RESUMEN
BACKGROUND: Radiation is currently used to be a mainstay of salvage therapy for nasopharyngeal carcinoma (NPC), however, development of radioresistance largely limits the radiation efficacy. Circular RNAs (circRNAs) have been shown to affect NPC progression, but its role in radioresistance remain unclear. METHODS: The circular structure of circFIP1L1(circ_0069740) was verified by RNA-sequencing, RT-PCR based on gDNA or cDNA, RNase R treatment, and actinomycin D treatment. Cellular localization of circFIP1L1 and miR-1253 was detected by nucleoplasmic separation and/or fluorescence in situ hybridization. Expression of non-coding RNAs and mRNAs was detected by qRT-PCR, protein expression was detected by Western blot. Functionally, EdU, CCK-8, and colony formation experiments were employed to assess cell proliferation, flow cytometry was adopted to estimate cell cycle and apoptosis. Xenograft tumor growth was performed to detect the role of circFIP1L1 in vivo. Mechanistically, we examined the interplay between miR-1253 and circFIP1L1 or EIF4A3 through dual-luciferase reporter assay. The potential regulatory impacts of EIF4A3 on circFIP1L1 or PTEN was examined by RNA immunoprecipitation and RNA pull-down assays. RESULTS: CircFIP1L1 overexpression and miR-1253 knockdown repressed NPC cell proliferation, facilitated NPC cell apoptosis, and enhanced NPC radiosensitivity. Mechanistically, circFIP1L1 was revealed to repress miR-1253 by binding to it, and EIF4A3 is a target gene of miR-1253. CircFIP1L1 regulated NPC proliferation, apoptosis, and radiosensitivity through miR-1253/EIF4A3. Moreover, we found that EIF4A3 bound to FIP1L1 mRNA transcript and induced circFIP1L1 formation, and thus stabilizing PTEN mRNA. CONCLUSION: Our findings suggested that EIF4A3-induced circFIP1L1 repressed NPC cell proliferation, facilitated NPC cell apoptosis, and enhanced NPC radiosensitivity by miR-1253.
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MicroARNs , Neoplasias Nasofaríngeas , Línea Celular Tumoral , Proliferación Celular/genética , ARN Helicasas DEAD-box/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , MicroARNs/genética , MicroARNs/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/radioterapia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/radioterapia , ARN Mensajero , Tolerancia a Radiación/genéticaRESUMEN
BACKGROUND/AIMS: EMT plays an essential role in tumor progression and metastasis. Hyperthermia is a potent approach for cancers with low side effects. However, the effect of hyperthermia on EMT of cancer cells is unknown. METHODOLOGY: Cells were treated with TGF-ß1 and epidermal growth factor for 96 h and then exposed to hyperthermia at 43°C for 0.5 h. Cell morphology was observed. Expressions of E-cadherin and vimentin were determined by Western blot. The protein and mRNA expressions of Snail were detected with Western blot and RT-PCR. Cell migratory capacity was evaluated. RESULTS: TGF-ß1 induced EMT in HepG2 cells, which was evidenced by morphological, molecular and functional changes, including the formation of spindle shape and the loss of cell contact. The expression of E-cadherin was decreased but the expression of vimentin increased; also, the migratory capability was increased by 2.1±0.19-fold as compared with untreated cells. However, those effects were inhibited by the treatment of hyperthermia. Furthermore, the protein and mRNA expressions of Snail induced by TGF-ß1 were also significantly inhibited by hyperthermia treatment CONCLUSIONS: Hyperthermia can inhibit TGF-ß1-induced EMT in HepG2 cells, suggesting that hyperthermia may alter the properties of metastatic potential in cancer cells and inhibit tumor metastasis.
