RESUMEN
Interferon-α2b (IFN-α2b) reduces proliferation and increases apoptosis in hepatocellular carcinoma cells by decreasing ß-catenin/TCF4/Smads interaction. Forkhead box O-class 3a (FoxO3a) participates in proliferation and apoptosis and interacts with ß-catenin and Smads. FoxO3a is inhibited by Akt, IκB kinase ß (IKKß), and extracellular-signal-regulated kinase (Erk), which promote FoxO3a sequestration in the cytosol, and accumulates in the nucleus upon phosphorylation by c-Jun N-terminal kinase (JNK) and p38 mitogen-activated kinase (p38 MAPK). We analyzed FoxO3a subcellular localization, the participating kinases, FoxO3a/ß-catenin/Smads association, and FoxO3a target gene expression in IFN-α2b-stimulated HepG2/C3A and Huh7 cells. Total FoxO3a and Akt-phosphorylated FoxO3a levels decreased in the cytosol, whereas total FoxO3a levels increased in the nucleus upon IFN-α2b stimulus. IFN-α2b reduced Akt, IKKß, and Erk activation, and increased JNK and p38 MAPK activation. p38 MAPK inhibition blocked IFN-α2b-induced FoxO3a nuclear localization. IFN-α2b enhanced FoxO3a association with ß-catenin and Smad2/3/7. Two-step coimmunoprecipitation experiments suggest that these proteins coexist in the same complex. The expression of several FoxO3a target genes increased with IFN-α2b. FoxO3a knockdown prevented the induction of these genes, suggesting that FoxO3a acts as mediator of IFN-α2b action. Results suggest a ß-catenin/Smads switch from TCF4 to FoxO3a. Such events would contribute to the IFN-α2b-mediated effects on cellular proliferation and apoptosis. These results demonstrate new mechanisms for IFN-α action, showing the importance of its application in antitumorigenic therapies.
Asunto(s)
Carcinoma Hepatocelular/terapia , Núcleo Celular/metabolismo , Factores de Transcripción Forkhead/metabolismo , Inmunoterapia/métodos , Interferón-alfa/farmacología , Proteínas Smad/metabolismo , beta Catenina/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Carcinoma Hepatocelular/inmunología , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Células Hep G2 , Humanos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/genética , Factor de Transcripción 4 , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
SCOPE: Quercetin is the most abundant flavonoid in human diet. It has special interest as it holds anticancerous properties. This study aims to clarify the mechanisms involved in quercetin effects during the occurrence of preneoplastic lesions in rat liver. METHODS AND RESULTS: Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis (initiated-promoted group). Initiated-promoted animals also received quercetin 10 and 20 mg/kg body weight (IPQ10 and IPQ20 groups, respectively). Antioxidant defenses were modified by quercetin administration at both doses. However, only IPQ20 group showed a reduction in number and volume of preneoplastic lesions. This group showed increased apoptosis and a reduction in the proliferative index. In addition, IPQ20 group displayed a reduction of cell percentages in G1 and S phases, accumulation in G2, and decrease in M phase, with reduced expression of cyclin D1, cyclin A, cyclin B, and cyclin-dependent kinase 1. Interestingly, peroxisome proliferator activated receptor-α levels were reduced in IPQ20 group. CONCLUSION: The outcomes of this study represent a significant contribution to the current understanding on the preventive mechanisms of quercetin during the early stages of liver cancer development, demonstrating that in addition to its known proapoptotic characteristics, the flavonoid modulates the expression of critical cell cycle regulators and peroxisome proliferator activated receptor-α activity.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinogénesis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Hepáticas/prevención & control , Quercetina/farmacología , Animales , Proteína Quinasa CDC2/metabolismo , División Celular/efectos de los fármacos , Ciclina D1/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , PPAR alfa/metabolismo , Ratas , Ratas WistarRESUMEN
Wnt/ß-catenin pathway is often dysregulated in hepatocellular carcinoma (HCC). Activated ß-catenin accumulates in the cytosol and nucleus and forms a nuclear complex with TCF/LEF factors like TCF4. Interferon-α (IFN-α) has recently been recognized to harbor therapeutic potential in prevention and treatment of HCC. Transforming Growth Factor-ß1 (TGF-ß1) is a mediator of apoptosis, exerting its effects via Smads proteins. One mode of interaction between Wnt/ß-catenin and TGF-ß1/Smads pathways is the association of Smads with ß-catenin/TCF4. In this study we analyzed the effects of IFN-α2b and TGF-ß1 treatments on Wnt/ß-catenin pathway, Smads proteins levels, ß-catenin/TCF4/Smads interaction and proliferation and apoptotic death in HepG2/C3A and Huh7 cell lines. IFN-α2b and TGF-ß1 attenuated Wnt/ß-catenin signal by decreasing ß-catenin and Frizzled7 receptor proteins contents and the interaction of ß-catenin with TCF4. Truncated ß-catenin form present in C3A cell line also diminished after treatments. Both cytokines declined Smads proteins and their interaction with TCF4. The overall cellular response to cytokines was the decrease in proliferation and increase in apoptotic death. Treatment with Wnt3a, which elevates ß-catenin protein levels, also generated the increment of Smads proteins contents when comparing with untreated cells. In conclusion, IFN-α2b and TGF-ß1 proved to be effective as modulators of Wnt/ß-catenin pathway in HCC cell lines holding both wild-type and truncated ß-catenin. Since the inhibition of ß-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, IFN-α2b and TGF-ß1 could be useful as potential treatments in patients with HCC.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Interferón-alfa/farmacología , Neoplasias Hepáticas/metabolismo , Proteínas Smad/fisiología , Factor de Crecimiento Transformador beta1/farmacología , Proteínas Wnt/fisiología , beta Catenina/fisiología , Apoptosis/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Interferón alfa-2 , Neoplasias Hepáticas/patología , Proteínas Recombinantes/farmacología , Transducción de Señal , Factor de Transcripción 4 , Factores de Transcripción/fisiología , Proteína Wnt3A/farmacologíaRESUMEN
