Incidence and risk factors of hepatocellular carcinoma in patients with hepatitis C who achieved a sustained virological response through direct-acting antiviral agents among the working population in Japan.

Background and Aim: The development of hepatocarcinogenesis after a sustained virological response (SVR) remains an important issue affecting the balance between treatment and occupational life of workers with chronic hepatitis C virus (HCV) infection in Japan. Here, we aimed to evaluate the hepatocellular carcinoma (HCC) reducing effect and risk factors for developing HCC after SVR in patients treated with direct-acting antiviral agents (DAAs) among the working population. Methods: We studied 2579 working patients with chronic HCV infection who achieved SVR after antiviral treatment. We compared the difference in the cumulative incidence of post-SVR HCC between the interferon (IFN)-based n = 1615 and DAA (n = 964) groups. The risk factors for post-SVR HCC development were determined in the DAA group. Results: After propensity score matching (n = 644 in each group), the HCC development rates were not significantly different between the groups (P = 0.186). Multivariate Cox regression and the cutoff values determined by the receiver operating characteristic curve analyses revealed that age ≥61 years, diabetes, lower serum albumin levels <4.0 g/dL at 24 weeks after the end of treatment (EOT), and higher serum α-fetoprotein levels ≥4.1 ng/mL at 24 weeks after the EOT were associated with the development of HCC. Conclusion: The HCC suppressing effect after SVR through DAA treatment is equivalent to that of IFN treatment in patients in the working population. Intensive follow-up is required after SVR with DAA treatment in Japanese workers with these risk factors to ensure the promotion of health and employment support.

[A case of eosinophilic gastroenteritis caused after the use of Seprafilm®].

Eosinophilic gastroenteritis is a fairly uncommon condition. It has been suggested that allergic reactions may have played a role in the development of this illness. The case of a 66-year-old woman who had a total hysterectomy due to a right ovarian tumor is described here. At this operation, a sodium hyaluronate carboxymethylcellulose bioresorbable membrane (Seprafilm®) was used. She was admitted to our hospital 47 days after the operation with abdominal pain. Laboratory data indicated elevated WBC (29450/µl) and eosinophilia (69.2%), and CT scan showed thickening of intestinal wall and ascites around there. Ascites cytology showed a significant increase of eosinophils (94.0%). She began taking oral steroids after being diagnosed with eosinophilic gastroenteritis, and her symptoms improved quickly. Despite the fact that Seprafilm® was thought to be a reliable and safe tool, it was suggested that a foreign body reaction to Seprafilm® could lead to eosinophilic gastroenteritis.

Significance of in vitro photodynamic cytodiagnosis with 5-aminolevulinic acid in biliary brush cytology for malignant biliary stricture.

BACKGROUND: For the early diagnosis of malignant biliary stricture due to biliary-pancreatic carcinoma, conventional biliary brush cytology with endoscopic retrograde cholangiopancreatography (ERCP; the conventional method) is not sensitive enough. METHODS: Two hundred nine patients with biliary stricture who were admitted between September 2015 and June 2020 were enrolled in this study. Biliary brush cytology was performed on all patients. Samples were diagnosed independently by an expert pathologist and medical doctor with conventional cytology and photodynamic diagnosis (PDD) with 5-aminolevulinic acid. RESULTS: The definitive diagnoses were 49 benign and 160 malignant diseases. The conventional method had a sensitivity of 77.5% (124/160) and specificity of 100% (49/49). The PDD method had a sensitivity of 77.5% (124/160) and specificity of 67.3% (33/49). The conventional method identified 36 malignant diseases as false negatives, while the PDD method enabled successful diagnoses of malignant diseases in 19 of these 36 patients. When PDD was combined with the conventional method, the sensitivity significantly increased to 89.4% (143/160, P = 0.006), and for biliary tract diseases only, the sensitivity increased to 95.6% (88/92, P = 0.001). CONCLUSIONS: Malignant biliary stricture can be diagnosed effectively and safely with the in vitro PDD method. The sensitivity could be further increased by combining PDD with the conventional method.

Oral Zinc Supplementation Decreases the Risk of HCC Development in Patients With HCV Eradicated by DAA.

