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The cytokine transforming growth factor‑β (TGF‑β) serves a key role in hepatic fibrosis and has cytostatic effects on hepatocytes. The present study investigated the anti‑fibrogenic and regenerative effects of the TGF‑β receptor type I kinase inhibitor galunisertib (LY2157299) in mice with carbon tetrachloride (CCl4)‑induced liver cirrhosis and in vitro. Mice were intraperitoneally treated with CCl4 for 8 weeks. At week 5, the mice were divided randomly into four treatment groups: Vehicle‑treated; and treated with low‑; middle‑; and high‑dose galunisertib, which was administered from weeks 5‑8. The mice were sacrificed after 8 weeks of CCl4 treatment. Liver fibrosis, as evaluated by histology and determination of hydroxyproline content, progressed during week 4‑8 of CCl4 treatment in the vehicle‑treated mice. Galunisertib treatment dose‑dependently prevented liver fibrosis, as demonstrated by the direct inhibition of α‑smooth muscle actin‑positive activated hepatic stellate cells (HSCs) after 8 weeks of CCl4 treatment. The levels of active matrix metalloproteinase (MMP)‑9 in galunisertib‑treated livers were significantly increased compared with the vehicle‑treated livers. In the high‑dose group, the number of PCNA‑positive hepatocytes and endothelial cells markedly increased compared with the vehicle group. Reverse transcription‑quantitative PCR analysis verified that interleukin‑6 and epiregulin expression levels were significantly increased in livers from the group treated with high‑dose galunisertib compared with the vehicle‑treated group. Galunisertib inhibited the proliferation of activated HSCs and collagen synthesis in addition to restoring MMP activity. Moreover, galunisertib promoted liver remodeling by proliferating hepatocytes and vascular endothelial cells, while significantly increasing liver weight. These results are consistent with the cytostatic action of TGF‑β that negatively regulates liver regeneration, and demonstrated that galunisertib inhibited TGF‑β signaling, halted liver fibrosis progression and promoted hepatic regeneration. The results of the present study suggest that galunisertib may be an effective treatment for liver cirrhosis.

The aim of the present study was to quantify calprotectin levels using an enzyme-linked immunosorbent assay (ELISA) and a point-of-care test (POCT) in patients with inflammatory bowel disease. Overall, 113 patients with ulcerative colitis (UC; 51 men and 62 women) and 42 patients with Crohn's disease (CD; 29 men and 13 women), who were scheduled to undergo a colonoscopy, were prospectively enrolled and scored endoscopically and clinically. An additional 96 healthy, age-matched subjects served as the normal controls. Feces and blood samples from the patients with UC and CD, and the normal controls were analyzed. These patients had received adequate medical treatment. The tissue distribution of calprotectin was investigated using immunohistochemistry. The fecal calprotectin levels, as measured using an ELISA, were correlated with the endoscopic and clinical disease activities and laboratory parameters, including serum levels of hemoglobin (Hb), albumin and C-reactive protein, and erythrocyte sedimentation rate, particularly among the patients with UC. The fecal Hb level was close to that of the fecal calprotectin level (r=0.57; P<0.0001). The fecal calprotectin level measured using an ELISA was well-correlated with the fecal calprotectin level measured using the POCT (r=0.81; P<0.0001), but was not correlated with the serum calprotectin level (r=0.1013; P=0.47). An immunohistochemical investigation revealed that patients with both UC and CD had higher neutrophil and monocyte/macrophage calprotectin-positive cell expression levels, compared with those in the normal controls. Fecal calprotectin was considered a reliable marker for disease activity, and the assessment of fecal calprotectin via POCT showed potential as a rapid and simple measurement in clinical settings.

