RESUMO
We previously reported that impaired retinoid signaling causes hepatocellular carcinoma (HCC) through oxidative stress. However, the interaction between oxidative stress and retinoid signaling has not been fully understood. To address this issue, the effects of hydrogen peroxide on the transcriptional activity of RAR/RXR heterodimers, RARα and RXRα proteins and intracellular signaling pathways were examined. The transcriptional activity of RAR/RXR examined by the DR5-tk-Luc reporter assay was significantly suppressed. The RARα protein level began to decrease at 6 h after treatment and declined thereafter. However, RARα mRNA were not changed. Activation of extracellular regulated kinases (ERK), p38, c-Jun N-terminal kinase (JNK) and Akt was observed after treatment of hydrogen peroxide. SP600125, an inhibitor of JNK, reversed the RARα protein level reduced by hydrogen peroxide. Anisomycin, an activator of JNK, reduced RARα protein. Transfection of wild-type JNK-constitutive actively expressing plasmid, but not kinase-negative JNK-expressing plasmid caused reduction of RARα protein. Proteasomal degradation of RARα was observed after anisomycin treatment; however, the mutant RARα, of which phosphorylation sites are replaced with alanines, was not degradated. In hepatitis C virus (HCV)-related human liver tissues, phospho-JNK and RARα reciprocally expressed with the progression of liver disease. Finally, the staining of 8-OHdG and thioredoxin was increased with the disease progression. These data indicate that JNK activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of RARα, suggesting that a vicious cycle between aberrant retinoid signaling and oxidative stress accelerates hepatocarcinogenesis.
Assuntos
Ativação Enzimática/fisiologia , Hepatócitos/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Western Blotting , Humanos , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Oxidantes/farmacologia , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides/metabolismo , Retinoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
BACKGROUND & AIMS: We previously reported that impaired retinoid signaling in the liver causes steatohepatitis and hepatocellular carcinoma. Recently, oxidative stress induced by hepatic iron overload has emerged as an important factor for the progression of liver disease in patients with chronic hepatitis C, alcoholic liver disease, and nonalcoholic steatohepatitis. In this study, the relationship between retinoid signaling and iron metabolism in the liver was investigated. METHODS: The effect of retinoids on the iron metabolism was examined in HuH7 cells treated with all-trans retinoic acid and acyclic retinoid NIK-333. In in vivo experiments, we used the mice expressing the dominant negative form of retinoic acid receptor alpha gene under the control of albumin enhancer/promoter (RAR-E Tg) and iron-overloaded wild mice fed with retinoid-deficient and retinoid-excess diets. RESULTS: Hepatic iron accumulation and increased expression of hemojuvelin were observed in RAR-E Tg mouse liver. Retinoid treatment significantly suppressed expression of hemojuvelin and mildly suppressed expression of transferrin receptor type 2 and hepcidin, accompanied by decreased hepatic iron content and iron-induced oxidative stress in vitro and in vivo. Overexpression of hemojuvelin in HuH7 hepatoma cells led to a significant increase in cellular iron content. CONCLUSIONS: Our results suggest that retinoids are involved in hepatic iron metabolism through transcriptional regulation of hemojuvelin. This study demonstrated a novel functional role of retinoids in preventing iron-induced oxidative stress in the liver.
Assuntos
Ferro/metabolismo , Fígado/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Retinoides/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Hepatite C Crônica/metabolismo , Hepcidinas , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/análise , Receptores do Ácido Retinoico/fisiologia , Receptores da Transferrina/genética , Receptor alfa de Ácido Retinoico , Retinoides/administração & dosagemRESUMO
Retinoids play an important role in the regulation of cell growth and death. Synthetic retinoid CD437 reportedly induces apoptosis in various cancer cell lines. However, the mechanism of inducing apoptosis in hepatocellular carcinoma (HCC) cells by this agent remains to be clarified. In this study, we investigated the signaling pathway by which CD437 induces apoptosis in HCC cell lines. Apoptosis of six human HCC cell lines was induced by treatment with CD437. Caspase-3 and -9 were activated by CD437, suggesting that the apoptosis is mediated by mitochondrial pathways. Consistent with these findings, the treatment with CD437 upregulated Bax protein, downregulated Bcl-2 protein and released cytochrome c into the cytoplasm. Moreover, rhodamine123 staining revealed mitochondrial depolarization in the cells treated with CD437. These data of the present study suggest that CD437 induces apoptosis in HCC cells via mitochondrial pathways.
Assuntos
Apoptose , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Mitocôndrias/efeitos dos fármacos , Retinoides/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Humanos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
A synthetic retinoid, CD437, has been shown to exert potent anti-tumor activity against various types of cancer cell lines, regardless of their sensitivities to natural retinoids. We herein demonstrate that CD437 induces endoplasmic reticulum (ER) stress, including the up-regulation of CHOP, BIP and GADD34 mRNA through ER stress transducer (PERK and IRE1alpha) activation in an ovarian adenocarcinoma cell line, SKOV3. It was also shown that CD437 induced the CHOP and GADD34 expressions in another four ovarian adenocarcinoma cell lines, indicating that CD437 functions as an ER stress inducer in these cell lines. Moreover, the siRNA-mediated knockdown of inducible CHOP expression prevented the cytotoxic effect of CD437. These results suggest that ER stress plays an important role in the mechanism by which CD437 induces apoptosis in ovarian adenocarcinoma cells.