We have reported that the plasma zinc concentration gradually decreases with the progression of fibrosis and is related to hepatocellular carcinoma (HCC) development. The aim of this study was to examine the impact of the zinc concentration on HCC development (study 1) and the relationship between zinc intake and HCC development (study 2) in patients with hepatitis C virus (HCV) eradicated by direct-acting antivirals (DAAs). A total of 599 sustained virological response (SVR) patients treated with DAAs without a history of HCC were retrospectively analyzed in this study. Eighty patients received supplemental zinc (Zn treatment group), and 519 patients did not receive zinc (no Zn treatment group). In study 1, the cumulative incidence rate of HCC was compared between the Zn treatment group and the no Zn treatment group. In study 2, the risk factors for HCC development were examined in the no Zn treatment group. In study 1, in the Zn treatment group, HCC did not develop during follow-up, and the cumulative risk of HCC was significantly lower in the Zn treatment group than in the no Zn treatment group (P = 0.048). In study 2, the 1-year and 3-year cumulative incidence rates of HCC were 1.8% and 5.6%, respectively. The risk factors for HCC identified by multivariate analysis were male sex, cirrhosis, low platelet count before treatment, and low serum zinc concentration 12 weeks after the end of DAA therapy. Conclusion: The Zn concentration is related to HCC development in patients with HCV eradicated by DAA therapy. Oral zinc supplementation is recommended as a means of suppressing HCC development in patients who have achieved SVR.

Early Administration of Tolvaptan Can Improve Survival in Patients with Cirrhotic Ascites.

(1) Backgrounds and aim: Tolvaptan, a selective vasopressin type 2 receptor antagonist, was approved for ascites, and its short-term efficacy and safety have been confirmed. However, it is still unclear whether this novel drug may improve long-term survival rates in cirrhotic patients with ascites. (2) Patients and methods: A total of 206 patients who responded insufficiently to conventional diuretics and were hospitalized for refractory ascites for the first time were retrospectively enrolled in this study. Among them, the first 57 consecutive patients were treated with conventional diuretics (the conventional therapy group); the latter 149 consecutive patients were treated with tolvaptan in addition to the conventional therapy (the tolvaptan group). (3) Results: The exacerbation of renal function was significantly milder in the tolvaptan group than in the conventional therapy group. The prognostic factors for survival in the tolvaptan group were being male, having hyperbilirubinemia, having a high blood urea nitrogen (BUN), and receiving high-dose furosemide at the start of tolvaptan treatment. The one-year and three-year cumulative survival rates were 67.8 and 45.3%, respectively, in patients with low-dose furosemide (<40 mg/day) at the start of tolvaptan treatment. The prognosis was significantly better in the tolvaptan group with low-dose furosemide than in the conventional therapy group (p < 0.001). (4) Conclusion: Tolvaptan can improve survival in patients with cirrhotic ascites, especially when tolvaptan is started before high-dose furosemide administration.

Significance of in vitro photodynamic cytodiagnosis using 5-aminolevulinic acid in solid pancreatic tumors extracted by endoscopic ultrasound-guided fine-needle aspiration.

BACKGROUND: Recently, photodynamic diagnosis using 5-aminolevulinic acid (5-ALA) has gained attention for the diagnosis of neoplastic diseases. In the present study, an in vitro method of photodynamic cytodiagnosis (PDCD) performed using the reagent 5-ALA in the cytodiagnosis of solid pancreatic tumors was developed. Here, we assess the accuracy of PDCD for malignancy. MATERIALS AND METHODS: EUS-FNA was performed from September 2015 to March 2018 in patients with solid pancreatic tumors at Osaka Rosai Hospital. Samples were diagnosed independently by an expert pathologist and a medical doctor with conventional cytology and PDCD. RESULTS: A total of 53 patients (35 males, average age: 70.2 years old) were enrolled. The definitive diagnoses were 7 benign lesions and 46 malignant lesions. Using the in vitro PDCD method, the detection of reddish fluorescence in cell samples indicated cancer cells. PDCD had a sensitivity of 91.3% (42/46) and a specificity of 100% (7/7), while conventional cytology had a sensitivity of 93.5% (43/46) and a specificity of 85.7% (6/7). Two patients were successfully diagnosed with malignancy only by the PDCD method. CONCLUSIONS: In vitro PDCD performed using the 5-ALA method can effectively and safely identify a diagnosis of pancreatic cancer without requiring an expert pathologist. The sensitivity of this technique could be increased in the diagnosis of pancreatic malignancy by combining it with the conventional method.

