RESUMEN
CONTEXT: 5-Caffeoylquinic acid (5-CQA) is one of the most abundant compounds found in natural foods including coffee. OBJECTIVE: We investigated whether 5-CQA had a cytoprotective effect through the NF-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) signalling pathway. MATERIALS AND METHODS: Nrf2 activation in response to 5-CQA treatment at the concentration of 10-100 µM is evaluated by Western blotting of Nrf2 and ARE reporter gene assay as well as its target gene expression in HepG2 cells. Intracellular reactive oxygen species (ROS) and glutathione (GSH) levels were measured in the tert-butyl hydroperoxide-induced hepatocytes to examined cytoprotective effect of 5-CQA (10-100 µM). The specific role of 5-CQA on Nrf2 activation was examined using Nrf2 knockout cells or Nrf2 specific inhibitor, ML-385. RESULTS: Nuclear translocation of Nrf2 is increased by 5-CQA in HepG2 cells which peaked at 6 h. Consequently, 5-CQA significantly increases the ARE reporter gene activity and downstream antioxidant proteins, including glutamate cysteine ligase (GCL), hemeoxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1, and Sestrin2. Nrf2 deficiency or inhibition completely antagonized ability of 5-CQA to induce HO-1 and GCL expression. Cells pre-treated with 5-CQA were rescued from tert-butyl hydroperoxide-induced ROS production and GSH depletion. Nrf2 activation by 5-CQA was due to increased phosphorylation of MAPKs, AMPK and PKCδ. DISCUSSION AND CONCLUSIONS: Taken together, our results demonstrate that as a novel Nrf2 activator, 5-CQA, may be a promising candidate against oxidative stress-mediated liver injury. Additional efforts are needed to assess 5-CQA, as a potential therapeutic in liver diseases in vivo and in humans.
Asunto(s)
Muerte Celular/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ácido Quínico/análogos & derivados , Elementos de Respuesta Antioxidante/efectos de los fármacos , Antioxidantes/metabolismo , Relación Dosis-Respuesta a Droga , Técnicas de Inactivación de Genes , Glutatión/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Sustancias Protectoras/administración & dosificación , Sustancias Protectoras/farmacología , Ácido Quínico/administración & dosificación , Ácido Quínico/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Lipin1 was identified as a phosphatidate phosphatase enzyme, and it plays a key role in lipid metabolism. Since free radicals contribute to metabolic diseases in the liver, this study investigated the effects of free radicals on the regulation of Lipin1 expression in Huh7 and AML12 cells. Hydrogen peroxide induced mRNA and protein expression of Lipin1 in Huh7 cells, which was assayed by quantitative RT-PCR and immunoblotting, respectively. Induction of Lipin1 by hydrogen peroxide was confirmed in AML12 cells. Hydrogen peroxide treatment significantly increased expression of sterol regulatory element-binding protein (SREBP)-2, but not SREBP-1. Moreover, nuclear translocation of SREBP-2 was detected after hydrogen peroxide treatment. Hydrogen peroxide-induced Lipin1 or SREBP-2 expression was significantly reduced by N-acetyl-l-cysteine treatment, indicating that reactive oxygen species (ROS) were implicated in Lipin1 expression. Next, we investigated whether the hypoxic environments that cause endogenous ROS production in mitochondria in metabolic diseases affect the expression of Lipin1. Exposure to hypoxia also increased Lipin1 expression. In contrast, pretreatment with antioxidants attenuated hypoxia-induced Lipin1 expression. Collectively, our results show that ROS activate SREBP-2, which induces Lipin1 expression.