Whole-wheat intake is known to reduce the risk of metabolic syndrome. However, the active component remains unclear. Recently, we identified bioactive peptides [leucine-arginine-proline (LRP) and leucine-glutamine‑proline (LQP)] from wheat bran autolytic hydrolysate. The present study aimed to investigate the effects of LRP and LQP on non-alcoholic steatohepatitis (NASH) in a mouse model. We also evaluated the effects of these peptides on oxidative stress and on the AMP-activated protein kinase (AMPK) signaling pathway, two major pathogenic factors of NASH. Seven‑week-old male C57BL/6 mice were fed a high-fat diet for 10 weeks and administered water supplemented with 0.05% LRP, 0.20% LRP, 0.05% LQP, or 0.20% LQP (each n=5) or distilled water (control; n=5) ad libitum. Oxidative stress was evaluated by measuring the serum levels of diacron reactive oxygen metabolite (d-ROM) and biological antioxidant potential (BAP). Hepatic expression of phosphorylated AMPK and phosphorylated acetyl-CoA carboxylase (ACC) were evaluated by immunoblotting. The result showed that non‑alcoholic fatty liver disease activity score was significantly decreased in all types of treatment. Serum d-ROM levels were significantly decreased in the 0.20% LRP group, but not in the 0.05% LRP, 0.05% LQP, and 0.20% LQP groups. Serum BAP levels were significantly increased in the 0.05% LRP and 0.20% LRP groups, but not in the 0.05% LQP and 0.20% LQP groups. Immunoblotting analysis revealed that the expression of phospho-AMPK was increased whereas that of phospho-ACC was decreased in the 0.20% LQP group. In conclusion, we demonstrated that both LRP and LQP alleviated the severity of NASH in a high-fat diet-induced NASH mouse model. In addition, we showed that LRP and LQP modulated oxidative stress and upregulated AMPK/ACC, respectively. Thus, LRP and LQP may constitute clinically applicable therapeutic agents for NASH.

Ulcerative colitis (UC) patients are well known to carry a higher risk of developing colorectal dysplasia/cancer. However, it is hard to detect the lesion in the early phase during colonoscopy. This pilot study was conducted to analyze the endoscopic characteristics of neoplastic lesions associated with UC using advanced imaging techniques. This is a retrospective analysis of 15 colorectal neoplastic lesion obtained from 11 UC patients during remission who underwent white-light- and advanced endoscopic imaging techniques, including chromoendoscopy, narrow-band imaging and autofluorescence imaging (AFI), and were treated with surgery. These lesions were analyzed for histology, location, size, shape, color and endoscopic features. The green/red ratio was also assessed to quantify the AFI intensity. All 11 patients had extensive colitis with the median disease duration of 14.0 years. A total of 15 lesions, consisting of 8 high-grade dysplasia and 7 cancer, was mostly located in the distal colon (86.7%, 13/15) with the mean size of 8.6 mm. The shape was protruding in 46.7% (7/15), flat elevated in 40.0% (6/15) and flat in 13.3% (2/15) and the color was red in 60.0% (9/15), same colored in 33.3% (5/15) and discolored in 6.7% (1/15). The lesion predominantly showed Kudo's neoplastic pit pattern in 86.7% (13/15; 5 type IIIL, 7 type IV and 1 type VI) on chromoendoscopy and Sano's neoplastic capillary pattern (type IIIa) in 63.6% (7/11) on narrow-band imaging, but were colored purple as neoplastic lesions in only 37.5% (3/8) on AFI. Of note, the AFI green/red ratio was significantly lower in the neoplastic lesions than UC-involved areas (p=0.00014) and UC-uninvolved areas (p=0.00651) irrespective of the lesion's size and histological type. In conclusion, endoscopic analysis based on advanced imaging, in particular AFI quantitation, may be helpful to detect early stage neoplastic lesions in long standing UC. Large-scale, prospective studies are needed.

