RESUMEN
UNLABELLED: Liver fibrosis expanding from portal tracts and vascular remodeling are determinant factors in the progression of liver diseases to cirrhosis. In the present study, we examined the potential contribution of portal myofibroblasts (PMFs) to the vascular changes leading to cirrhosis. The analyses of liver cells based on the transcriptome of rat PMFs, compared to hepatic stellate cell HSC-derived myofibroblasts in culture, identified collagen, type XV, alpha 1 (COL15A1) as a marker of PMFs. Normal liver contained rare COL15A1-immunoreactive cells adjacent to the bile ducts and canals of Hering in the portal area. A marked increase in COL15A1 expression occurred together with that of the endothelial marker, von Willebrand factor, in human and rat liver tissue, at advanced stages of fibrosis caused by either biliary or hepatocellular injury. In cirrhotic liver, COL15A1-expressing PMFs adopted a perivascular distribution outlining vascular capillaries proximal to reactive ductules, within large fibrotic septa. The effect of PMFs on endothelial cells (ECs) was evaluated by in vitro and in vivo angiogenesis assays. PMF-conditioned medium increased the migration and tubulogenesis of liver ECs as well as human umbilical vein ECs and triggered angiogenesis within Matrigel plugs in mice. In coculture, PMFs developed intercellular junctions with ECs and enhanced the formation of vascular structures. PMFs released vascular endothelial growth factor (VEGF)A-containing microparticles, which activated VEGF receptor 2 in ECs and largely mediated their proangiogenic effect. Cholangiocytes potentiated the angiogenic properties of PMFs by increasing VEGFA expression and microparticle shedding in these cells. CONCLUSION: PMFs are key cells in hepatic vascular remodeling. They signal to ECs through VEGFA-laden microparticles and act as mural cells for newly formed vessels, driving scar progression from portal tracts into the parenchyma.
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Micropartículas Derivadas de Células/metabolismo , Cirrosis Hepática/etiología , Hígado/citología , Miofibroblastos/fisiología , Remodelación Vascular , Animales , Colágeno/análisis , Humanos , Cirrosis Hepática/patología , Masculino , Neovascularización Fisiológica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Ratas , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
UNLABELLED: Microvillous inclusion disease (MVID) is a congenital disorder of the enterocyte related to mutations in the MYO5B gene, leading to intractable diarrhea often necessitating intestinal transplantation (ITx). Among our cohort of 28 MVID patients, 8 developed a cholestatic liver disease akin to progressive familial intrahepatic cholestasis (PFIC). Our aim was to investigate the mechanisms by which MYO5B mutations affect hepatic biliary function and lead to cholestasis in MVID patients. Clinical and biological features and outcome were reviewed. Pretransplant liver biopsies were analyzed by immunostaining and electron microscopy. Cholestasis occurred before (n = 5) or after (n = 3) ITx and was characterized by intermittent jaundice, intractable pruritus, increased serum bile acid (BA) levels, and normal gamma-glutamyl transpeptidase activity. Liver histology showed canalicular cholestasis, mild-to-moderate fibrosis, and ultrastructural abnormalities of bile canaliculi. Portal fibrosis progressed in 5 patients. No mutation in ABCB11/BSEP or ATP8B1/FIC1 genes were identified. Immunohistochemical studies demonstrated abnormal cytoplasmic distribution of MYO5B, RAB11A, and BSEP in hepatocytes. Interruption of enterohepatic BA cycling after partial external biliary diversion or graft removal proved the most effective to ensure long-term remission. CONCLUSION: MVID patients are at risk of developing a PFIC-like liver disease that may hamper outcome after ITx. Our results suggest that cholestasis in MVID patients results from (1) impairment of the MYO5B/RAB11A apical recycling endosome pathway in hepatocytes, (2) altered targeting of BSEP to the canalicular membrane, and (3) increased ileal BA absorption. Because cholestasis worsens after ITx, indication of a combined liver ITx should be discussed in MVID patients with severe cholestasis. Future studies will need to address more specifically the effect of MYO5B dysfunction in BA homeostasis.