In this work we investigated the role of nitric oxide (NO) in the angiogenesis mediated by vascular endothelial growth factor (VEGF) during rat liver regeneration after two-thirds partial hepatectomy. Sham operated (Sh) and partially hepatectomized (PH) male Wistar rats were randomized in three experimental groups: control (treated with vehicle); pre-treated with sodium nitroprusside (SNP: 0.25 mg/kg body weight, i.v. at a rate of 1 ml/h) and pre-treated with the preferential iNOS inhibitor, aminoguanidine (AG, 100 mg/kg body weight, i.p.). Animals were killed at 5, 24 and 72 h after surgery. At 5 h post-surgery, NO production was estimated by EPR (Sh-Control: 37.65+/-10.70; PH-Control: 88.13+/-1.60(); Sh-SNP: 90.35+/-3.11(); PH-SNP: 119.5+/-12.10()(#); Sh-AG: 33.27+/-5.23, PH-AG: 36.80+/-3.40(#)) (p<0.05 vs Sh-Control; (#)p<0.05 vs PH-Control). At 24 h after PH, VEGF levels showed no difference between PH-Control and PH-SNP animals. However, after 72 h, VEGF protein levels in PH-SNP animals were found to be increased (above 300%) with respect to PH-Control. On the other hand, aminoguanidine (AG) pre-treatment blocked the rise of inhibition of NO generation and decreased VEGF expression. Our results demonstrated that NO plays a role in modulating VEGF protein expression after hepatectomy in rats.
Asunto(s)
Regeneración Hepática/fisiología , Hígado , Neovascularización Fisiológica/fisiología , Óxido Nítrico/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Western Blotting , Espectroscopía de Resonancia por Spin del Electrón , Hígado/irrigación sanguínea , Hígado/metabolismo , Hígado/fisiología , Regeneración Hepática/efectos de los fármacos , Masculino , Neovascularización Fisiológica/efectos de los fármacos , Nitratos/metabolismo , Óxido Nítrico/biosíntesis , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitroprusiato/farmacología , Ratas , Ratas Wistar , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Glutathione S-transferases (GSTs) are involved in the detoxification of xenobiotics, such as several cytostatic drugs, through conjugation with glutathione (GSH). Pi class GST (GST P) liver expression is associated with preneoplastic and neoplastic development and contributes with the drug-resistance phenotype. Ethacrynic acid (EA) is an inhibitor of rat and human GSTs. In addition, causes lipid peroxidation in isolated rat hepatocytes. Therefore, we decided to evaluate the role of the GST/GSH system in isolated hepatocytes from preneoplastic rat livers (IP) in the presence of EA and determine the cytotoxicity of the drug. Our results showed a resistance to the toxic effects of EA since viability and cellular integrity values were significantly higher than control. Initial levels of thiobarbituric acid reactive substances (TBARS) in IP hepatocytes were significantly higher than control and the presence of EA did not change TBARS levels. A diminution in intracellular total GSH was observed by treating with EA isolated hepatocytes from both groups. However, the initial total GSH levels were higher in IP hepatocytes than in control. Immunoblotting analysis showed the presence of GST P in IP animals only. Although alpha and mu class isoenzymes levels were decreased in IP hepatocytes, total GST activity was 1.5-fold higher than in control. In addition, multidrug-resistance protein 2 (Mrp2) showed fivefold decreased levels in IP hepatocytes. In conclusion, increased total GSH, decreased Mrp2 levels and the presence of GST P could be critical factors involved in the resistance of IP hepatocytes to the toxicity of EA.
Asunto(s)
Diuréticos/toxicidad , Ácido Etacrínico/toxicidad , Glutatión Transferasa/metabolismo , Hepatocitos/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , Lesiones Precancerosas/metabolismo , 2-Acetilaminofluoreno/toxicidad , Transportadoras de Casetes de Unión a ATP/metabolismo , Alquilantes/toxicidad , Animales , Carcinógenos/toxicidad , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dietilnitrosamina/toxicidad , Resistencia a Medicamentos , Glutatión/metabolismo , Hepatocitos/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Neoplasias Hepáticas/inducido químicamente , Masculino , Lesiones Precancerosas/inducido químicamente , Ratas , Ratas Wistar , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
We have already demonstrated that interferon alfa-2b (IFN-alpha2b) induces apoptosis in isolated hepatocytes from preneoplastic rat livers via the secretion of transforming growth factor beta(1) (TGF-beta(1)), and this process is accompanied by caspase-3 activation. The aim of this study was to further investigate the mechanism of this activation. Isolated hepatocytes from preneoplastic livers induced DNA fragmentation in response to IFN-alpha2b, which was completely blocked when anti-TGF-beta(1) was added to the culture media. IFN-alpha2b mediated radical oxygen species (ROS) production that preceded the loss of mitochondrial transmembrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-3. Bax levels increased in a time-dependent fashion, and Bcl-x(L) was down-regulated in the early hours of IFN-alpha2b treatment. The delayed translocation of Bid into the mitochondria was in concordance with late caspase-8 activation. In conclusion, endogenous TGF-beta(1) secreted under IFN-alpha2b stimulus seems to induce cytochrome c release through a mechanism related to Bcl-2 family members and loss of mitochondrial DeltaPsi. Bax protein could be responsible of the release of cytochrome c during the initial hours of IFN-alpha2b-induced apoptosis via TGF-beta(1). Activated Bid by caspases could amplificate the mitochondrial events, enhancing the release of cytochrome c.