Survival rates are low in patients with poorly differentiated hepatocellular carcinoma (HCC). Fucoidan, a sulfated polysaccharide derived from brown seaweed, has anticancer activity; however, the effects of fucoidan on poorly differentiated HCC remain unclear. In this study, we investigated the effects of fucoidan on AMP-activated protein kinase (AMPK), a proliferation regulator, and its downstream metabolism- and cell cycle-related molecules in a poorly differentiated human hepatoma HLF cell line. HLF cells were treated with fucoidan (10, 50, or 100 µg/ml; n=4) or phosphate buffered saline (control; n=4) for 96 h. Proliferation was evaluated by counting cells every 24 h. AMPK, TSC2, mTOR, GSK3β, acetyl-CoA carboxylase (ACC), ATP-citrate lyase, p53, cyclin D1, cyclin-dependent kinase (CDK) 4, and CDK6 expression and/or phosphorylation were examined by immunoblotting 24 h after treatment with 100 µg/ml fucoidan. Cell cycle progression was analyzed by fluorescence-activated cell sorter 48 h after treatment. Treatment with 50 or 100 µg/ml fucoidan significantly and dose- and time-dependently suppressed HLF cell proliferation (P<0.0001). Fucoidan induced AMPK phosphorylation on Ser172 24 h after treatment. Although no differences were seen in expression and phosphorylation levels of TSC2, mTOR, GSK3β, ATP-citrate lyase, and p53 between the control and fucoidan-treated HLF cells, fucoidan induced ACC phosphorylation on Ser79. Moreover, fucoidan decreased cyclin D1, CDK4 and CDK6 expression 24 h after treatment. Furthermore, HLF cells were arrested in the G1/S phase 48 h after fucoidan treatment. We demonstrated that fucoidan suppressed HLF cell proliferation with AMPK phosphorylation. We showed that fucoidan phosphorylated ACC and downregulated cyclin D1, CDK4 and CDK6 expression. Our findings suggest that fucoidan inhibits proliferation through AMPK-associated suppression of fatty acid synthesis and G1/S transition in HLF cells.

Catechins, a major component of green tea extract, have anti-hyperlipidemic effects. The present study investigated the effects of consumption of green tea with high-density catechins in non-alcoholic fatty liver disease (NAFLD) patients. Seventeen patients with NAFLD consumed green tea with high-density catechins, low-density catechins or a placebo for 12 weeks in a randomized double-blind study. Ultrasonography and computed tomography (CT) were performed at baseline and after 12 weeks. Serum alanine aminotransferase (ALT) levels and urine 8-isoprostane were monitored and compared to baseline at 4, 8 and 12 weeks. Body fat was significantly decreased in the high-density catechin group compared with the placebo and low-density catechin groups after 12 weeks of consumption. All the patients in the high-density catechin group showed a significantly improved liver-to-spleen CT attenuation ratio compared with the placebo and low-density catechin groups after 12 weeks of consumption. The high-density catechin group significantly decreased serum ALT levels and reduced urinary 8-isoprostane excretion compared with the placebo and low-density catechin group after 12 weeks of consumption. Based on a reduced proportion of body fat as estimated by bioimpedance measurement, increased liver-to-spleen CT attenuation ratio, decreased serum ALT levels and reduced urinary 8-isoprostane excretion, we concluded that 12 weeks of 700 ml per day of green tea containing >1 g catechin improved liver fat content and inflammation by reducing oxidative stress in patients with NAFLD.

Although overt hepatitis B virus (HBV) infection promotes the onset of hepatocellular carcinoma (HCC) in hepatitis C virus (HCV)-infected patients, the effect of occult HBV infection remains unclear. The aim of this study was to investigate the effect of occult HBV infection on the early-onset of HCC in HCV-infected patients. A total of 173 HCC patients with HCV infection were enrolled and classified into 2 groups according to the median age of HCC onset: the early-onset group (n=91; 61.1±5.6 years) and the late-onset group (n=82; 73.8±3.7 years). Independent factors associated with the early-onset of HCC were assessed by multivariate analysis. In the overall analysis, independent risk factors for the early-onset of HCC were the white blood cell count and alanine aminotransferase level, but not the presence of HBV DNA. In a stratification analysis according to albumin levels of ≥3.5 g/dl, the presence of HBV DNA was a significant independent risk factor for the early-onset of HCC (OR 145.18, 95% CI 1.38-15296.61, P=0.036), whereas the presence of antibodies against hepatitis B core antigen was not found to be a risk factor. The presence of HBV DNA was not a risk factor for the early-onset of HCC in the overall analysis. However, its presence was an independent factor for the early-onset of HCC in HCV-infected patients with an albumin level of ≥3.5 g/dl. Thus, occult HBV infection may accelerate hepatocarcino-genesis in HCV-infected patients with relatively low carcinogenic potential.