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Ácidos y Sales Biliares/metabolismo , Colestasis , Síndromes de Malabsorción , Microvellosidades/patología , Mucolipidosis , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Biopsia , Preescolar , Colestasis/genética , Colestasis/metabolismo , Colestasis/patología , Diarrea Infantil/genética , Diarrea Infantil/metabolismo , Diarrea Infantil/patología , Endosomas/metabolismo , Endosomas/patología , Enterocitos/metabolismo , Enterocitos/patología , Femenino , Hepatocitos/metabolismo , Hepatocitos/patología , Heterocigoto , Homocigoto , Humanos , Lactante , Síndromes de Malabsorción/genética , Síndromes de Malabsorción/metabolismo , Síndromes de Malabsorción/patología , Masculino , Microvellosidades/genética , Microvellosidades/metabolismo , Mucolipidosis/genética , Mucolipidosis/metabolismo , Mucolipidosis/patología , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Autophagy is a regulated process that can be involved in the elimination of intracellular microorganisms and in antigen presentation. Some in vitro studies have shown an altered autophagic response in hepatitis C virus infected hepatocytes. The present study aimed at evaluating the autophagic process in the liver of chronic hepatitis C (CHC) patients. Fifty-six CHC patients and 47 control patients (8 with nonalcoholic steatohepatitis or alcoholic liver disease, 18 with chronic heptatitis B virus infection, and 21 with no or mild liver abnormalities at histological examination) were included. Autophagy was assessed by means of electron microscopy and microtubule-associated protein light chain 3 immunoblotting. Using light chain 3 immunoblotting, the form present on autophagic vesicle (light chain 3-II) was significantly higher in CHC patients than in controls (P < 0.05). Using quantitative electron microscopy analysis, the median number of autophagic vesicles observed in hepatocytes from CHC patients was sixfold higher than in overall controls (P < 0.001). In contrast, there was no difference between CHC patients and controls in the number of mature lysosomes with electron-dense contents arguing in favor of a lack of fusion between autophagosome and lysosome. Neither genotype nor viral load influenced the autophagy level. In conclusion, autophagy is altered in hepatocytes from CHC patients, likely due to a blockade of the last step of the autophagic process.
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Autofagia , Hepacivirus/fisiología , Hepatitis C Crónica/patología , Hepatitis C Crónica/virología , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Hepacivirus/genética , Hepatocitos/patología , Hepatocitos/ultraestructura , Humanos , Immunoblotting , Hígado/metabolismo , Hígado/patología , Hígado/virología , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Carga Viral , Adulto JovenRESUMEN
In hepatocytes, the accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and the unfolded protein response (UPR), mediated by the ER-resident stress sensors ATF-6, IRE1, and PERK. UPR-responsive genes are involved in the fate of ER-stressed cells. Cells carrying hepatitis C virus (HCV) subgenomic replicons exhibit in vitro ER stress and suggest that HCV inhibits the UPR. Since in vivo ER homeostasis is unknown in livers with chronic HCV infection, we investigated ER stress and the UPR in liver samples from untreated patients with chronic hepatitis C (CHC), in comparison with normal livers. Electron microscopy, western blotting, and real-time RT-PCR were used in liver biopsy specimens. Electron microscopy identified features showing ER stress in hepatocyte samples from patients with CHC; however, 'ER-stressed' hepatocytes were found in clusters (3-5 cells) that were scattered in the liver parenchyma. Western blot analysis confirmed the existence of hepatic ER stress by showing activation of the three ER stress sensors ATF-6, IRE1, and PERK in CHC. Real-time RT-PCR showed no significant induction of UPR-responsive genes in CHC. In contrast, genes involved in the control of diffuse processes such as liver proliferation, inflammation, and apoptosis were significantly induced in CHC. In conclusion, livers from patients with untreated CHC exhibit in vivo hepatocyte ER stress and activation of the three UPR sensors without apparent induction of UPR-responsive genes. This lack of gene induction may be explained by the inhibiting action of HCV per se (as suggested by in vitro studies) and/or by our finding of the localized nature of hepatocyte ER stress.
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Retículo Endoplásmico/ultraestructura , Hepatitis C Crónica/patología , Hepatocitos/ultraestructura , Factor de Transcripción Activador 6/metabolismo , Adulto , Apoptosis/genética , Proliferación Celular , Retículo Endoplásmico/metabolismo , Endorribonucleasas/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Hepatitis C Crónica/metabolismo , Hepatitis C Crónica/fisiopatología , Hepatocitos/metabolismo , Humanos , Cirrosis Hepática/patología , Cirrosis Hepática/fisiopatología , Cirrosis Hepática/virología , Masculino , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transducción de Señal/fisiología , Estrés Fisiológico/fisiología , Respuesta de Proteína Desplegada/genética , Respuesta de Proteína Desplegada/fisiología , eIF-2 Quinasa/metabolismoRESUMEN
BACKGROUND & AIMS: Acute liver insufficiency is a rare complication of anorexia nervosa. The mechanisms for this complication are unclear. The aim of this study was to describe patient characteristics and clarify the mechanisms involved. METHODS: Liver specimens from 12 patients (median age, 24 years; median body mass index, 11.3 kg/m(2)), with a prothrombin index <50% and/or an International Normalized Ratio >1.7 and anorexia nervosa as the only cause for acute liver injury were analyzed. A detailed pathologic examination was performed, including under electron microscopy. RESULTS: Liver cell glycogen depletion was a constant finding. There was a contrast between the increase in serum alanine aminotransferase (56 times normal on average; 1,904 IU/L) and the absence of significant hepatocyte necrosis on histology. Centrilobular changes (trabecular atrophy and/or sinusoidal fibrosis) were observed in 6 patients. There were rare or no (<5%) terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive hepatocytes, suggesting that apoptosis was not the primary mechanism. Hepatocytes from 4 patients showed numerous autophagosomes, a morphologic hallmark of autophagy, on electron microscopy. In contrast, the mitochondria, endoplasmic reticulum, and nuclei were normal in most cells. These features were absent in 11 control patients. The outcome was favorable in all patients, with a rapid return to normal liver function. CONCLUSIONS: Anorexia nervosa with extremely poor nutritional status should be added to the list of conditions causing acute liver insufficiency. Our findings show that starvation-induced autophagy in the human liver may be involved in liver cell death during anorexia nervosa, even though other mechanisms of liver cell damage could also play a role.