RESUMEN
5-Fluorouracil (5-FU) is a chemotherapeutic agent used in the treatment of colorectal cancer. In this study, we investigated whether 5-FU induces Sestrin2 (SESN2), an antioxidant enzyme, and the role of SESN2 in 5-FU action in colon cancer cells. We found that 5-FU upregulated SESN2 protein expression in both HCT116 and HT29 cells. It also increased transcripts of SESN1 and SESN2, but not of SESN3. Furthermore, we investigated whether production of reactive oxygen species (ROS) was involved in 5-FU-induced SESN2 expression. 5-FU did not increase ROS production nor affect Nrf2 phosphorylation and expression levels. Moreover, SESN2 upregulation by 5-FU was not prevented by pretreatment with antioxidants. Next, we investigated p53 levels after 5-FU treatment to elucidate the regulation of SESN2 by 5-FU. An increase in p53 levels was detected following 5-FU treatment; pifithrin-α, an inhibitor of p53 activation, reversed 5-FU-induced SESN2 expression. 5-FU prevented serum-induced in vitro cell migration, but knockdown of SESN2 or treatment with pifithrin-α reversed a 5-FU-mediated decrease in cell migration. Taken together, our results suggest that 5-FU increases SESN2 levels via a p53-dependent pathway, which contributes to inhibition of cancer cell migration in vitro.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Fluorouracilo/farmacología , Proteínas Nucleares/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antioxidantes/farmacología , Benzotiazoles/farmacología , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HT29 , Proteínas de Choque Térmico/genética , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Nucleares/genética , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Tolueno/análogos & derivados , Tolueno/farmacología , Activación Transcripcional , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Regulación hacia ArribaRESUMEN
Isorhamnetin is a flavonoid metabolite of quercetin and isolated from water dropwort (Oenanthe javanica, Umbelliferae). It has been reported that isorhamnetin exerts beneficial effects including antioxidant, anti-inflammatory, and anti-proliferative activities. The present study investigated whether the antioxidant activity of isorhamnetin is correlated with its anti-cancer effects on colorectal cancer cells. Isorhamnetin significantly repressed cobalt chloride (CoCl2)- or hypoxia-induced hypoxia inducible factor-1α (HIF-1α) accumulation in HCT116 and HT29 cells. When compared with quercetin, isorhamnetin showed potent inhibition of HIF-1α. Moreover, it inhibited CoCl2-induced activity of hypoxia response element reporter gene and HIF-1α-dependent transcription of genes such as glucose transporter 1, lactate dehydrogenase A, carbonic anhydrase-IX, and pyruvate dehydrogenase kinase 1. Isorhamnetin also blocked hydrogen peroxide (H2O2)-induced HIF-1α accumulation. The antioxidant effects of isorhamnetin were confirmed by observation of CoCl2- or H2O2-induced reactive oxygen species (ROS) production. Consistently, overexpressed HIF-1α was decreased by isorhamnetin or N-acetyl-L-cysteine in HEK293 cells. In vitro migration and invasion assay further confirmed the inhibitory effects of isorhamnetin on cancer cells. Collectively, these results demonstrate that isorhamnetin inhibits ROS-mediated HIF-1α accumulation, which contributes to its anti-metastatic efficacy.
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Antineoplásicos/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Quercetina/análogos & derivados , Movimiento Celular/efectos de los fármacos , Cobalto , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Quercetina/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Sestrin2 (SESN2) is an antioxidant protein that modulates cellular redox homeostasis through regeneration of peroxiredoxins. It has beneficial effects in oxidative or metabolic stress conditions as an upstream regulator of AMP-activated protein kinase (AMPK). Since hypoxia causes oxidative and metabolic stress, this study investigated the effect of SESN2 on signaling pathways altered by hypoxia in colon cancer cells. SESN2 overexpression in HEK293 cells inhibited hypoxia-inducible factor-1α (HIF-1α), which plays a crucial role in tumor growth and development in hypoxia. Moreover, infection with adenovirus-SESN2 (Ad-SESN2) decreased hypoxia or CoCl2-induced HIF-1α accumulation in colorectal cancer cells. Ad-SESN2 also reduced CoCl2-induced hypoxia response element (HRE)-luciferase activity and mRNA level of HIF-1α-driven genes. Furthermore, Ad-SESN2 infected cells showed anti-metastatic effects in serum-induced cell migration and invasion in vitro. Ad-SESN2 facilitated the ubiquitination of HIF-1α protein and increased hydroxyl-HIF-1α (OH-HIF-1α) level. In contrast, treatment with dimethyloxalylglycine (DMOG), an inhibitor of prolyl hydroxylase (PHD), reversed Ad-SESN2-induced OH-HIF-1α and subsequently suppressed HIF-1α level. The inhibitory effects of SESN2 on the serum-induced in vitro cell migration and invasion were also abrogated by DMOG treatment. Furthermore, knockdown of AMPKα reversed Ad-SESN2-mediated increase of OH-HIF-1α and inhibition of HIF-1α. Dominant-negative form of AMPK also restored the Ad-SESN2 mediated decrease in HIF-1α accumulation. Lastly, Ad-SESN2 suppressed tumor growth in a mouse xenograft model. Taken together, these results suggest that SESN2 increases degradation of HIF-1α via AMPK-PHD regulation that contributes to inhibition of in vitro and in vivo tumorigenesis.