In order to improve the prognosis of patients with unresectable pancreatic cancer, there is an urgent need for enhancement of the anticancer effect of gemcitabine (Gem), a first-line drug for the disease. Here, we demonstrated that ligands for peroxisome proliferator-activated receptor γ (PPARγ) such as pioglitazone (Pio) and rosiglitazone potentiated the cytotoxic action of Gem on human pancreatic cancer cells in a dosage-dependent manner. Notably, the synergistic effect was PPARγ-dependent, since the effect was augmented by PPARγ overexpression and was attenuated by both a PPARγ inhibitor (GW9662) and PPARγ-specific siRNA. To further increase the collaborative effect, the histone deacetylase (HDAC) inhibitor valproic acid (VPA), a known potentiator for PPARγ function, was added to the combinatorial treatment, robustly inducing apoptosis mediated by highly expressed death receptors, including Fas/CD95 and DR5. In xenograft tumor experiments in nude mice, Gem plus Pio significantly suppressed tumor growth as compared with the control treatment, while Gem-only treatment did not. Triple treatment with Gem, Pio, and VPA failed to demonstrate a significant antitumor effect when compared with Gem plus Pio in the current setting. Considered together, Gem plus PPARγ ligands, including Pio, may have therapeutic advantage in the treatment of advanced pancreatic cancer. Since Pio is widely used in the treatment of diabetes mellitus, it may become a feasible partner of Gem-based chemotherapy, fine-tuning the strength of the therapy in a dosage-dependent fashion.

In recent years, the number of elderly patients with hepatocellular carcinoma (HCC) has been increasing. The aim of this study was to compare the liver function and the background factors of HCC patients with hepatitis C virus (HCV) infection by generation and to examine the characteristics of this disease in the elderly. A total of 1096 patients (776 men and 320 women) diagnosed with HCV-related HCC at our institution from 1995 to 2006 were divided into 4 groups as follows: D group, 75 years of age or older; C group, 65-74 years of age; B group, 55-64 years of age; A group, 54 years of age or younger, and the liver function and other clinical characteristics were compared among these 4 groups. The average age at initial diagnosis of HCV-related HCC was 66.9 years of age. The A, B, C and D groups were comprised of 87, 363, 514 and 132 patients, respectively. The rate of Child-Pugh class A patients in the D group was significantly higher than that of the other groups (P<0.05). The average levels of ALT, TB and PT-INR in the D group were significantly lower than the levels in the other groups (P<0.05). The average Alb level in the D group was significantly higher than that in the other groups (P<0.05). In conclusion, we found that HCV-related HCC in the elderly occurred against a background of chronic liver disease with mild inflammation and fibrosis.

We examined whether or not epigallocatechin-3-gallate (EGCG) improves liver injury of nonalcoholic steatohepatitis (NASH) model mice expressing nuclear sterol regulatory element-binding protein 1c (nSREBP-1c) in adipose tissue. nSREBP-1c transgenic C57BL6 mice aged 30 weeks were divided into group 1 (no treatment), group 2 (ascorbic acid alone), group 3 (ascorbic acid and 0.05% EGCG), and group 4 (ascorbic acid and 0.1% EGCG). At 42 weeks, we performed measurement of liver weight to body weight, biochemical assays, morphometry of liver specimens, immunohistochemistry for 8-hydro-2'-deoxyguanosine (8-OhdG), and Western blotting for insulin and TNF-alpha signalings. Ratio of liver weight to body weight in the high dose EGCG-treated group (group 4) was significantly lower than those of groups 1 and 2 (p<0.05 and <0.01, respectively). Blood ALT, glucose, total cholesterol, and triglyceride levels of group 4 were significantly low compared with those of the EGCG-non-treated group (groups 1 and 2) (p<0.05, respectively). The degrees of steatosis, inflammation, ballooning hepatocytes and Mallory-Denk bodies in group 4 significantly improved compared with those in other groups (p<0.05, respectively). The 8-OhdG immunolocalization in liver tissues of the group 4 obviously decreased compared with those of groups 2 and 3. For Western blotting, the expressions of insulin receptor substrate-1 (IRS-1) and phosphorylated IRS-1 (pIRS-1) in liver tissues of group 4 increased compared with those of groups 2 and 3. On the other hand, the expressions of pAkt, pIKKbeta and pNF-kappaB decreased compared with those of groups 2 and 3. From these results, EGCG reduces inflammation, insulin resistance and oxidative stress, and suppresses liver injury in nSREBP-1c transgenic mice.