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Anorexia Nerviosa/patología , Autofagia , Insuficiencia Hepática/patología , Hepatocitos/patología , Inanición/patología , Enfermedad Aguda , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adolescente , Adulto , Anorexia Nerviosa/complicaciones , Anorexia Nerviosa/metabolismo , Anorexia Nerviosa/fisiopatología , Apoptosis , Proteínas de Arabidopsis/metabolismo , Beclina-1 , Retículo Endoplásmico/ultraestructura , Chaperón BiP del Retículo Endoplásmico , Femenino , Proteínas de Choque Térmico/metabolismo , Insuficiencia Hepática/etiología , Insuficiencia Hepática/metabolismo , Hepatocitos/metabolismo , Hepatocitos/fisiología , Hepatocitos/ultraestructura , Humanos , Etiquetado Corte-Fin in Situ , Glucógeno Hepático/metabolismo , Persona de Mediana Edad , Chaperonas Moleculares/metabolismo , Oligopéptidos/metabolismo , Señales de Clasificación de Proteína , Inanición/metabolismo , Inanición/fisiopatologíaRESUMEN
Alcohol consumption increases reactive oxygen species formation and lipid peroxidation, whose products can damage mitochondrial DNA (mtDNA) and alter mitochondrial function. A possible role of manganese superoxide dismutase (MnSOD) on these effects has not been investigated. To test whether MnSOD overexpression modulates alcohol-induced mitochondrial alterations, we added ethanol to the drinking water of transgenic MnSOD-overexpressing (TgMnSOD) mice and their wild type (WT) littermates for 7 weeks. In TgMnSOD mice, alcohol administration further increased the activity of MnSOD, but decreased cytosolic glutathione as well as cytosolic glutathione peroxidase activity and peroxisomal catalase activity. Whereas ethanol increased cytochrome P-450 2E1 and mitochondrial ROS generation in both WT and TgMnSOD mice, hepatic iron, lipid peroxidation products and respiratory complex I protein carbonyls were only increased in ethanol-treated TgMnSOD mice but not in WT mice. In ethanol-fed TgMnSOD mice, but not ethanol-fed WT mice, mtDNA was depleted, and mtDNA lesions blocked the progress of polymerases. The iron chelator, DFO prevented hepatic iron accumulation, lipid peroxidation, protein carbonyl formation and mtDNA depletion in alcohol-treated TgMnSOD mice. Alcohol markedly decreased the activities of complexes I, IV and V of the respiratory chain in TgMnSOD, with absent or lesser effects in WT mice. There was no inflammation, apoptosis or necrosis, and steatosis was similar in ethanol-treated WT and TgMnSOD mice. In conclusion, prolonged alcohol administration selectively triggers iron accumulation, lipid peroxidation, respiratory complex I protein carbonylation, mtDNA lesions blocking the progress of polymerases, mtDNA depletion and respiratory complex dysfunction in TgMnSOD mice but not in WT mice.