Influence of increased physical activity without body weight loss on hepatic inflammation in patients with nonalcoholic fatty liver disease.

BACKGROUND: Physical activity (PA) that includes an accumulated exercise regimen that meets or exceeds a certain intensity reduces intrahepatic fat, leading to the improvement of nonalcoholic fatty liver disease (NAFLD) in afflicted patients. However, whether an increase in comprehensive PA, including activities of daily living, contributes to ameliorating the pathophysiology of NAFLD remains unclear. This study aimed to examine whether PA improves liver function in patients with NAFLD. METHODS: The study included 45 patients with NAFLD who underwent follow-up examinations at least 6 months-but no later than 1 year-after their baseline examinations. The patients were interviewed about their daily activities and exercise habits to determine whether they had engaged in at least 3 metabolic equivalents (METs) per day during the previous 6 months; the quantity of PA, expressed in Ekusasaizu (Ex) units, was calculated as METs multiplied by hours. Patients who had achieved at least a 1-Ex increase in PA per week compared to baseline at the time of their follow-up interview (the PA increase group) were compared to those whose PA was the same or lower at the time of follow-up (the PA non-increase group). RESULTS: There were no significant changes in all blood and biochemical parameters in the PA non-increase group at the time of follow-up when compared with baseline levels. In the PA increase group, aspartate aminotransferase, alanine aminotransferase, and γ-guanosine triphosphate levels were all significantly lower at follow-up than they were at baseline. Body weight did not change significantly from baseline to follow-up in both groups. CONCLUSIONS: In the present study, hepatic inflammation improvement was accompanied by increased PA but not decreased body weight. Increasing PA may be effective for the improvement of hepatic inflammation even without body weight loss. Our results indicate the effectiveness of PA monitoring for the management of NAFLD. TRIAL REGISTRATION: UMIN-CTR, UMIN000038530.

Effects of Zinc Acetate on Serum Zinc Concentrations in Chronic Liver Diseases: a Multicenter, Double-Blind, Randomized, Placebo-Controlled Trial and a Dose Adjustment Trial.

The essential trace element zinc maintains liver functions. Liver diseases can alter overall zinc concentrations, and hypozincemia is associated with various hepatic pathologies. Modulating systemic zinc through dietary supplementation is potentially useful for liver diseases. We evaluated the usefulness of zinc (NPC-02; acetate formulation) supplementation. We conducted two NPC-02 studies on zinc-deficient patients (serum zinc < 70 µg/dL). Study 1: double-blind, randomized, placebo-controlled trial on 57 subjects with chronic liver diseases comparing serum zinc in patients given NPC-02 (NPC-02 group) versus placebo (Placebo group). Study 2: dose adjustment study on 43 subjects with/without liver diseases to determine proportions maintaining serum zinc target (≥ 80 µg/dL but < 200 µg/dL). In study 1, NPC-02 subjects had higher serum zinc concentrations at week 8 than Placebo subjects (83.2 ± 20.2 and 61.3 ± 12.0, respectively; P < 0.0001), and more NPC-02 than Placebo subjects achieved the serum zinc target (15/27 vs. 1/26). In study 2, the NPC-02-induced serum zinc increase was dose-dependent in subjects both with and without liver diseases (r = 0.5143, P = 0.0022 and r = 0.5753, P = 0.0005, respectively). Interestingly, there was a marginally positive correlation between serum zinc and albumin levels in subjects with but not in those without liver diseases (r = 0.4028, P = 0.0631 and r = 0.1360, P = 0.5567, respectively). NPC-02 dose-dependently increases serum zinc in hypozincemic patients, regardless of liver disease. NPC-02 is a potentially effective therapy for liver cirrhosis, in which zinc deficiency is common. Clinical trial registry number: NCT02337569, NCT02321865.

