosignaling integrate into the overall context of bile acid transport, growth factor signaling and the execution of apoptotic programs.
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Apoptosis , Ácidos y Sales Biliares/metabolismo , Hígado/citología , Hígado/metabolismo , Animales , Transporte Biológico , Tamaño de la Célula , Receptores ErbB/metabolismo , Receptores ErbB/fisiología , Integrinas/metabolismo , Integrinas/fisiología , Ósmosis , Transducción de Señal , Receptor fas/metabolismo , Receptor fas/fisiologíaRESUMEN
In perfused rat liver, hyperosmolarity induces Mrp2- (Kubitz, R., D'urso, D., Keppler, D., and Häussinger, D. (1997) Gastroenterology 113, 1438-1442) and Bsep retrieval (Schmitt, M., Kubitz, R., Lizun, S., Wettstein, M., and Häussinger, D. (2001) Hepatology 33, 509-518) from the canalicular membrane leading to cholestasis. The aim of this study was to elucidate the underlying signaling events. Hyperosmolarity-induced retrieval of Mrp2 and Bsep from the canalicular membrane in perfused rat liver was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes but not of c-Src. Both hyperosmotic transporter retrieval and Src kinase activation were sensitive to apocynin (300 µmol/liter), N-acetylcysteine (NAC; 10 mmol/liter), and SU6656 (1 µmol/liter). Also PP-2 (250 nmol/liter), which inhibited hyperosmotic Fyn but not Yes activation, prevented hyperosmotic transporter retrieval from the canalicular membrane, suggesting that Fyn but not Yes mediates hyperosmotic Bsep and Mrp2 retrieval. Neither hyperosmotic Fyn activation nor Bsep/Mrp2 retrieval was observed in livers from p47(phox) knock-out mice. Hyperosmotic activation of JNKs was sensitive to apocynin and NAC but insensitive to SU6656 and PP-2, indicating that JNKs are not involved in transporter retrieval, as also evidenced by experiments using the JNK inhibitors L-JNKI-1 and SP6001255, respectively. Hyperosmotic transporter retrieval was accompanied by a NAC and Fyn knockdown-sensitive inhibition of biliary excretion of the glutathione conjugate of 1-chloro-2,4-dinitrobenzene in perfused rat liver and of cholyl-L-lysyl-fluorescein secretion into the pseudocanaliculi formed by hepatocyte couplets. Hyperosmolarity triggered an association between Fyn and cortactin and increased the amount of phosphorylated cortactin underneath the canalicular membrane. It is concluded that the hyperosmotic cholestasis is triggered by a NADPH oxidase-driven reactive oxygen species formation that mediates Fyn-dependent retrieval of the Mrp2 and Bsep from the canalicular membrane, which may involve an increased cortactin phosphorylation.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Canalículos Biliares/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Acetilcisteína/farmacología , Animales , Canalículos Biliares/patología , Colestasis/genética , Colestasis/metabolismo , Colestasis/patología , Cortactina/genética , Cortactina/metabolismo , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Hepatocitos/metabolismo , Hepatocitos/patología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Noqueados , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Presión Osmótica/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/genética , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fyn/genética , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95; the present studies have determined how sorafenib and vorinostat individually contribute to CD95 activation. Sorafenib (3-6 micromol/L) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib-induced (3 micromol/L) CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 micromol/L), or sorafenib (3 micromol/L) and vorinostat (500 nmol/L) treatment promoted higher levels of CD95 phosphorylation which correlated with DISC formation, receptor surface localization, and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to localize plasma membrane or induce autophagy. Knockdown/knockout of Src family kinases abolished sorafenib-induced CD95 tyrosine phosphorylation, DISC formation, and the induction of cell death and autophagy. Knockdown of platelet-ived growth factor receptor-beta enhanced Src Y416 and CD95 tyrosine phosphorylation, which correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased reactive oxygen species levels, and in a delayed NF kappa B-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95; however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by approximately 50%. Thus, sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation, whereas vorinostat contributes to CD95 activation via the initial facilitation of reactive oxygen species generation and subsequently of FAS-L expression.