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Consumo de Bebidas Alcohólicas/efectos adversos , Daño del ADN , ADN Mitocondrial/metabolismo , Etanol/toxicidad , Hígado/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Animales , Peso Corporal , Caspasa 3/metabolismo , Catalasa/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Proteínas de Unión al ADN/metabolismo , Deferoxamina/farmacología , Regulación hacia Abajo , Complejo I de Transporte de Electrón/metabolismo , Etanol/sangre , Etanol/metabolismo , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Hierro/metabolismo , Quelantes del Hierro/farmacología , Peroxidación de Lípido/efectos de los fármacos , Hígado/enzimología , Hígado/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias Hepáticas/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/genética , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Fas stimulation recruits neutrophils and activates macrophages that secrete tumor necrosis factor-alpha (TNF-alpha), which aggravates Fas-mediated liver injury. To determine whether nonsteroidal anti-inflammatory drugs modify these processes, we challenged 24-hour-fasted mice with the agonistic Jo2 anti-Fas antibody (4 microg/mouse), and treated the animals 1 h later with saline or ibuprofen (250 mg/kg), a dual cyclooxygenase (COX)-1 and COX-2 inhibitor. Ibuprofen attenuated the Jo2-mediated recruitment/activation of myeloperoxidase-secreting neutrophils/macrophages in the liver, and attenuated the surge in serum TNF-alpha. Ibuprofen also minimized hepatic glutathione depletion, Bid truncation, caspase activation, outer mitochondrial membrane rupture, hepatocyte apoptosis and the increase in serum alanine aminotransferase (ALT) activity 5 h after Jo2 administration, to finally decrease mouse mortality at later times. The concomitant administration of pentoxifylline (decreasing TNF-alpha secretion) and infliximab (trapping TNF-alpha) likewise attenuated the Jo2-mediated increase in TNF-alpha, the decrease in hepatic glutathione, and the increase in serum ALT activity 5 h after Jo2 administration. The concomitant administration of the COX-1 inhibitor, SC-560 (10 mg/kg) and the COX-2 inhibitor, celecoxib (40 mg/kg) 1 h after Jo2 administration, also decreased liver injury 5 h after Jo2 administration. In contrast, SC-560 (10 mg/kg) or celecoxib (40 or 160 mg/kg) given alone had no significant protective effects. In conclusion, secondary TNF-alpha secretion plays an important role in Jo2-mediated glutathione depletion and liver injury. The combined inhibition of COX-1 and COX-2 by ibuprofen attenuates TNF-alpha secretion, glutathione depletion, mitochondrial alterations, hepatic apoptosis and mortality in Jo2-treated fasted mice.
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Apoptosis/fisiología , Glutatión/deficiencia , Hepatitis/metabolismo , Ibuprofeno/administración & dosificación , Factor de Necrosis Tumoral alfa/sangre , Receptor fas/toxicidad , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Hepatitis/enzimología , Hepatitis/mortalidad , Hepatitis/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Ibuprofeno/farmacología , Masculino , Ratones , Ratones Endogámicos ICR , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
IL-4 is overexpressed in liver grafts during severe recurrent hepatitis C and rejection. Hepatocyte apoptosis is involved in both these phenomena. We therefore examined the proapoptotic effect of IL-4 on HepG2 cells and human hepatocytes in vitro, together with the underlying mechanisms. We first measured IL-4 receptor expression, STAT6 activation by IL-4, and STAT6 inhibition by an anti-IL-4 antibody or by STAT6 siRNA transfection. We then focused on the pathways involved in IL-4-mediated apoptosis and the role of STAT6 activation in apoptosis initiation. The IL-4 receptor was expressed on both cell types, and STAT6 was activated by IL-4. Both anti-IL-4 and STAT-6 siRNA inhibited this activation. IL-4 induced apoptosis of both HepG2 cells (P=0.008 vs. untreated control) and human hepatocytes (P<0.001 vs. untreated control). IL-4 reduced the mitochondrial membrane potential, activated Bid and Bax, and augmented caspase 3, 8, and 9 activity. STAT6 blockade inhibited IL-4-induced apoptosis. Expression of Fas and Fas ligand was unaffected when HepG2 cells and hepatocytes were cultured with IL-4, and Fas/FasL pathway blockade failed to inhibit IL-4-induced apoptosis. These results show that IL-4 induces apoptosis of human hepatocytes through IL-4 receptor binding, STAT6 activation, decreased mitochondrial membrane potential, and increased caspase activation, independently of the Fas pathway. IL-4 might thus contribute to the progression of severe liver graft damage.
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Apoptosis/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Interleucina-4/farmacología , Receptor fas/metabolismo , Secuencia de Bases , Caspasas/metabolismo , Línea Celular Tumoral , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Hepatocitos/citología , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Interleucina-4/genética , ARN Mensajero/genética , Receptores de Interleucina-4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT6/antagonistas & inhibidoresRESUMEN
