RESUMEN
The antiepileptic drug phenobarbital (PB) exerts hepatic effect based on indirect activation of the constitutive androstane receptor (CAR) via inhibition of the epidermal growth factor receptor (EGFR) and the kinase Src. It has furthermore been observed that in mice PB suppresses the growth of hepatocellular carcinoma with overactive signaling through the oncogenic Wnt/ß-catenin pathway, thus suggesting an interference of PB with ß-catenin signaling. The present work was aimed to characterize effects of PB on ß-catenin signaling at different cellular levels and to elucidate molecular details of the interaction of PB and ß-catenin in an in vitro system of mouse hepatoma cells. PB efficiently inhibited signaling through ß-catenin. This phenomenon was in-depth characterized at the levels of ß-catenin protein accumulation and transcriptional activity. Mechanistic analyses revealed that the effect of PB on ß-catenin signaling was independent of the activation of CAR and also independent of the cytosolic multi-protein complex responsible for physiological post-translation control of the ß-catenin pathway via initiation of ß-catenin degradation. Instead, evidence is provided that PB diminishes ß-catenin protein production by inhibition of protein synthesis via signal transduction through EGFR and Src. The proposed mechanism is well in agreement with previously published activities of PB at the EGFR and Src-mediated regulation of ß-catenin mRNA translation. Inhibition of ß-catenin signaling by PB through the proposed mechanism might explain the inhibitory effect of PB on the growth of specific sub-populations of mouse liver tumors. In conclusion, the present data comprehensively characterize the effect of PB on ß-catenin signaling in mouse hepatoma cells in vitro and provides mechanistic insight into the molecular processes underlying the observed effect.
Asunto(s)
Anticonvulsivantes/farmacología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Fenobarbital/farmacología , Transducción de Señal , beta Catenina/metabolismo , Animales , Línea Celular Tumoral , Receptor de Androstano Constitutivo , Relación Dosis-Respuesta a Droga , Receptores ErbB/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Receptores Citoplasmáticos y Nucleares/metabolismo , Vía de Señalización Wnt , beta Catenina/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
Hepatocellular carcinoma (HCC) represents the second leading cause of cancer-related deaths and is reported to be resistant to chemotherapy caused by tumor-initiating cells. These tumor-initiating cells express stem cell markers. An accumulation of tumor-initiating cells can be found in 2% to 50% of all HCC and is correlated with a poor prognosis. Mechanisms that mediate chemoresistance include drug export, increased metabolism, and quiescence. Importantly, the mechanisms that regulate quiescence in tumor-initiating cells have not been analyzed in detail so far. In this research we have developed a single cell tracking method to follow up the fate of tumor-initiating cells during chemotherapy. Thereby, we were able to demonstrate that mCXCL1 exerts cellular state-specific effects regulating the resistance to chemotherapeutics. mCXCL1 is the mouse homolog of the human IL8, a chemokine that correlates with poor prognosis in HCC patients. We found that mCXCL1 blocks differentiation of premalignant cells and activates quiescence in tumor-initiating cells. This process depends on the activation of the mTORC1 kinase. Blocking of the mTORC1 kinase induces differentiation of tumor-initiating cells and allows their subsequent depletion using the chemotherapeutic drug doxorubicin. Our work deciphers the mCXCL1-mTORC1 pathway as crucial in liver cancer stem cell maintenance and highlights it as a novel target in combination with conventional chemotherapy. Cancer Res; 76(18); 5550-61. ©2016 AACR.
Asunto(s)
Carcinoma Hepatocelular/patología , Diferenciación Celular/fisiología , Quimiocina CXCL1/metabolismo , Neoplasias Hepáticas/patología , Complejos Multiproteicos/metabolismo , Células Madre Neoplásicas/patología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular Tumoral , Proteínas Cullin , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Reacción en Cadena de la PolimerasaRESUMEN
Depending on their position within the liver lobule, hepatocytes fulfill different metabolic functions. Cytochrome P450 (CYP) 2E1 is a drug-metabolizing enzyme which is exclusively expressed in hepatocytes surrounding branches of the hepatic central vein. Previous publications have shown that signaling through the Wnt/ß-catenin pathway, a major determinant of liver zonation, and the hepatocyte-enriched transcription factor HNF (hepatocyte nuclear factor) 1α participate in the regulation of the gene. This study was aimed to decipher the molecular mechanisms by which the two transcription factors, ß-catenin and HNF1α, jointly regulate CYP2E1 at the gene promoter level. Chromatin immunoprecipitation identified a conserved Wnt/ß-catenin-responsive site (WRE) in the murine Cyp2e1 promoter adjacent to a known HNF1α response element (HNF1-RE). In vitro analyses demonstrated that both, activated ß-catenin and HNF1α, are needed for the full response of the promoter. The WRE was dispensable for ß-catenin-mediated effects on the Cyp2e1 promoter, while activity of ß-catenin was integrated into the promoter response via the HNF1-RE. Physical interaction of ß-catenin and HNF1α was demonstrated by co-immunoprecipitation. In conclusion, present data the first time identify and characterize the interplay of HNF1α and ß-catenin and elucidate molecular determinants of CYP2E1 expression in the liver.