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Carcinoma Hepatocelular/metabolismo , Transición Epitelial-Mesenquimal , Hipertermia Inducida , Neoplasias Hepáticas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Antígenos CD , Western Blotting , Cadherinas/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Movimiento Celular , Forma de la Célula , Factor de Crecimiento Epidérmico/metabolismo , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Invasividad Neoplásica , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia Snail , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vimentina/metabolismoRESUMEN
BACKGROUND: 12-lipoxygenase (12-LOX) has been reported to be an important gene in cancer cell proliferation and survival, and tumor metastasis. However, its role in hepatocellular carcinoma (HCC) cells remains unknown. METHODS: Expression of 12-LOX was assessed in a diethyl-nitrosamine-induced rat HCC model, and in SMMC-7721, HepG2 and L-02 cells using immunohistochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR). GST-π and Ki-67 were determined in vivo by immunohistochemical staining. Apoptosis was evaluated by TUNEL assay. Cell viability and apoptosis were determined by MTT assay and flow cytometry, respectively. Apoptosis-related proteins in SMMC-7721 and HepG2 cells were detected by Western blotting. RESULTS: Immunohistochemical staining and RT-PCR showed that 12-LOX was over-expressed in rat HCC and two HCC cell lines, while the expression was inhibited by baicalein, a specific inhibitor of 12-LOX. Baicalein inhibited cell proliferation and induced apoptosis in rat HCC and both cell lines in a dose- and time-dependent manner. Our in vivo study demonstrated that baicalein also reduced neoplastic nodules. Mechanistically, baicalein reduced Bcl-2 protein expression coupled with a slight increase of the expression of Bax and activation of caspase-3. Furthermore, baicalein inhibited the activation of ERK-1/2 (phosphorylated). Interestingly, the effects of baicalein were reversed by 12(S)-HETE, a metabolite of 12-LOX. CONCLUSIONS: Inhibition of 12-LOX leads to reduced numbers of HCC cells, partially caused by increased apoptosis. 12-LOX may be a potential molecular target for HCC prevention and treatment.
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Apoptosis/efectos de los fármacos , Araquidonato 12-Lipooxigenasa/efectos de los fármacos , Carcinoma Hepatocelular/patología , Proliferación Celular/efectos de los fármacos , Flavanonas/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Neoplasias Hepáticas/patología , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Carcinoma Hepatocelular/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/metabolismo , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Wistar , Factores de Tiempo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
AIM: To investigate whether autophagic cell death is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells, and to explore the underlying mechanism. METHODS: Human hepatocellular carcinoma cells were treated with hyperthermia and ionizing radiation. MTT and clonogenic assays were performed to determine cell survival. Cell autophagy was detected using acridine orange staining and flow cytometric analysis, and the expression of autophagy-associated proteins, LC3 and p62, was determined by Western blot analysis. Intracellular reactive oxygen species (ROS) were quantified using the fluorescent probe DCFH-DA. RESULTS: Treatment with hyperthermia and ionizing radiation significantly decreased cell viability and surviving fraction as compared with hyperthermia or ionizing radiation alone. Cell autophagy was significantly increased after ionizing radiation combined with hyperthermia treatment, as evidenced by increased formation of acidic vesicular organelles, increased expression of LC3II and decreased expression of p62. Intracellular ROS were also increased after combined treatment with hyperthermia and ionizing radiation. Pretreatment with N-acetylcysteine, an ROS scavenger, markedly inhibited the cytotoxicity and cell autophagy induced by hyperthermia and ionizing radiation. CONCLUSION: Autophagic cell death is involved in hyperthermic sensitization of cancer cells to ionizing radiation, and its induction may be due to the increased intracellular ROS.
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Autofagia/efectos de la radiación , Carcinoma Hepatocelular/radioterapia , Hipertermia Inducida , Neoplasias Hepáticas/radioterapia , Radiación Ionizante , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Autofagia/efectos de los fármacos , Carcinoma Hepatocelular/patología , Supervivencia Celular/efectos de la radiación , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
AIM: To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-kappaB (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha) expression in the liver. METHODS: Female Sprague-Dawley rats were given fish oil (0.5 mL) along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase (ALT) activity and pathological analysis. Liver malondialdehyde (MDA), nitric oxide contents, iNOS and eNOS activity were determined. NF-kappaB p65ìiNOS, eNOS and TNF-alpha protein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration (6 wk) enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-kappaB p65, iNOS and TNF-alpha protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-kappaB, and TNF-alpha mRNA expression. CONCLUSION: iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-kappaB and TNF-alpha expression. eNOS activity is reduced, but its mRNA expression is not affected.