OBJECTIVE: Stavudine (d4T), a nucleoside reverse-transcriptase inhibitor (NRTI), can induce lipoatrophy, fatty liver, hyperlactataemia and abnormal liver tests. NRTI toxicity is usually ascribed to mitochondrial DNA (mtDNA) depletion and impaired mitochondrial respiration. However, NRTIs could have effects unrelated to mtDNA. Recently, we reported that 100 mg/kg/day of d4T stimulated fatty acid oxidation (FAO) in mouse liver, and reduced body fatness without depleting white adipose tissue (WAT) mtDNA. We hypothesized that higher d4T doses could further reduce adiposity, while inhibiting hepatic FAO. METHODS: Mice were treated for 2 weeks with d4T (500 mg/kg/day), L-carnitine (200 mg/kg/day) or both drugs concomitantly. Body fatness was assessed by dual energy X-ray absorptiometry, and investigations were performed in plasma, liver, muscle and WAT. RESULTS: D4T reduced the gain of body adiposity, WAT leptin, whole body FAO and plasma ketone bodies, and increased liver triglycerides and plasma aminotransferases with mild ultrastructural abnormalities in hepatocytes. Plasma lactate and respiratory chain activities in tissues were unchanged. Stearoyl-CoA desaturase (SCD-1), an enzyme negatively regulated by leptin, was overexpressed in liver. High doses of beta-aminoisobutyric acid (BAIBA), a d4T catabolite, increased plasma ketone bodies. Although L-carnitine did not correct body adiposity, it prevented d4T-induced impairment of FAO and liver abnormalities. CONCLUSIONS: D4T overdosage triggers fat wasting, leptin insufficiency and mild liver damage, without causing respiratory chain dysfunction. Overexpression of SCD-1 reduces fatty acid oxidation and overcomes the stimulating effect of BAIBA on hepatic FAO. L-carnitine does not correct leptin insufficiency but prevents d4T-induced impairment of FAO and liver damage.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Lipodistrofia/inducido químicamente , Mitocondrias Hepáticas/metabolismo , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Estavudina/administración & dosificación , Síndrome Debilitante/inducido químicamente , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Administración Oral , Ácidos Aminoisobutíricos/metabolismo , Animales , Carnitina/administración & dosificación , Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Cuerpos Cetónicos/sangre , Cuerpos Cetónicos/metabolismo , Leptina/análisis , Leptina/metabolismo , Lipodistrofia/sangre , Lipodistrofia/metabolismo , Hígado/metabolismo , Hígado/fisiopatología , Hepatopatías/sangre , Hepatopatías/metabolismo , Masculino , Ratones , Inhibidores de la Transcriptasa Inversa/efectos adversos , Estavudina/efectos adversos , Estearoil-CoA Desaturasa/metabolismo , Transaminasas/sangre , Transaminasas/metabolismo , Complejo Vitamínico B/administración & dosificación , Síndrome Debilitante/metabolismoRESUMEN
Doxorubicin, cis-diamminedichloroplatinum (II) and 5-fluorouracil used in chemotherapy induce apoptosis in Hep3B cells in the absence of p53, p73, and functional Fas. Since mediators remain unknown, the requirement of PKC delta (PKCdelta) and c-Abl was investigated. Suppression of c-Abl or PKCdelta expression using SiRNAs impaired PARP cleavage, Gleevec and/or rottlerin inhibited the induction of the subG1 phase and the increase of reactive oxygen species level. Co-precipitations and phosphorylations to mitochondria of c-Abl, PKCdelta and Bcl-X(L/s) were induced. A depolarization of the mitochondrial membrane and activations of caspase-2 and -9 were observed. We propose that, in the absence of p53, p73 and Fas, genotoxic drugs could require both PKCdelta and c-Abl to induce apoptosis through the mitochondrial pathway.
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Apoptosis/efectos de los fármacos , Fluorouracilo/toxicidad , Mitocondrias/metabolismo , Mutágenos/toxicidad , Proteína Quinasa C-delta/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Caspasa 2 , Caspasa 9 , Caspasas/metabolismo , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cisplatino/toxicidad , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Doxorrubicina/toxicidad , Humanos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Proteínas Nucleares/deficiencia , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Proteína Quinasa C-delta/genética , Proteínas Proto-Oncogénicas c-abl/genética , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/metabolismo , Proteína bcl-X/metabolismo , Receptor fas/metabolismoRESUMEN
Response to interferon-gamma (IFN-gamma)-induced apoptosis of human hepatoma cell lines (HHCLs) is variable. We analyzed this different behavior in Hep3B, Chang-liver, HepG2, and HuH7 cells. We studied (1) IFN-gamma-induced apoptosis, (2) protein expression of Stat1, (3) binding of nuclear proteins to IFN-gamma activated sequence (GAS), (4) mRNA and expression of proteins acting in apoptosis, and (5) HuH7 sensitivity after inducible nitric oxide synthase (iNOS) siRNA transfection. IFN-gamma induced apoptosis in Hep3B and Chang-liver cells only. In all HHCLs, Stat1 protein increased. Binding of proteins and transactivation activity of GAS increased much more in HuH7. In all HHCLs, caspase activity and apoptotic proteins were not implicated in resistance or sensitivity. iNOS mRNA and protein expression increased in HuH7, disappeared in Hep3B, and remained unchanged in Chang-liver and HepG2. We compared the role of iNOS in Hep3B and HuH7. The iNOS inhibitor, L-NAME, sensitized HuH7 to IFN-gamma, Hep3B/HuH7 coculture partially inhibited Hep3B apoptosis, and HuH7 transfection with iNOS siRNA induced a 50% inhibition of iNOS protein and cell apoptosis. GAS activity and overexpression of iNOS in HuH7, but not in the other HHCLs, suggest that this enzyme could play an important role in the resistance of HuH7 to IFN-gamma-induced apoptosis, perhaps by the antiapoptotic action of NO.