Asunto(s)
Citocromo P-450 CYP2E1/metabolismo , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , beta Catenina/metabolismo , Animales , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Regiones Promotoras GenéticasRESUMEN
Enzymatic conversion of most xenobiotic compounds is accomplished by hepatocytes in the liver, which are also an important target for the manifestation of the toxic effects of foreign compounds. Most cell lines derived from hepatocytes lack important toxifying or detoxifying enzymes or are defective in signaling pathways that regulate expression and activity of these enzymes. On the other hand, the use of primary human hepatocytes is complicated by scarce availability of cells and high interdonor variability. Thus, analyses of drug metabolism and hepatotoxicity in vitro are a difficult task. The cell line HC-AFW1 was isolated from a pediatric hepatocellular carcinoma and so far has been used for tumorigenicity and chemotherapy resistance studies. Here, a comprehensive characterization of xenobiotic metabolism in HC-AFW1 cells is presented along with studies on the functionality of the most important transcriptional regulators of drug-metabolizing enzymes. Results from HC-AFW1 cells were compared with commercially available HepaRG cells and cultured primary human hepatocytes. Data show that the nuclear receptors and xenosensors AHR (aryl hydrocarbon receptor), CAR (constitutive androstane receptor), PXR (pregnane-X-receptor), NRF2 [nuclear factor (erythroid-derived 2)-like 2], and PPARα (peroxisome proliferator-activated receptor α) are functional in HC-AFW1 cells, comparable to HepaRG and primary cells. HC-AFW1 cells possess considerable activities of different cytochrome P450 enzymes, which, however, are lower than corresponding enzyme activities in HepaRG cells or primary hepatocytes. In summary, HC-AFW1 are a new promising tool for studying the mechanisms of the regulation of drug metabolism in human liver cells in vitro.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Sistema Enzimático del Citocromo P-450/biosíntesis , Regulación Enzimológica de la Expresión Génica , Neoplasias Hepáticas/enzimología , Receptores Citoplasmáticos y Nucleares/agonistas , Animales , Sistema Enzimático del Citocromo P-450/genética , Dimetilsulfóxido/farmacología , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/fisiología , Humanos , Dibenzodioxinas Policloradas/farmacologíaRESUMEN
BACKGROUND: Serotonin plays a critical role in the regulation of food intake. The solute carrier family 6 member 14 (SLC6A14) and serotonin receptor 2C (5-HTR2C) genes are involved in the bioavailability and action of this neurotransmitter. OBJECTIVE: Evaluation of the association of six polymorphisms in these genes with food intake and nutritional status in children followed to 7-8years of age. DESIGN: Blood samples and the biodemographic data of 344 children were collected at three development stages, in a cross-sectional study undertaken with the cohort from a randomized trial. Polymorphisms were analyzed using polymerase chain reaction-based techniques. RESULTS: At 7 to 8years of age, carriers of the A alleles for both the SLC6A14 rs2312054 and SLC6A14 rs12391221 polymorphisms showed higher food intake, except for the sugar-dense food (SDF) intake parameter, than T/T and C/C homozygotes, respectively. Boy carriers of the C allele of rs2071877 had a higher sum of triceps and subscapular folds than T allele carriers at 7 to 8years old. For 5-HTR2C gene variants, A allele carriers (rs3813928) and T allele carriers (rs3813929) had higher food intake at 3 to 4years old than G/G and C/C homozygotes, respectively, except for SDF. At this age, the intake of energy-dense foods was higher in carriers of the T allele (rs3813929) than in C/C homozygotes. CONCLUSION: This study provides evidence that genetic variants of these proteins might be involved in the determination of food intake and nutritional status in children.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/genética , Ingestión de Alimentos/genética , Estado Nutricional/genética , Polimorfismo de Nucleótido Simple , Receptor de Serotonina 5-HT2C/genética , Alelos , Sistemas de Transporte de Aminoácidos , Niño , Preescolar , Estudios Transversales , Femenino , Expresión Génica , Frecuencia de los Genes , Genotipo , Heterocigoto , Homocigoto , Humanos , Lactante , Masculino , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Genes involved in detoxification of foreign compounds exhibit complex spatiotemporal expression patterns in liver. Cytochrome P450 1A1 (CYP1A1), for example, is restricted to the pericentral region of liver lobules in response to the interplay between aryl hydrocarbon receptor (AhR) and Wnt/ß-catenin signaling pathways. However, the mechanisms by which the two pathways orchestrate gene expression are still poorly understood. With the help of 29 mutant constructs of the human CYP1A1 promoter and a mathematical model that combines Wnt/ß-catenin and AhR signaling with the statistical mechanics of the promoter, we systematically quantified the regulatory influence of different transcription factor binding sites on gene induction within the promoter. The model unveils how different binding sites cooperate and how they establish the promoter logic; it quantitatively predicts two-dimensional stimulus-response curves. Furthermore, it shows that crosstalk between Wnt/ß-catenin and AhR signaling is crucial to understand the complex zonated expression patterns found in liver lobules. This study exemplifies how statistical mechanical modeling together with combinatorial reporter assays has the capacity to disentangle the promoter logic that establishes physiological gene expression patterns.