Assuntos
Adenocarcinoma/metabolismo , Apoptose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Neoplasias Ovarianas/metabolismo , Retinoides/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Feminino , Humanos , Estresse Oxidativo/efeitos dos fármacosRESUMO
Mesenchymal stem cells (MSCs) are an attractive cell source for cell therapy. Based on our hypothesis that suppression of Wnt/ß-catenin signal enhances hepatic differentiation of human MSCs, we developed human mesenchymal stem cell-engineered hepatic cell sheets by a small molecule compound. Screening of 10 small molecule compounds was performed by WST assay, TCF reporter assay, and albumin mRNA expression. Consequently, hexachlorophene suppressed TCF reporter activity in time- and concentration-dependent manner. Hexachlorophene rapidly induced hepatic differentiation of human MSCs judging from expression of liver-specific genes and proteins, PAS staining, and urea production. The effect of orthotopic transplantation of human mesenchymal stem cell-engineered hepatic cell sheets against acute liver injury was examined in one-layered to three-layered cell sheets system. Transplantation of human mesenchymal stem cell-engineered hepatic cell sheets enhanced liver regeneration and suppressed liver injury. The survival rates of the mice were significantly improved. High expression of complement C3 and its downstream signals including C5a, NF-κB, and IL-6/STAT-3 pathway was observed in hepatic cell sheets-grafted tissues. Expression of phosphorylated EGFR and thioredoxin is enhanced, resulting in reduction of oxidative stress. These findings suggest that orthotopic transplantation of hepatic cell sheets manufactured from MSCs accelerates liver regeneration through complement C3, EGFR and thioredoxin.
Assuntos
Hepatócitos/transplante , Regeneração Hepática/fisiologia , Células-Tronco Mesenquimais/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/mortalidade , Doença Hepática Induzida por Substâncias e Drogas/terapia , Complemento C3/metabolismo , Complemento C5a/metabolismo , Citocinas/metabolismo , Receptores ErbB/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Hexaclorofeno/farmacologia , Humanos , Interleucina-6/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Taxa de Sobrevida , Tiorredoxinas/metabolismo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
AIM: We have recently reported that hyperdynamic state of retinoid metabolism, which may lead to the shortage of retinoid, is observed in patients with non-alcoholic fatty liver disease (NAFLD). Hepatic iron overload, which causes production of reactive oxygen species (ROS), is also frequently seen in NAFLD patients. The aim of the study is to examine iron state and retinoid metabolic state simultaneously, and to clarify the relationship between two disorders. METHODS: Thirty-six persons, comprising 17 patients with simple steatosis (SS), 11 with NASH, and 8 normal controls (N), were examined on hepatic expression of iron metabolism-related genes including hemojuvelin (HJV), hepcidin (HEPC), transferrin receptor 1 and 2 (TfR1, TfR2), ferroportin (FPN), neogenin (NEO) and ferritin heavy chain (FtH) and hepatic iron contents in addition to expression 51 genes which is involved in retinoid metabolism and antioxidative action. RESULTS: In patients with NAFLD, expression of HJV, TfR2, FPN, TfR1, FtH, SOD and catalase was increased, compared with that in N. In addition, hepatic iron content, which was increased in NASH, was correlated with expression level of TfR2. Expression of cellular retinoid binding protein (CRBP1), alcohol dehydrogenase 1 (ADH1) and cytochrome P450 26A1(CYP26A1) was significantly correlated with that of HJV, TfR2 and FPN, respectively. CONCLUSION: The results of the present study suggest that the reasons responsible for iron accumulation in NASH in the present study may partly be due to enhanced expression of TfRs, especially TfR2, and hyperdynamic state of retinoid metabolism is closely related to iron metabolism in the disease.
RESUMO
AIM: The patients with non-alcoholic fatty liver disease (NAFLD) have been reported to be at greater risk for progression to chronic liver disease including liver cirrhosis (LC). To examine the mechanisms for the progression of NAFLD, a genetic analysis of hepatic expression profile in retinoid metabolism in NAFLD was performed since the loss of retinoid signaling is associated with the progression of liver disease via reactive oxygen species (ROS) generation. METHODS: Fifty-one genes, which are associated with retinoid metabolism and action, were examined in thirty six subjects including 17 patients with simple steatosis, 11 with non-alcoholic steatohepatitis (NASH) and eight controls were examined by real-time reverse transcriptase polymerase chain reaction. Immunohistochemical study was also done by 3 kinds of antibodies. RESULTS: Higher expression of CRBP1 LRAT, DGT1/2 and CES1 in NAFLD suggests that mutual conversion between retinyl ester and retinal occurs actively. Expression of ADH1/2/3, RDH5/10/11, DHRS3 and RALDH1/3 was increased in NAFLD, suggesting that oxidation process from retinol to all-trans retinoic acid (ATRA) was enhanced. Importantly, greater expression of CYP26A1 indicated that degradation of ATRA was enhanced in NAFLD. Further, expression of SOD1/2, catalase, thioredoxin and uncoupling protein 2 was also enhanced. CONCLUSION: Hyperdynamic state of retinoid metabolism is present in the liver tissues with NAFLD, which may be a putative mechanism by which NAFLD progresses to chronic liver disease including LC.