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Cirrosis Hepática Alcohólica/enzimología , Cirrosis Hepática Alcohólica/genética , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Secuencia de Bases , Etanol/toxicidad , Femenino , Expresión Génica/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Cirrosis Hepática Alcohólica/patología , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Transcripción ReIA/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Tea polyphenols have been shown to protect against carbon tetrachloride (CCl4)-induced liver injury, liver fibrosis, hepatic ischemia-reperfusion injury. In this study, we examined the effect of tea polyphenols on lipopolysaccharide (LPS)-induced liver injury, and explored its mechanisms. METHODS: Sprague-Dawley rats received tea polyphenols (100 mg.kg-1.d-1) or vehicle (water) intragastrically by gavage for 14 days, followed by LPS (5 mg/kg) or saline injection intraperitoneally. Liver injury was assessed by biochemical assay and pathological analysis. Serum tumor necrosis factor-alpha (TNF-alpha) levels and liver malondialdehyde (MDA) contents were determined. Inducible nitric oxide synthase (iNOS) protein and TNF-alpha, iNOS and endothelial nitric oxide synthase (eNOS) mRNA expressions in the liver were detected by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: Administration of LPS resulted in liver injury in rats, evidenced by elevated activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), hepatocellular necrosis, and neutrophil infiltration in the liver. These responses were associated with increased serum TNF-alpha levels, induced iNOS protein, expressions of TNF-alpha, iNOS mRNA in the liver and elevated lipid peroxidation at 90 minutes or 6 hours after LPS injection. Pretreatment with tea polyphenols attenuated LPS-induced liver injury, and blunted the rises of serum TNF-alpha levels and lipid peroxidation and the induction of expressions of TNF-alpha, iNOS in the liver. CONCLUSION: Tea polyphenols prevent LPS-induced liver injury, and the mechanisms may involve the reduction of serum TNF-alpha levels and lipid peroxidation and the suppression of TNF-alpha, iNOS expressions in the liver.
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Flavonoides/farmacología , Hepatopatías/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fenoles/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Análisis de Varianza , Animales , Secuencia de Bases , Biopsia con Aguja , Enfermedad Hepática Inducida por Sustancias y Drogas , Modelos Animales de Enfermedad , Inmunohistoquímica , Lipopolisacáridos , Pruebas de Función Hepática , Masculino , Datos de Secuencia Molecular , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacos , Polifenoles , Probabilidad , ARN Mensajero/análisis , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Técnicas de Cultivo de Tejidos , Factor de Necrosis Tumoral alfa/efectos de los fármacosRESUMEN
AIM: To investigate effects of ischemic pre-conditioning on the liver endogenous oxidant-antioxidant system during ischemia/reperfusion injury. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into sham-operated (Sham), ischemia/reperfusion (I/R), ischemic pre-conditioning plus ischemia/reperfusion (IPC) groups. Serum ALT, AST and hyaluronic acid levels were assayed and pathologic alterations observed. Liver malondialdehyde (MDA) contents, endogenous antioxidant enzymes, superoxidase dismutase (SOD), catalase (CAT), gultathionine peroxidase (GSH-Px) activities, neutrophils accumulation marker, myeloperoxidase (MPO) activities were measured respectively. RESULTS: Compared with I/R group, sinusoidal endothelial cells as well as hepatocytes damages, as assessed biochemically and histochemically, were improved significantly in IPC group; neutrophils infiltration was also markedly reduced. In IPC group, liver peroxidation, as measured by MDA contents, was significantly decreased when compared with I/R group; endogenous antioxidant enzymes, SOD, CAT and GSH-Px activities were markedly higher than that in I/R group. CONCLUSION: Ischemic pre-conditioning exerts protective effects on both hepatic sinusoidal endothelial cells and hepatocytes during liver I/R injury. Its mechanisms may involve dimunition of neutrophils infiltration and modulation of the imbalance of endogenous oxidant-antioxidant system in the organism.