Long-Term Zinc Supplementation Improves Liver Function and Decreases the Risk of Developing Hepatocellular Carcinoma.

Zinc plays a pivotal role in various zinc enzymes, which are crucial in the maintenance of liver function. Patients with chronic liver diseases (CLDs) usually have lower concentrations of zinc, which decrease further as liver fibrosis progresses. Whether long-term zinc supplementation improves liver function and reduces the risk of hepatocellular carcinoma (HCC) development remains unknown. Two hundred and sixty-seven patients with CLDs who received a zinc preparation (Zn-group; 196 patients), or who did not receive zinc (no Zn-treatment group; 71 patients), were retrospectively analyzed in this study. The Zn-group was divided into 4 groups according to their serum Zn concentrations at 6 months after the start of Zn treatment. Liver function significantly deteriorated in the no Zn-treatment group, while no notable change was observed in the Zn-group. The cumulative incidence rates of events and HCC at 3 years were observed to be lower in the Zn-group (9.5%, 7.6%) than in the no Zn-treatment group (24.9%, 19.2%) (p < 0.001). According to serum Zn concentrations, the cumulative incidence rates of events and HCC were significantly decreased in patients with Zn concentrations ≥ 70 µg/dL (p < 0.001). Zinc supplementation appears to be effective at maintaining liver function and suppressing events and HCC development, especially among patients whose Zn concentration is greater than 70 µg/dL.

Signal transducer and activator of transcription 5 plays a crucial role in hepatic lipid metabolism through regulation of CD36 expression.

AIM: Liver-specific signal transducer and activator of transcription (STAT)5-deficient mice (STAT5KO) show lipid accumulation in the liver. We investigated the role of hepatic STAT5 in lipid metabolism in vitro and in vivo. METHODS AND RESULTS: High expression of CD36, one of the receptors for free fatty acids, is associated with a high concentration of hepatic triglyceride (TG) in STAT5KO mice. Peroxisome proliferator-activated receptor (PPAR)γ, one of the regulatory factors of CD36, was upregulated and microRNA (miR)-20b was downregulated in STAT5KO mice. Reporter assays revealed direct regulation involving miR-20b and the 3'-untranslated region of CD36 mRNA. Treatment with free fatty acids enhanced accumulation of TG in STAT5-deleted hepatoma cells, and this was partially canceled by introduction of siRNA for PPARγ and/or pre-miR-20b through inhibition of CD36 expression. In vivo, STAT5/CD36 double knockout mice displayed hepatic TG was decreased compared to STAT5KO mice and it was also reduced by treatment with PPARγ antagonists, GW9662, and/or pre-miR-20b. CONCLUSIONS: Signal transducer and activator of transcription 5 plays an important role in hepatic fat metabolism through regulation of CD36, and is a potential therapeutic candidate for liver steatosis.

Ultra-deep sequencing analysis of resistance-associated variants during retreatment with simeprevir-based triple therapy after failure of telaprevir-based triple therapy in patients with genotype 1 hepatitis C virus infection.

AIM: Simeprevir (SMV)-based triple therapy is an effective retreatment option following failure of telaprevir (TVR)-based triple therapy. However, it is unclear whether the persistence of resistance-associated variants (RAVs) induced by TVR-based therapy may reduce the treatment effect of SMV-based therapy. METHODS: The factors associated with the treatment effect, including RAVs in the NS3 region, were examined in 21 patients with genotype 1b HCV infection who were treated with SMV-based therapy after failure of TVR-based therapy. Ultra-deep sequencing was carried out to detect RAVs. RESULTS: With the exception of one patient who discontinued treatment owing to adverse events, the sustained virologic response (SVR) rate was 50% (10/20). Ultra-deep sequencing at the start of SMV-based therapy revealed that TVR-resistant variants were detected in six patients (29%), and no variants were observed at position 168. Cross-resistance between TVR and SMV with low frequency was detected in only one patient, and this patient achieved SVR. Higher SVRs for SMV-based therapy were attained in patients who discontinued treatment owing to the adverse effects of prior TVR-based therapy (discontinuation 100% vs. non-discontinuation 29%, P = 0.005), and patients who relapsed following prior pegylated interferon plus ribavirin therapy (relapse 100% vs. non-response 20%, P = 0.007). CONCLUSIONS: In this study, ultra-deep sequencing analysis revealed that TVR and/or SMV-resistant variants may have no influence on the effect of SMV-based therapy after failure of TVR-based therapy. Patients who discontinued treatment owing to adverse effects of TVR-based therapy and relapsers to previous pegylated interferon/ribavirin therapy would be good candidates for retreatment with SMV-based therapy.