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Autofagia/efectos de los fármacos , Bencenosulfonatos/farmacología , Neoplasias Gastrointestinales/enzimología , Neoplasias Gastrointestinales/patología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Receptor fas/metabolismo , Familia-src Quinasas/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Fosfotirosina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Sorafenib , VorinostatRESUMEN
High concentrations of urea were shown to induce a paradoxical regulatory volume decrease response with K(+) channel opening and subsequent hepatocyte shrinkage (Hallbrucker, C., vom Dahl, S., Ritter, M., Lang, F., and Häussinger, D. (1994) Pflügers Arch. 428, 552-560), although the hepatocyte plasma membrane is thought to be freely permeable to urea. The underlying mechanisms remained unclear. As shown in the present study, urea (100 mmol/liter) induced within 1 min an activation of ß(1) integrins followed by an activation of focal adhesion kinase, c-Src, p38(MAPK), extracellular signal-regulated kinases, and c-Jun N-terminal kinase. Because α(5)ß(1) integrin is known to act as a volume/osmosensor in hepatocytes, which becomes activated in response to hepatocyte swelling, the findings suggest that urea at high concentrations induces a nonosmotic activating perturbation of this osmosensor, thereby triggering a volume regulatory K(+) efflux. In line with this, similar to hypo-osmotic hepatocyte swelling, urea induced an inhibition of hepatic proteolysis, which was sensitive to p38(MAPK) inhibition. Molecular dynamics simulations of a three-dimensional model of the ectodomain of α(5)ß(1) integrin in water, urea, or thiourea solutions revealed significant conformational changes of α(5)ß(1) integrin in urea and thiourea solutions, in contrast to the simulation of α(5)ß(1) in water. These changes lead to an unbending of the integrin structure around the genu, which may suggest activation, whereas the structures of single domains remained essentially unchanged. It is concluded that urea at high concentrations affects hepatic metabolism through direct activation of the α(5)ß(1) integrin system.
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Integrina alfa5beta1/metabolismo , Hígado/metabolismo , Urea/farmacología , Animales , Western Blotting , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Técnicas In Vitro , Integrina alfa5beta1/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/efectos de los fármacos , Masculino , Modelos Moleculares , Simulación de Dinámica Molecular , Perfusión , Multimerización de Proteína , Estructura Secundaria de Proteína , Ratas , Ratas Wistar , Tiourea/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study, we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat, but not the individual drugs, rapidly increased reactive oxygen species (ROS), Ca(2+), and ceramide levels in gastrointestinal tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation, and cell killing was also blocked by quenching of induced Ca(2+) levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS; however, combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca(2+). Quenching of ROS did not affect drug-induced ceramide/dihydro-ceramide levels, whereas quenching of Ca(2+) reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knockdown of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings show that induction of cytosolic Ca(2+) by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Bencenosulfonatos/farmacología , Calcio/metabolismo , Neoplasias Gastrointestinales/tratamiento farmacológico , Ácidos Hidroxámicos/farmacología , Piridinas/farmacología , Receptor fas/metabolismo , Animales , Bencenosulfonatos/administración & dosificación , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Hidrolasas de Éster Carboxílico/metabolismo , Línea Celular Tumoral , Ceramidas/metabolismo , Neoplasias Gastrointestinales/metabolismo , Humanos , Ácidos Hidroxámicos/administración & dosificación , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Piridinas/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Sorafenib , VorinostatRESUMEN