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Proteínas Quinasas Activadas por AMP/genética , Neoplasias Colorrectales/terapia , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Nucleares/genética , Prolil Hidroxilasas/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Aminoácidos Dicarboxílicos/farmacología , Animales , Hipoxia de la Célula/genética , Movimiento Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Proteínas Nucleares/metabolismo , Prolil Hidroxilasas/metabolismo , Elementos de Respuesta , Transducción de Señal , UbiquitinaciónRESUMEN
Compound C is a widely used chemical inhibitor that down-regulates AMP-activated protein kinase (AMPK) activity. However, it has been suggested that compound C exerts AMPK-independent effects in various cells. Here, we investigated whether compound C induces Sestrin2 (SESN2), an antioxidant enzyme induced by diverse stress. In addition, the mechanism responsible for SESN2 induction by compound C was determined. Our results showed that compound C increased SESN2 protein expression in HepG2 cells in a concentration- and time-dependent manner. The induction of SESN2 mRNA was also observed in cells treated with compound C. Increase of SESN2 luciferase activity confirmed transcriptional regulation by compound C and this substance also increased nuclear factor erythroid 2 (NF-E2)-related factor-2 (Nrf2) phosphorylation, which implies that Nrf2 was involved in SESN2 induction. Next, we sought to demonstrate whether production of reactive oxygen species (ROS) accompanied SESN2 expression. Compound C increased ROS production, but this effect was prevented by pretreatment with antioxidants or the mitochondrial complex I inhibitor. Moreover, cyclosporin A, an inhibitor of pore formation in the mitochondrial membrane, attenuated compound C-induced SESN2 induction. However, overexpression of a constitutively active form of AMPK was not able to abolish SESN2 induction by compound C, which implies that its action is independent of AMPK inhibition. In conclusion, this is the first study demonstrating that compound C alters mitochondrial function and induces ROS production, which ultimately leads to phosphorylation of Nrf2 and induction of SESN2.
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Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Mitocondrias/efectos de los fármacos , Proteínas Nucleares/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Células Hep G2 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/fisiología , Proteínas Nucleares/genética , ARN Mensajero/metabolismoRESUMEN
Sestrin2 (SESN2) regulates redox-homeostasis and apoptosis in response to various stresses. Although the antioxidant effects of SESN2 have been well established, the roles of SESN2 in mitochondrial function and metabolic stress have not yet been elucidated. In this study, we investigated the role of SESN2 in mitochondrial dysfunction under glucose deprivation and related signaling mechanisms. Glucose deprivation significantly upregulated SESN2 expression in hepatocyte-derived cells. Antioxidant treatments repressed SESN2 induction under glucose deprivation, this result suggested that reactive oxygen species (ROS) production was involved in SESN2 induction. Moreover, NF-E2-related factor-2 (Nrf2) phosphorylation was accompanied in induction of SESN2 by glucose deprivation. To elucidate the functional role of SESN2, we examined cells that stably overexpressed SESN2. Overexpression of SESN2 inhibited glucose deprivation-induced ROS production and cell death. In addition, under glucose deprivation, the changes in mitochondrial membrane potential, ADP/ATP ratio, and mitochondrial DNA content were significantly restored in SESN2-overexpressing cells. Moreover, siRNA knockdown of SESN2 failed to prevent mitochondrial permeability transition by glucose depletion. Mechanistic investigation showed that glucose deprivation significantly increased AMP-activated protein kinase (AMPK) activation. The recovery of mitochondrial function under glucose deprivation in SESN2-overexpressing cells was not seen in SESN2-overexpressing cells transfected with a dominant-negative AMPK; this result suggested that AMPK activation was responsible for SESN2-mediated mitochondrial protection against glucose deprivation. Treatment with 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR, an AMPK activator) also provided cytoprotective effects against glucose deprivation. Our findings provide evidence for the functional importance of SESN2-AMPK activation in the protection of mitochondria and cells against glucose deprivation-induced metabolic stress.