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Antioxidantes/metabolismo , Precondicionamiento Isquémico , Hígado/metabolismo , Daño por Reperfusión/metabolismo , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Ácido Hialurónico/sangre , Hígado/patología , Masculino , Malondialdehído/metabolismo , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Superóxido Dismutasa/metabolismoRESUMEN
AIM: To investigate effects of pioglitazone on rat hepatic fibrosis and to explore its mechanism. METHODS: Rat hepatic fibrosis was induced by carbon tetrachloride (CCl(4)). Forty Sprague-Dawley rats were divided randomly into 4 groups: control, model, and two treatment (PI, PII) groups. Except for rats in control group, all rats were given subcutaneous injection of 400 mL/L CCl(4), twice a wk for 8 wk. Rats in PI and PII groups were also treated with pioglitazone of 3 mg/kg, daily via gastrogavage beginning on the 1(st) day and at the end of the 2(nd) week, administration of CCl(4) respectively. Liver functions (ALT, AST), serum fibrotic markers (HA, LN, PCIII) and hepatic hydroxyproline (HP) concentration were determined respectively. Histochemical staining of formalin-fixed liver sections with HE, Masson-Trichrome, and immunohistochemical staining for alpha-smooth muscle actin (alpha-SMA) were performed. Modified Knodell and Chevallier semi-quantitative scoring system (SSS) was used to evaluate necroinflammatory activity and fibrosis degree. RESULTS: Compared with model group, pioglitazone significantly reduced the serum levels of ALT, AST, HA, LN and PCIII (P<0.05 or <0.01). The HP concentrations in PI (210.90+/-24.07 microg/g), and PII (257.36+/-30.55 microg/g) groups were also lower than those in model group (317.80+/-36.44 microg/g) (P<0.01). Histologic examination showed that PI and PII groups had milder hepatocellular degeneration, necrosis and infiltration of inflammatory cells, and thinner or less fibrotic septa than did model group. The scores for necroinflammation in PI (2.80+/-1.03), and PII (3.00+/-1.05) groups were significantly reduced as compared with model group (4.88+/-2.30) (P<0.05 or <0.01); the fibrosis scores in PI (3.40+/-1.65), and PII (4.60+/-1.35) groups were also markedly lower than those in model group (7.00+/-3.21) (P<0.05 or <0.01). Immunohistochemical staining showed that expression of alpha-SMA in PI and PII groups was ameliorated dramatically compared with model group. CONCLUSION: PPARgamma agonist pioglitazone greatly retards the progression of rat hepatic fibrosis induced by CCl(4) through inhibition of HSC activation and amelioration of hepatocyte necroinflammation in rats.
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Cirrosis Hepática/patología , Cirrosis Hepática/fisiopatología , Receptores Citoplasmáticos y Nucleares/agonistas , Tiazolidinedionas/farmacología , Factores de Transcripción/agonistas , Animales , Hidroxiprolina/metabolismo , Hígado/metabolismo , Hígado/patología , Hígado/fisiopatología , Cirrosis Hepática/metabolismo , Masculino , Pioglitazona , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To investigate the clinical features of alcoholic liver disease (ALD) in hospitalized Chinese patients, and their differences compared with western countries. METHODS: Four hundred and eight hospitalized patients with ALD at First Affiliated Hospital of Zhejiang University College of Medicine, Hangzhou, from January 2008 to December 2010 were studied retrospectively. Clinical data were analyzed and compared with western countries through literature review. RESULTS: The patients with ALD accounted for 7.8% of all hospitalized patients with liver diseases. These patients comprised 400 men and 8 women, aged between 45 and 55 years. Among the patients, there were 318 patients with alcoholic cirrhosis (77.9%), 48 patients with alcoholic hepatitis (11.8%), 9 patients with fatty liver (2.2%), and 33 patients with mild alcoholic injury (8.1%). The abstinence rate in these patients was 37.7%. Logistic-regression analysis showed that daily intake amount, duration of drinking, drinking hard liquors and smoking were the risk factors for alcoholic cirrhosis, but abstinence was the favorable factor for treatment. Compared with western countries, Chinese patients had a lower constituent ratio of ALD among liver diseases, lower proportions of females, and rate of concomitant hepatitis C infection; but the drinking status, clinical manifestations, and abstinence rate were similar between them. CONCLUSION: There are differences as well as similarities between China and western countries in the clinical features of ALD.