Glucose intolerance frequently is found in hepatocellular carcinoma (HCC) patients with hepatitis C virus (HCV) infection; however, the significance of glucose intolerance remains unclear. In addition, SH2 domain-containing inositol phosphatase (SHIP) 2 is a negative regulator of intracellular insulin signaling; however, changes in SHIP2 expression have not been investigated in HCC. To assess the significance of glucose intolerance, we analyzed 118 HCC patients with HCV infection. Twenty HCC specimens were used for immunoblotting and immunostaining for SHIP2. Patients were classified into two groups: a glucose intolerance group (n=39) and a normal glucose tolerance group (n=79). There was no significant difference in the disease-free survival (P=0.838) or long-term survival (P=0.091) between the groups. However, for males, the cumulative survival rate was significantly lower in the glucose intolerance group (n=22) than that in the normal glucose tolerance group (n=52) (P=0.036). In multivariate analysis, Child-Pugh class (P=0.0003) and glucose intolerance (P=0.036) were identified as statistically significant and independent prognostic factors in males. SHIP2 expression level decreased in HCC compared to that in nontumor tissues. In conclusion, this study is the first to demonstrate the significance of glucose intolerance in prognosis of male HCC patients and down-regulation of SHIP2 expression in HCC.

Hepatocarcinogenesis is closely related to hepatic fibrosis. In this study, we investigated the relationship of type II transforming growth factor-beta receptor (T beta RII) to hepatic fibrosis and hepatocellular carcinoma (HCC). In vivo: liver tissues were obtained from 30 patients (10 chronic hepatitis, 7 cirrhosis, 13 HCC). Protein expression and immunolocalization of T beta RII were examined by Western blot analysis and immunohistochemistry. In vitro: T beta RII protein expression in hepatoma cell lines (HepG2, Hep3B, HLE, HLF and Huh7) was examined by Western blot analysis. Next, we transfected T beta RII cDNA to Huh7, and compared the change of cell number and observed the induction of apoptosis after TGF-beta1 treatment using a FACScan flow cytometer. In vivo: T beta RII immunolocalization in liver tissues was significantly decreased in patients with HCC compared with that of patients with chronic hepatitis or liver cirrhosis. In Western blot analysis, T beta RII expression in tissues attenuated in comparison with that in non-tumor tissues in some patients with HCC. In vitro: T beta RII protein expression in HLE, HLF and Huh7 cells was weaker than that in HepG2 and Hep3B cells. In Huh7 cells transfected T beta RII cDNA, cell arrest and apoptosis were obviously induced. These results indicated that human HCC has a reduced expression of T beta RII for TGF-beta1. This may provide a selective growth advantage to HCC to escape the inhibitory growth signals of TGF-beta1, and may be linked with critical steps in the growth of hepatoma cells.