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Proteínas Quinasas Activadas por AMP/metabolismo , Glucosa/farmacología , Mitocondrias/efectos de los fármacos , Proteínas Nucleares/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas Activadas por AMP/química , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Apoptosis/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Células Hep G2 , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Mitocondrias/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ribonucleótidos/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Degradation of glucose is aberrantly increased in hyperglycemia, which causes various harmful effects on the liver. Methylglyoxal is produced during glucose degradation and the levels of methylglyoxal are increased in diabetes patients. In this study we investigated whether methylglyoxal induces mitochondrial impairment and apoptosis in HepG2 cells and induces liver toxicity in vivo. Methylglyoxal caused apoptotic cell death in HepG2 cells. Moreover, methylglyoxal significantly promoted the production of reactive oxygen species (ROS) and depleted glutathione (GSH) content. Pretreatment with antioxidants caused a marked decrease in methylglyoxal-induced apoptosis, indicating that oxidant species are involved in the apoptotic process. Methylglyoxal treatment induced mitochondrial permeability transition, which represents mitochondrial impairment. However, pretreatment with cyclosporin A, an inhibitor of the formation of the permeability transition pore, partially inhibited methylglyoxal-induced cell death. Furthermore, acute treatment of mice with methylglyoxal increased the plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicating liver toxicity. Collectively, our results showed that methylglyoxal increases cell death and induces liver toxicity, which results from ROS-mediated mitochondrial dysfunction and oxidative stress.
RESUMEN
Methylglyoxal is found in high levels in the blood and other tissues of diabetic patients and exerts deleterious effects on cells and tissues. Previously, we reported that resveratrol, a polyphenol in grapes, induced the expression of Sestrin2 (SESN2), a novel antioxidant protein, and inhibited hepatic lipogenesis. This study investigated whether resveratrol protects cells from the methylglyoxal-induced toxicity via SESN2 induction. Methylglyoxal significantly induced cell death in HepG2 cells. However, cells pretreated with resveratrol were rescued from methylglyoxal-induced apoptosis. Resveratrol attenuated glutathione (GSH) depletion and ROS production promoted by methylglyoxal. Moreover, mitochondrial damage was observed by methylglyoxal treatment, but resveratrol restored mitochondrial function, as evidenced by the observed lack of mitochondrial permeability transition and increased ADP/ATP ratio. Resveratrol treatment inhibited SESN2 depletion elicited by methylglyoxal. SESN2 overexpression repressed methylglyoxal-induced mitochondrial dysfunction and apoptosis. Likewise, rotenone-induced cytotoxicity was not observed in SESN2 overexpressed cells. Furthermore, siRNA knockdown of SESN2 reduced the ability of resveratrol to prevent methylglyoxal-induced mitochondrial permeability transition. In addition, when mice were exposed to methylglyoxal after infection of Ad-SESN2, the plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and GSH depletion by methylglyoxal in liver was reduced in Ad-SESN2 infected mice. Our results demonstrated that resveratrol is capable of protecting cells from methylglyoxal-induced mitochondrial dysfunction and oxidative stress via SESN2 induction.