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Hepatopatías Alcohólicas/fisiopatología , China , Europa (Continente) , Humanos , Estudios RetrospectivosRESUMEN
We investigated the effect of casticin on apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). We found that casticin potentiated TRAIL-induced apoptosis in human colon cancer cells. Casticin downregulated cell survival proteins including Bcl-xL, Bcl-2, survivin, XIAP and cFLIP, and induced death receptor 5 (DR5), but had no effect on DR4 and decoy receptors (DcR1 or DcR2). Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and casticin. In addition, casticin induced reactive oxygen species (ROS) generation in a dose-dependent manner. Collectively, the present study showed that casticin potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and induction of DR5 mediated by ROS.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias del Colon/metabolismo , Flavonoides/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Análisis de Varianza , Regulación hacia Abajo/efectos de los fármacos , Proteínas Ligadas a GPI/metabolismo , Silenciador del Gen , Células HCT116 , Células HT29 , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Miembro 10c de Receptores del Factor de Necrosis Tumoral , Survivin , Receptores Señuelo del Factor de Necrosis Tumoral/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína bcl-X/metabolismoRESUMEN
AIM: To investigate the effect of hyperthermia on hypoxia-induced epithelial-mesenchymal transition (EMT) in HepG2 hepatocellular carcinoma (HCC) cells, and its mechanism. METHODS: Cells were treated with hyperthermia at 43â °C for 0.5 h, followed by incubation under hypoxic or normoxic conditions for 72 h. Cell morphology was observed. Expressions of E-cadherin and vimentin were determined by immunofluorescence assay or Western blot. The protein and mRNA expressions of Snail were also determined by Western blot and reverse transcription-polymerase chain reaction. Cell migratory capacity was evaluated. RESULTS: Hypoxia induced EMT in HepG2 cells, which was evidenced by morphological, molecular and functional changes, including the formation of a spindle shape and the loss of cell contact. The expression of E-cadherin was decreased but the expression of vimentin was increased; also, the migratory capability was increased by 2.2 ± 0.20-fold as compared with normoxia. However, those effects were inhibited by hyperthermia pretreatment. Furthermore, protein synthesis and mRNA expression of Snail in the cells were enhanced by hypoxia as compared with normoxia, and also significantly inhibited by hyperthermia pretreatment. CONCLUSION: Hyperthermia may inhibit hypoxia-induced EMT in HepG2 HCC cells, and the mechanism may involve inhibition of induced expression of Snail.
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Hipoxia de la Célula , Transición Epitelial-Mesenquimal , Hipertermia Inducida , Cadherinas/análisis , Movimiento Celular , Células Hep G2 , Humanos , Factores de Transcripción de la Familia Snail , Factores de Transcripción/análisis , Vimentina/análisisRESUMEN
5-Lipoxygenase (5-LOX) has been implicated in the development and progression of lung, pancreatic and esophageal cancers. However, its role in hepatocellular carcinoma (HCC) remains unclear. This study aimed to explore the role of 5-LOX in the pathogenesis of HCC. The expression of 5-LOX was detected in human HCC, HepG2 cells and diethylnitrosamine (DEN)-induced rat HCC using immunohistochemistry (IHC) staining or reverse transcriptase-polymerase chain reaction. Apoptosis in rat HCC was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. Cell viability and apoptosis were determined in HepG2 cells by MTT assay and flow cytometry, respectively. IHC staining showed that the 5-LOX protein was highly expressed in human HCC, HepG2 cells and rat HCC, but not in the normal liver tissues. 5-LOX mRNA expression in human and rat HCC was also significantly increased compared to normal liver tissues. Zileuton, a 5-LOX inhibitor, reduced the nodule incidence and the mean number of nodules per nodule-bearing liver in DEN-induced rats. Further study using TUNEL assay showed that zileuton treatment induced apoptosis in the liver as the result of inhibition on 5-LOX levels. This result is consistent with our observation of significantly higher apoptotic indices in rats treated with DEN/zileuton, which were significantly higher compared to those from the control groups. In addition, zileuton reduced cell viability and induced apoptosis in a concentration- and time-dependent manner as detected using HepG2 cells in our in vitro analysis. In conclusion, 5-LOX is expressed in HCC, and the inhibition of 5-LOX blocks the development of HCC via the induction of apoptosis in tumor cells.