We evaluated the effects of angiogenesis inhibitor, TNP-470, on hepatocellular carcinoma (HCCs) induced by a choline-deficient L-amino acid defined (CDAA) diet in rats. Male Fisher 344 rats were fed CDAA for 68 weeks. Rats were treated by subcutaneous injection of TNP-470 (15 mg/kg) or saline (control) three times per week from 53 to 68 weeks. At the end of the experiment, we determined the frequency and size of HCCs and glutathione S-transferase placental form (GSTP)-positive lesions, histology of liver cirrhosis, liver function, and liver weight per body weight. We also determined, using histologic and immunohistochemical semiquantification analyses, the degree of vascularity, apoptosis and proliferation in HCC and adjacent tissues. Treatment with TNP-470 resulted in a reduction of the size and frequency of HCC compared to untreated rats. However, TNP-470 did not influence the histology of liver cirrhosis and liver function. The liver weight per body weight of TNP-470-treated rats was slightly heavier in comparison with that of the controls. Treatment with TNP-470 significantly reduced tumor vascularity relative to the controls. There were no significant differences in the Ki-67 labeling index of HCCs between TNP-470 treated and control rats. The frequency of apoptotic hepatoma cells in TNP-470-treated rats was higher than in control rats. Our results indicate that TNP-470 suppresses the progression of CDAA-induced HCCs in rats through inhibition of angiogenesis, and suggest that TNP-470 might be useful clinically for HCCs.

Kupffer cells are major matrix metalloproteinase-producing cells in the liver. The production of metalloproteinases in Kupffer cells may contribute to the improvement of liver fibrosis inducing liver cirrhosis. In this study, we examined the effect of the OK-432 (a biological response modifier) on the dimethylnitrosamine-induced liver cirrhosis in rats. Dimethylnitrosamine (10 microg/ml) was injected intraperitoneally into 20 male Wistar rats 3x/week for 4 weeks. For the subsequent 4 weeks, the animals were injected with saline (1 ml, 1x/week) (Group I, n=10) or OK-432 (1 Klinishe Einheit, 1x/week) (Group II, n=10). The control rats were injected with 1 ml saline for the initial 4 weeks and subsequent 4 weeks (Group III, n=10). The degree of hepatic fibrosis, the immunolocalization of type IV collagen, hyaluronic acid, and alpha-smooth muscle actin, and the mRNA expression by Northern blotting and the activity by gelatin zymography of metalloproteinase-9 were evaluated. Serum aminotransferase, hyaluronic acid, interleukin-1beta and tumor necrosis factor-alpha levels were measured. The deposition of á-smooth muscle actin and extracellular matrix containing type IV collagen and hyaluronic acid was markedly suppressed by OK-432. The mRNA expression and the activity of metalloproteinase-9 were markedly increased by OK-432. The serum aminotransferase and hyaluronic acid levels were decreased by OK-432. The serum interleukin-1 and tumor necrosis factor-alpha values were lower than the detectable limit in all samples from all three groups. These results indicate that OK-432 increased the production of metalloproteinase-9 and improved the rat dimethylnitrosamine-induced liver cirrhosis. OK-432 is suggested to be useful for the treatment of liver cirrhosis.

Carboxy-terminal telopeptide of type I collagen (ICTP) is a degradation product of type I collagen. In this study, we investigated the usefulness of measuring the serum ICTP concentration for diagnosing and monitoring bone metastasis from hepatocellular carcinoma (HCC). The serum concentrations of ICTP, type I procollagen carboxy-terminal propeptide (PICP), type III procollagen aminoterminal propeptide (PIIIP), type IV collagen (Ty IV), type IV collagen 7S-domain (7S), and hyaluronic acid (HA) were measured in patients with liver cirrhosis, HCC with or HCC without bone metastasis, and in healthy controls. The diagnostic efficiency of the serum ICTP and fibrosis marker levels in the HCC patients with and without bone metastasis was evaluated using receiver operating characteristic curves. We also retrospectively examined the changes in the serum ICTP levels before and after bone metastasis in the HCC patients. The serum ICTP level was significantly higher in the HCC patients with bone metastasis than in the patients with other diseases and the healthy controls. The serum PICP, PIIIP, Ty IV, 7S and HA levels of the HCC patients with bone metastasis did not differ significantly from those of the patients without bone metastasis. The diagnostic efficiency for HCC with bone metastasis was 87% for ICTP, 51% for PICP, 65% for Ty IV, 55% for PIIIP and 51% for HA. During the follow-up, the changes in the serum ICTP values paralleled the behavior of bone metastasis. These results indicate that the measurement of serum ICTP concentration is useful for detecting and monitoring HCC patients with bone metastasis.