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Apoptosis , Resistencia a Medicamentos , Interferón gamma/farmacología , Óxido Nítrico Sintasa de Tipo II/fisiología , Apoptosis/genética , Carcinoma Hepatocelular , Línea Celular Tumoral , Técnicas de Cocultivo , Resistencia a Medicamentos/genética , Inhibidores Enzimáticos/farmacología , Humanos , Molsidomina/análogos & derivados , Molsidomina/farmacología , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/genética , ARN Mensajero/análisis , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Activación TranscripcionalRESUMEN
The study examined the effects of various progesterone and mifepristone concentrations on the proliferation and apoptosis of the human ovarian cancer cell line, OVCAR-3. OVCAR-3 cells were incubated with progesterone and mifepristone at concentrations ranging from 10(-3) to 10(-9) M. Proliferation and apoptosis were studied by means of inverted optical microscopy, DAPI staining, and crystal violet assay. Immunoblotting was used to study the regulation of the apoptosis-related proteins, bcl-2, caspase-3 and PARP, after incubation with various reagents. OVCAR-3 cell density was increased by progesterone concentrations of 10(-5) M or less, and decreased by 10(-3) M progesterone. DAPI staining showed no apoptotic bodies. Mifepristone concentrations of 10(-3) and 10(-4) M reduced the OVCAR-3 cell density. Immunoblotting showed PARP cleavage in the presence of mifepristone 10(-4) M. Caspase-3 and bcl-2 expression was reduced by mifepristone 10(-4) and 10(-7) M. These results suggest that progesterone has a paradoxical effect on OVCAR-3 cell proliferation, stimulating it at low concentrations and inhibiting it at high concentrations, potentially through a caspase-independent non-apoptotic death pathway. Mifepristone seems to inhibit OVCAR-3 cell proliferation by down-regulating bcl-2 and up-regulating caspase-3 activity. These preliminary results suggest that progesterone and mifepristone have beneficial effects in ovarian cancer.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Mifepristona/farmacología , Progesterona/farmacología , Caspasa 3 , Caspasas/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Violeta de Genciana , Antagonistas de Hormonas/farmacología , Humanos , Immunoblotting , Indoles , Microscopía Fluorescente , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Apoptosis or programmed cell death occurs in the liver as in other organs. In the normal state it is not a frequent mode of hepatic cell destruction. Morphological and biochemical characteristics of liver cell apoptosis do not differ from what is observed in other cells. The Fas receptor pathway, a frequent hepatic apoptotic pathway among various others, involves intra-cellular signals amplified by mitochondria. Although hepatic apoptosis may occur by following several others pathways, Fas, which is abundantly expressed in the plasma membrane of hepatocytes, is very often involved in hepatocyte demise during B or C viral hepatitis irrespective of their clinical form, alcoholic hepatitis, cholestasis due to accumulation of hepatic biliary salts, or certain types of drug-induced hepatitis. Fas is also probably responsible for the death of biliary cells in primary biliary cirrhosis. In contrast one of the causes of resistance to apoptosis of hepatic cancerous cells could be related to an alteration of the Fas receptor. This is why much experimental work is presently performed to achieve inhibition of the Fas receptor either at the mRNA level or at the level of Fas-inductible proteolytic enzymes called caspases. One perspective is a specific treatment of apoptosis as an adjuvant treatment of liver diseases.
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Apoptosis , Hepatopatías/fisiopatología , Hígado/citología , Hígado/patología , Receptor fas/fisiología , Humanos , Hepatopatías/terapia , Transducción de Señal , Receptor fas/biosíntesis , Receptor fas/efectos de los fármacos , Receptor fas/genéticaRESUMEN
The response of three human leukemia cell lines, the proliferative promonocyte THP-1 and the promyeloid HL60 cells and the non-proliferative phorbol ester-treated HL60 cells (HL60/PMA), to oxidative stress induced by tert-butylhydroperoxide (t-BHP) treatment was analyzed by fluorescence microplate assay, anti-oxidant enzyme activity measurements, high performance liquid chromatography, yopro-1/PI incorporation, poly (ADP-ribose) polymerase and caspase 3 cleavages. After t-BHP treatment, the non-proliferative HL60/PMA cells exhibited a weak increase in reactive oxygen species (ROS) production, a better preservation of thiol content, a decrease of glutathione peroxidase activity and a high ability to undergo necrosis rather than apoptosis. Submitted to the same treatment, the proliferative HL60 and THP-1 cells exhibited a high increase of ROS production, a moderate thiol depletion and a high percentage of apoptosis. Under thiol depleting conditions, the oxidative treatment of the HL60/PMA cells resulted in a high ROS production that reached levels similar to those of the two other cell lines and in cell death mainly by necrosis. In conclusion, these results that show proliferative phenotype is essential for cell response towards oxidative stress, are of particular interest in chemotherapy involving an oxidative mechanism.