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Araquidonato 5-Lipooxigenasa/fisiología , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Animales , Apoptosis , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/metabolismo , Carcinoma Hepatocelular/inducido químicamente , Supervivencia Celular , Dietilnitrosamina/toxicidad , Células Hep G2 , Humanos , Hidroxiurea/análogos & derivados , Hidroxiurea/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Neoplasias Hepáticas/inducido químicamente , ARN Mensajero/metabolismo , RatasRESUMEN
OBJECTIVE: To investigate whether hepatic oval cells are activated in diethylnitrosamine (DEN)-induced rat liver cirrhosis, and to explore its mechanism. METHODS: Liver cirrhosis was induced in rats (n=8) by weekly intraperitoneal injections of DEN at a dose of 50mg/kg body weight for 12 weeks followed by a 2-week wash out period. Rats (n=5) that received isovolumic vehicle served as the control group. Liver pathology was examined. Apoptotic hepatocytes were identified and quantified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. Oval cells were detected using immunohistochemical staining for pyruvate kinase type M2 (M2PK) and cytokeratin 19 (CK19). The work was carried out at Renmin Hospital of Wuhan University, Wuhan, Hubei, China from February to December 2009. RESULTS: Liver cirrhosis developed in rats subjected to DEN administration. The TUNEL and morphology assay showed that a substantial number of hepatocytes underwent apoptosis. The apoptotic index in rats subjected to DEN administration (0.75 0.15) was much higher than normal control rats (0.10 0.05). Both CK19 and M2PK were moderately expressed in the rat liver cirrhosis, and the expression was dispersed or forming small cords in the liver; but the expression was hardly detected in the liver tissue of normal control rats. CONCLUSION: In the DEN-induced rat liver cirrhosis, oval cells are activated and stimulated to proliferation, the mechanism of which may be related to substantial hepatocyte apoptosis in the model.
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Apoptosis , Hepatocitos/metabolismo , Cirrosis Hepática/metabolismo , Animales , Proliferación Celular , Dietilnitrosamina , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Queratina-19/metabolismo , Masculino , Piruvato Quinasa/metabolismo , Ratas , Ratas WistarRESUMEN
Ginkgo biloba extract has been shown to be hepatoprotective. However, its benefits when used in alcoholic liver injury have not been demonstrated. This study investigated the effects of Ginkgo biloba extract on alcohol-induced liver injury in a chronic alcohol plus fish oil gavage model in rats. Liver injury was evaluated histologically and by serum alanine aminotransferase (ALT). Liver tumor necrosis factor-alpha (TNF-alpha) protein levels, malondialdehyde (MDA) and glutathione (GSH) contents were determined. TNF-alpha mRNA expression in the liver was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). Rats given fish oil plus alcohol developed macrovesicular and microvesicular steatosis, spotty necrosis and mild inflammation in the liver and elevated serum ALT levels, which were accompanied by increased MDA contents, reduced GSH contents and elevated TNF-alpha protein and mRNA expressions in the liver. Supplementation with Ginkgo biloba extract (200 mg/kg, orally) improved the liver injury, blunted the rises of MDA contents and TNF-alpha expression, and restored the GSH content in the liver. Ginkgo biloba extract protects against alcohol-induced liver injury, and the mechanism may involve a reduction of lipid peroxidation, prevention of GSH depletion and inhibition of TNF-alpha expression in the liver.