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Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas Nucleares/fisiología , Piruvaldehído/toxicidad , Estilbenos/farmacología , Adenoviridae/genética , Animales , Productos Finales de Glicación Avanzada/metabolismo , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Mitocondrias/fisiología , Proteínas Nucleares/genética , Especies Reactivas de Oxígeno/metabolismo , ResveratrolRESUMEN
Previously, we reported that isorhamnentin, a 3'-O-methylated metabolite of quercetin, reduced inducible nitric oxide synthase (iNOS) expression and NO production. The present study further investigated the underlying mechanism of anti-inflammatory and antioxidant effects of isorhamnentin. Administration of isorhamnetin decreased the number of cyclooxygenase-2 (COX-2) positive cells in rats with carrageenan-induced paw edema. Isorhamnetin also suppressed lipopolysaccharide (LPS)-induced expression of COX-2 in cells. It is well known that LPS-induced reactive oxygen species (ROS) production leads to COX-2 induction. Isorhamnetin decreased LPS-induced ROS production and apoptosis. In addition, the basal expression of heme oxygenase-1 (HO-1) was increased by isorhamnetin treatment in agreement with the increase in nuclear translocation of NF-E2-related factor-2 (Nrf2), an essential transcription factor for the regulation of HO-1 expression. Moreover, pretreatment of tin protoporphyrin IX (SnPP), a chemical inhibitor of HO-1, reversed the ability of isothamnetin to inhibit COX-2 expression. These results demonstrate that induction of HO-1 by isorhamnetin leads to a reduction in ROS production and its antioxidant property might contribute to the inhibition of COX-2 expression in response to inflammation.
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Antioxidantes/farmacología , Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/biosíntesis , Hemo Oxigenasa (Desciclizante)/metabolismo , Quercetina/análogos & derivados , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Edema/inducido químicamente , Edema/tratamiento farmacológico , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo Oxigenasa (Desciclizante)/biosíntesis , Inflamación/inmunología , L-Lactato Deshidrogenasa/metabolismo , Lipopolisacáridos , Macrófagos/enzimología , Macrófagos/inmunología , Metaloporfirinas/farmacología , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Protoporfirinas/farmacología , Quercetina/farmacología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Liver X receptor-α (LXRα), a member of the nuclear receptor superfamily of ligand-activated transcription factors, regulates de novo fatty acid synthesis that leads to stimulate hepatic steatosis. Although, resveratrol has beneficial effects on metabolic disease, it is not known whether resveratrol affects LXRα-dependent lipogenic gene expression. This study investigated the effect of resveratrol in LXRα-mediated lipogenesis and the underlying molecular mechanism. Resveratrol inhibited the ability of LXRα to activate sterol regulatory element binding protein-1c (SREBP-1c) and thereby inhibited target gene expression in hepatocytes. Moreover, resveratrol decreased LXRα-RXRα DNA binding activity and LXRE-luciferase transactivation. Resveratrol is known to activate Sirtuin 1 (Sirt1) and AMP-activated protein kinase (AMPK), although its precise mechanism of action remains controversial. We found that the ability of resveratrol to repress T0901317-induced SREBP-1c expression was not dependent on AMPK and Sirt1. It is well established that hepatic steatosis is associated with antioxidant and redox signaling. Our data showing that expression of Sestrin2 (Sesn2), which is a novel antioxidant gene, was significantly down-regulated in the livers of high-fat diet-fed mice. Moreover, resveratrol up-regulated Sesn2 expression, but not Sesn1 and Sesn3. Sesn2 overexpression repressed LXRα-activated SREBP-1c expression and LXRE-luciferase activity. Finally, Sesn2 knockdown using siRNA abolished the effect of resveratrol in LXRα-induced FAS luciferase gene transactivation. We conclude that resveratrol affects Sesn2 gene induction and contributes to the inhibition of LXRα-mediated hepatic lipogenesis.