Asunto(s)
Citocromo P-450 CYP1A1/genética , Regiones Promotoras Genéticas , Activación Transcripcional , Vía de Señalización Wnt , Animales , Sitios de Unión , Línea Celular Tumoral , Células Cultivadas , Citocromo P-450 CYP1A1/biosíntesis , Humanos , Ratones , Modelos Estadísticos , Unión Proteica , Receptores de Hidrocarburo de Aril/metabolismo , Elementos de Respuesta , Termodinámica , Factores de Transcripción/metabolismoRESUMEN
The WNT/ß-catenin signaling pathway has been identified as an important endogenous regulator of hepatic cytochrome P450 (P450) expression in mouse liver. In particular, it is involved in the regulation of P450 expression in response to exposure to xenobiotic agonists of the nuclear receptors constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), and Nrf2. To systematically elucidate the effect of the WNT/ß-catenin pathway on the regulation and inducibility of major human P450 enzymes, HepaRG cells were treated with either the WNT/ß-catenin signaling pathway agonist, WNT3a, or with small interfering RNA directed against ß-catenin, alone or in combination with a panel of activating ligands for AhR [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)], CAR [6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO)], pregnane X receptor (PXR) [rifampicin], and peroxisome proliferator-activated receptor (PPAR) α [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY14,643)]. Assessment of P450 gene expression and enzymatic activity after downregulation or activation of the WNT/ß-catenin pathway revealed a requirement of ß-catenin in the AhR-, CAR-, and PXR-mediated induction of CYP1A, CYP2B6 and CYP3A4 (for CAR and PXR), and CYP2C8 (for PXR) gene expression. By contrast, activation of the WNT/ß-catenin pathway prevented PPARα-mediated induction of CYP1A, CYP2C8, CYP3A4, and CYP4A11 genes, suggesting a dominant-negative role of ß-catenin in PPARα-mediated regulation of these genes. Our data indicate a significant effect of the WNT/ß-catenin pathway on the regulation of P450 enzymes in human hepatocytes and reveal a novel crosstalk between ß-catenin and PPARα signaling pathways in the regulation of P450 expression.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , PPAR alfa/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Carcinoma Hepatocelular , Línea Celular Tumoral , Receptor de Androstano Constitutivo , Humanos , Isoenzimas/biosíntesis , Neoplasias Hepáticas , Receptor X de Pregnano , ARN Interferente Pequeño/metabolismo , Transducción de Señal , beta Catenina/genéticaRESUMEN
Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4'-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4'-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4'-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4'-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.
Asunto(s)
Luciérnagas/enzimología , Luciferasas de Luciérnaga/antagonistas & inhibidores , Luciferasas de Luciérnaga/metabolismo , Sustancias Luminiscentes/metabolismo , FN-kappa B/antagonistas & inhibidores , Estilbenos/farmacología , Animales , Línea Celular Tumoral , Copépodos/enzimología , Concentración 50 Inhibidora , Luciferasas/antagonistas & inhibidores , Luciferasas/metabolismo , Luciferasas de Renilla/antagonistas & inhibidores , Luciferasas de Renilla/metabolismo , Ratones , Renilla/enzimología , ResveratrolRESUMEN
At high concentrations, tert-butylhydroquinone (tBHQ), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. Here we describe that treatment of murine 3T3 cells with 300 µg/ml of tBHQ in 96-well culture plates induces the death of untreated cells in neighboring wells on the same plate. The mechanisms underlying that effect were investigated. Death of the seemingly untreated neighboring cells was caused by the more toxic and volatile tBHQ oxidation product tert-butyl-p-benzoquinone (tBQ) present at up to 3 µg/ml in the untreated neighboring wells. tBQ was formed from tBHQ in a non-enzymatic process involving copper ions and oxygen. The unexpected perturbation of cytotoxicity testing following treatment with tBHQ by its volatile metabolite tBQ shows that not only metabolic processes but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. Furthermore, our data show that even cells several wells away from the treated wells do not necessarily constitute proper "untreated" controls when cells are treated with the frequently used compound tBHQ. This might lead to an underestimation of the effects observed on the Nrf2 signaling pathway, where tBHQ is frequently used as an inductor in vitro.