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Leucemia/patología , Estrés Oxidativo , Apoptosis , Caspasa 3 , Caspasas/metabolismo , Muerte Celular , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Cromatografía Líquida de Alta Presión , Cisteína/química , Relación Dosis-Respuesta a Droga , Glutatión/química , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Células HL-60 , Humanos , Leucemia/metabolismo , Necrosis , Oxígeno/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Especies Reactivas de Oxígeno , Espectrometría de Fluorescencia , Compuestos de Sulfhidrilo/metabolismo , Superóxido Dismutasa/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
BACKGROUND: The BRCA1 caretaker gene is associated with poor prognostic features in hereditary breast cancer and may also play a role in sporadic breast cancer (SBC). HER-1 and HER-2 overexpression is associated with adverse prognosis in SBC. We studied whether BRCA1 expression was associated with HER1, HER2 and other prognostic features in SBC. PATIENTS AND METHODS: Fifty newly-diagnosed SBC patients were studied for prognostic features and immunohistochemical expressions of BRCA1, HER-1 and HER-2. RESULTS: Tumors were positive for BRCA1 in 26%, HER-1 in 32% and HER-2 in 20% of cases. Lack of BRCA1 expression was associated with node metastases and decreased estrogen receptor. HER-2 expression was associated with young age, HER-1, Ki67 and decreased hormone receptors. No correlation was observed between BRCA1 and HER-1 or HER-2. CONCLUSION: In SBC, the lack of BRCA1 expression was associated with poor prognostic features, but unrelated to HER-1 and HER-2. HER2 and HER-1 were, however, highly correlated.
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Proteína BRCA1/biosíntesis , Neoplasias de la Mama/metabolismo , Receptores ErbB/biosíntesis , Receptor ErbB-2/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Receptores ErbB/genética , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Pronóstico , Receptor ErbB-2/genéticaRESUMEN
BACKGROUND: Inherited mutations of the BRCA1 gene are responsible for hereditary breast and ovarian cancer syndrome. However, little is known of how disruption of BRCA1 functions preferentially increases cancer risk in hormone-dependent organs. We aimed to study whether BRCA1 was regulated by progesterone in the MCF7 breast cancer cell line. MATERIALS AND METHODS: MCF7 breast cancer cells were incubated with 10(-4) or 10(-10) M progesterone for 24 or 48 hours. BRCA1 expression, proliferation and apoptosis were analysed. RESULTS: 10(-4) M progesterone decreased cell proliferation, cell cycle progression and induced apoptosis. In addition, BRCA1 and cyclin A mRNA decreased. In contrast, none of these effects were observed in MCF7 cells incubated with 10(-10) M progesterone. CONCLUSION: The down-regulation of BRCA1 in MCF7 cells incubated with 10(-4) M progesterone seems to be a consequence of cell cycle alterations rather than a direct effect of the hormone on BRCA1.
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Proteína BRCA1/genética , Neoplasias de la Mama/genética , Progesterona/farmacología , Apoptosis/efectos de los fármacos , Proteína BRCA1/biosíntesis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclinas/biosíntesis , Ciclinas/genética , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Estrógenos/biosíntesis , Receptores de Progesterona/biosíntesisRESUMEN
Apoptosis is a physiological process by which multicellular organisms eliminate superfluous cells. Alterations in apoptosis play a key role in tumour development. The objective was to evaluate the immunohistochemical expression of p53, p21, bax, bak, fas, bcl-2 and bcl-x proteins in 10 endometriomas, 20 benign ovarian tumours (10 mucinous, 10 serous) and 30 malignant ovarian tumours (9 mucinous, 19 serous; 2 endometrioids). p53 positive cells (mean+/-SD) in endometriomas, and benign and malignant tumours were 1.9+/-3.2, 0 and 16.2+/-33.0, respectively. The difference was significant between benign tumours and endometriomas (P=0.003) but not between endometriomas and malignant tumours. P21 expression in endometriomas and benign and malignant tumours was 19.5+/-27.8, 1.7+/-6.7 and 4.1+/-8.6, respectively. Increased p21 expression was found in endometriomas compared with benign (P=0.001) and malignant (P=0.01) tumours. Bax expression was higher in endometriomas than in benign tumours (P=0.01), but no difference was found between endometriomas and malignant tumours. No difference in bak, fas, bcl-2 or bcl-x expression was observed among the groups. In endometriomas, a negative correlation was found between p53 and fas expression (P=0.04, r=0.66). Although endometriomas have histological features of benign ovarian tumours, endometriomas share with malignant ovarian tumours certain alterations in apoptosis-related proteins.