The aim of the study was to analyze whether the proliferative effects of insulin in rat liver involve cross-signaling toward the epidermal growth factor receptor (EGFR) and whether this is mediated by insulin-induced hepatocyte swelling. Studies were performed in the perfused rat liver and in primary rat hepatocytes. Insulin (35 nmol/liter) induced phosphorylation of the EGFR at position Tyr(845) and Tyr(1173), but not at Tyr(1045), suggesting that EGF is not involved in insulin-induced EGFR activation. Insulin-induced EGFR phosphorylation and subsequent ERK1/2 phosphorylation were sensitive to bumetanide, indicating an involvement of insulin-induced hepatocyte swelling. In line with this, hypoosmotic (225 mosmol/liter) hepatocyte swelling also induced EGFR and ERK1/2 activation. Insulin- and hypoosmolarity-induced EGFR activation were sensitive to inhibition by an integrin-antagonistic RGD peptide, an integrin beta1 subtype-blocking antibody, and the c-Src inhibitor PP-2, indicating the involvement of the recently described integrin-dependent osmosensing/signaling pathway (Schliess, F., Reissmann, R., Reinehr, R., vom Dahl, S., and Häussinger, D. (2004) J. Biol. Chem. 279, 21294-21301). As shown by immunoprecipitation studies, insulin and hypoosmolarity induced a rapid, RGD peptide-, integrin beta1-blocking antibody and PP-2-sensitive association of c-Src with the EGFR. As for control, insulin-induced insulin receptor substrate-1 phosphorylation remained unaffected by the RGD peptide, PP-2, or inhibition of the EGFR tyrosine kinase activity by AG1478. Both insulin and hypoosmolarity induced a significant increase in BrdU uptake in primary rat hepatocytes, which was sensitive to RGD peptide-, integrin beta1-blocking antibody, PP-2, AG1478, and PD098059. It is concluded that insulin- or hypoosmolarity-induced hepatocyte swelling triggers an integrin- and c-Src kinase-dependent EGFR activation, which may explain the proliferative effects of insulin.
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Receptores ErbB/metabolismo , Hepatocitos/efectos de los fármacos , Insulina/farmacología , Hígado/metabolismo , Animales , Proteína Tirosina Quinasa CSK , Aumento de la Célula/efectos de los fármacos , Proliferación Celular , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatomegalia/inducido químicamente , Hepatomegalia/etiología , Integrinas/metabolismo , Hígado/patología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Oligopéptidos/farmacología , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Ratas , Familia-src QuinasasRESUMEN
The present studies determine in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin heat shock protein 90 inhibitors and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17-allylamino-17-demethoxygeldanamycin (17AAG) toxicity that was suppressed in cells deleted for mutant active RAS that were nontumorigenic but was magnified in isogenic tumorigenic cells expressing Harvey RAS V12 or Kirsten RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca(2+) levels and reduced GRP78/BiP expression in a Ca(2+)-dependent manner. GRP78/BiP overexpression, however, also suppressed drug-induced intracellular Ca(2+) levels. MEK1/2 inhibitor and 17AAG treatment increased reactive oxygen species (ROS) levels that were blocked by quenching Ca(2+) or overexpression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca(2+) quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced, which correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca(2+) levels. Thus, treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca(2+) and loss of GRP78/BiP function, leading to de novo ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation.
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Apoptosis/efectos de los fármacos , Benzoquinonas/farmacología , Calcio/farmacología , Carcinoma/patología , Neoplasias Gastrointestinales/patología , Proteínas de Choque Térmico/fisiología , Lactamas Macrocíclicas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzoquinonas/administración & dosificación , Calcio/metabolismo , Carcinoma/metabolismo , Ceramidas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Chaperón BiP del Retículo Endoplásmico , Neoplasias Gastrointestinales/metabolismo , Células HCT116 , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Lactamas Macrocíclicas/administración & dosificación , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Recent evidence suggests that epidermal growth factor receptor (EGFR)-dependent signaling contributes to liver cell proliferation, as well as to apoptotic liver cell death, and represents an important regulator of hepatic regeneration. This article summarizes recent findings on the molecular mechanisms involved in EGFR-mediated cell proliferation and apoptosis. The emphasis is on the interplay between EGFR and CD95 (Fas, APO-1) death receptor-signaling, which is determined by the signaling context and liver cell type investigated.