Impact of ribavirin dosage in chronic hepatitis C patients treated with simeprevir, pegylated interferon plus ribavirin combination therapy.

The factors associated with sustained virologic response (SVR) in chronic hepatitis C (CH-C) genotype 1 patients treated with simeprevir (SMV), pegylated interferon (Peg-IFN) plus ribavirin (RBV) triple therapy have not been fully investigated. Two hundred and twenty-nine treatment-naïve CH-C patients treated with SMV triple therapy were enrolled in this study. The overall SVR rate was 87% in per-protocol analysis. In multivariate analysis, the interleukin (IL) 28B genotype (rs8099917, TT vs. non-TT, odds ratio [OR]: 0.044, P = 0.001) and RBV dose (< 10/10-12/ ≥ 12 mg/kg/day, OR: 4.513, P = 0.041) were significant factors associated with SVR. In patients with the IL28B non-TT genotype, RBV dose affected SVR dose-dependently in stratified analysis of RBV dose (P = 0.015); it was 44% (8/18) for patients administered <10 mg/kg/day of RBV, 78% (14/18) for those administered 10-12 mg/kg/day of RBV, and 100% (3/3) for those administered ≥12 mg/kg/day of RBV, whereas in patients with the IL28B TT genotype, a significant correlation between SVR and RBV dose was not observed (P = 0.229). Regarding RBV dose reduction of less than 10 mg/kg/day, the inosine triphosphate pyrophosphatase (ITPA) genotype (rs1127354, CC vs. non-CC, OR: 0.239, P = 0.003) and age (by 1 y.o., OR: 1.084, P = 0.002) were significant independent factors. RBV dosage affected SVR dose-dependently in patients with the IL28B non-TT genotype treated with SMV triple therapy. Special attention to anemia progression and RBV dosage should be paid to aged patients with the ITPA CC genotype. J. Med. Virol. 88:1776-1784, 2016. © 2016 Wiley Periodicals, Inc.

Valine, the branched-chain amino acid, suppresses hepatitis C virus RNA replication but promotes infectious particle formation.

BACKGROUND & AIMS: Concentrations of the branched-chain amino acid (BCAA) in the serum of patients with liver cirrhosis correlate with their liver function. Oral administration of BCAA can ameliorate hypoalbuminemia and hepatic encephalopathy. In this study, we aim to clarify the role of BCAA in regulating the replication of the hepatitis C virus (HCV). METHODS: HCV sub-genomic replicon cells, genome-length replicon cells, and cells infected with cell culture-infectious HCV (HCVcc) were cultured in media supplemented with various concentrations of BCAA, followed by evaluation of the replicon or HCV abundance. RESULTS: BCAA was capable of suppressing the HCV replicon in a dose-dependent manner and the effect was independent of the mTOR pathway. Of the three BCAAs, valine was identified as being responsible for suppressing the HCV replicon. Surprisingly, an abundance of HJ3-5(YH/QL), an HCVcc, in Huh7 cells was augmented by BCAA supplementation. In contrast, BCAA suppressed an abundance of HJ3-5(wild), an HCVcc that cannot assemble virus particle in Huh7 cells. Internal ribosome entry site of HCV was shown to be a target of BCAA. Single-cycle virus production assays using Huh7-25 cells, which lacked CD81 expression, revealed that BCAA, especially valine, promoted infectious virus particle formation with minimal effect on virus secretion. Thus, BCAA was found to have two opposing effects on HCV production: suppression of the HCV genome RNA replication and promotion of infectious virus formation. CONCLUSIONS: BCAA accelerates HCV production through promotion of infectious virus formation in infected cells despite its suppressive effect on HCV genome replication.