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Apoptosis , Biomarcadores de Tumor/metabolismo , Endometriosis/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/metabolismo , Adulto , Anciano , Recuento de Células , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Endometriosis/patología , Femenino , Humanos , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2 , Proteína bcl-X , Receptor fas/metabolismoRESUMEN
OBJECTIVE: To compare the vascular parameters of uterine leiomyomas and normal myometrium, to correlate these parameters with vascular endothelial growth factor (VEGF) expression and clinical/pathological parameters, and to compare vascular parameters according to the endothelial markers used. DESIGN: An immunohistochemical technique was applied to formalin-fixed paraffin-embedded tissue samples, using antibodies against von Willebrand's factor (FvW), CD34, CD31, and VEGF. The intratumoral vascular area (VA), microvessel density (MVD), and vascular luminal area (VLA) were determined with an image analyser. SETTING: University teaching hospital. PATIENT(S): Thirty-two patients with uterine leiomyomas underwent conservative surgery. Twenty leiomyoma-free patients undergoing hysterectomy were the controls. INTERVENTION(S): Immunohistochemical and morphometrical analysis. MAIN OUTCOME MEASURE(S): Measurements of VA, MVD, and VLA. RESULT(S): The CD34 labeling showed decreased VA in myomas compared with myometrium. Decreased MVD and an increased VLA in myomas were found with FvW and CD34 labeling. The VA, MVD, and VLA were not related to VEGF expression or to clinical/pathological parameters. Similar results for VA and MVD were obtained with FvW and CD34 labeling. CONCLUSION(S): Leiomyomas have a smaller vascular area, a lower microvessel density, and a higher vascular luminal area than normal myometrium.
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Antígenos CD1/metabolismo , Antígenos CD34/metabolismo , Factores de Crecimiento Endotelial/metabolismo , Leiomioma/metabolismo , Linfocinas/metabolismo , Neoplasias Uterinas/metabolismo , Factor de von Willebrand/metabolismo , Antígenos CD/metabolismo , Femenino , Humanos , Inmunohistoquímica , Leiomioma/patología , Leiomioma/cirugía , Neoplasias Uterinas/patología , Neoplasias Uterinas/cirugía , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
OBJECTIVE: Previous studies have shown the importance of apoptosis in follicular atresia occurring especially in granulosa cells (GC) and its relation to the outcome of in vitro fertilization (IVF). The aim of this study was to evaluate the effects of a Gonadotropin-Releasing Hormone agonist (GnRHa) and of Follicle-Stimulating Hormone (FSH) on the apoptosis rate of human luteinized GC. STUDY DESIGN: GC were isolated from follicular fluids of 15 women undergoing IVF cycles, cultured for 1 day and then treated for 1 day in serum-free medium with triptorelin at 100 or 1000pg/ml or with FSH at 100 or 500ng/ml. GC cultured without any hormone addition were used as controls. Treatment of cultured GC with triptorelin 100pg/ml and FSH 100ng/ml was performed five times each. GC were analysed by flow cytometry after propidium iodide staining to measure the percentage of apoptotic GC. Some triptorelin-treated GC were also examined by electron microscopy. RESULTS: Percentages of GC apoptosis were after hormone treatment respectively: FSH: 100ng/ml, 2.9+/-0.6%; 500ng/ml, 2.9%; triptorelin: 100pg/ml, 18.6+/-2.8%; 1000pg/ml, 86.5% versus 9.8+/-1.8% in GC controls (FSH 100ng/ml versus control; triptorelin 100pg/ml versus control: P<10(-6)). Electron microscopy confirmed apoptosis of GC incubated with triptorelin. CONCLUSIONS: This study demonstrated that FSH decreased apoptosis in human luteinized GC. In contrast, triptorelin was possibly implicated in a dose-dependent increase in the incidence of apoptotic GC. This last result suggests that clinical use of GnRHa should perhaps be reconsidered in the context of its apoptosis-inducing effect.
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Apoptosis/efectos de los fármacos , Hormona Folículo Estimulante/farmacología , Células de la Granulosa/efectos de los fármacos , Luteinización , Pamoato de Triptorelina/farmacología , Adulto , Femenino , Fertilización In Vitro , Citometría de Flujo , Líquido Folicular/citología , Células de la Granulosa/ultraestructura , Humanos , Microscopía ElectrónicaRESUMEN
OBJECTIVE: The aim of this study was to investigate the relationship between induced apoptosis of human luteinized granulosa cells (GCs) and in vitro fertilization (IVF) outcome. STUDY DESIGN: We induced apoptosis with interferon gamma and an anti-human Fas antibody in cultured GCs isolated from follicular fluids coming from 25 different women undergoing 25 consecutive IVF cycles. After examination of 1000 GCs stained by DAPI with a fluorescent microscope, we determined the percentages of apoptotic GCs. Ovarian, endometrial and IVF parameters were recorded for every woman. RESULTS: We classified the women according to their induced GCs apoptosis percentages in two groups. Group 1 with a low percentage of apoptotic GCs (11.6+/-4.8%) had a significantly higher pregnancy rate (P<0.05) than group 2 with a high percentage of apoptotic GCs (59.5+/-14.8%). No other statistically significant differences were observed. CONCLUSION: Resistance of human GCs to apoptosis might be implicated in IVF outcome.