Autophagic cell death induced by reactive oxygen species is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells.

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.

Alcoholic liver disease. Are there any differences between China and Western countries in clinical features?

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.

Casticin, a flavonoid, potentiates TRAIL-induced apoptosis through modulation of anti-apoptotic proteins and death receptor 5 in colon cancer cells.

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.

Hyperthermia inhibits hypoxia-induced epithelial-mesenchymal transition in HepG2 hepatocellular carcinoma cells.

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 43 °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.

Hyperthermia inhibits transforming growth factor beta-induced epithelial-mesenchymal transition (EMT) in HepG2 hepatocellular carcinoma cells.

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.

Inhibition of 12-lipoxygenase reduces proliferation and induces apoptosis of hepatocellular carcinoma cells in vitro and in vivo.

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.

5-Lipoxygenase contributes to the progression of hepatocellular carcinoma.

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.

Hepatic oval cells activated by hepatocyte apoptosis in diethylnitrosamine-induced rat liver cirrhosis.

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.

Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanol-induced liver injury.

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.

Tea polyphenols inhibit expressions of iNOS and TNF-alpha and prevent lipopolysaccharide-induced liver injury in rats.

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.

Modulation of liver oxidant-antioxidant system by ischemic preconditioning during ischemia/reperfusion injury in rats.

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.

Effects of PPARg agonist pioglitazone on rat hepatic fibrosis.

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.