The combination therapy of α-galactosylceramide and 5-fluorouracil showed antitumor effect synergistically against liver tumor in mice.

α-Galactosylceramide (α-GalCer) has been reported to be therapeutic against metastatic liver tumors in mice. However, little is known regarding the efficacy of combined chemo-immunotherapy using α-GalCer and anticancer drugs. In this study, we evaluated the antitumor effect of the combination therapy of α-GalCer and 5-fluorouracil (5-FU) against liver tumors of MC38 colon cancer cells. The liver weights of tumor-bearing mice treated with the combination were significantly lower than those of nontreated mice and of mice treated with 5-FU or α-GalCer alone. No toxic effects on the liver and renal functions were observed in any of the treatment groups. α-GalCer treatment induced significant activation of liver NK cells in vivo, but 5-FU treatment did not. 5-FU treatment resulted in a significant upregulation of NKG2D activating molecules (Rae-1 and H60) and DNAM-1 ligands (CD112 and CD155) on MC38 cells, but α-GalCer did not. The cytolytic activity of α-GalCer-activated liver mononuclear cells against 5-FU-treated MC38 cells was significantly higher than that against nontreated cells. The increase of the cytolytic activity induced by 5-FU partially depended on NKG2D-Rae-1 or H60 signals. Depletion of NK cells significantly inhibited the antitumor efficacy of 5-FU against MC38 liver tumors, which suggested that the antitumor effect of 5-FU partially depended on the cytolytic activity of NK cells. These results demonstrated that the combination therapy of α-GalCer and 5-FU produced synergistic antitumor effects against liver tumors by increasing the expression of NK activating molecules on cancer cells. This study suggests a promising new chemo-immunotherapy against metastatic liver cancer.

Pancreatic STAT3 protects mice against caerulein-induced pancreatitis via PAP1 induction.

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that controls expressions of several genes involved in cell survival, proliferation and differentiation, and tissue inflammation. However, the significance of pancreatic STAT3 in acute pancreatitis remains unclear. We generated conditional STAT3 knockout (stat3(Δ/Δ)) mice by crossing stat3(flox/flox) mice with Pdx1-promoter Cre transgenic mice. Caerulein administration activated pancreatic STAT3 and induced acute pancreatitis as early as 3 hours in wild-type mice, and full recovery from the induced pancreatic injury was observed within 7 days. The levels of serum amylase and lipase and histologic scores of pancreatic necrosis and inflammatory cell infiltration were significantly higher at 3 hours in stat3(Δ/Δ) mice than in stat3(flox/flox) mice. Pancreatic recovery after pancreatitis was significantly delayed in stat3(Δ/Δ) mice compared with stat3(flox/flox) mice. Although stat3(flox/flox) mice had marked production in the pancreas of pancreatitis-associated protein 1 (PAP1), a serum acute phase protein, this induction was completely abrogated in stat3(Δ/Δ) mice. Enforced production of PAP1 by a hydrodynamic procedure in the liver significantly suppressed pancreatic necrosis and inflammation and also promoted pancreatic regeneration and recovery in stat3(Δ/Δ) mice to levels similar to those observed in stat3(flox/flox) mice. In conclusion, pancreatic STAT3 is indispensable for PAP1 production, and this STAT3/PAP1 pathway plays a protective role in caerulein-induced pancreatitis.

Interferon-α suppresses hepatitis B virus enhancer II activity via the protein kinase C pathway.

HBV has two enhancer (En) regions each of which promotes its own transcription. En II regulates production of pregenomic RNA, a key product of HBV replication, more strongly than En I. Although IFN-α has been found to suppress En I activity, its effect on En II activity has not been examined. Here we used luciferase assay to demonstrate that IFN-α suppresses En II activity. Analysis with several deletion/mutation constructs identified two major segments, nt 1703-1727 and nt 1746-1770, within the En II sequence as being responsible for the suppressive effects of IFN-α. Pre-treatment with protein kinase C (PKC) inhibitors blocked this effect regardless of the expression levels of phospho-STAT1 and Mx upon IFN-α stimulation. These results indicate that IFN-α suppresses En II activity via the PKC pathway, which may be an alternative suppressive pathway for HBV replication. (136 words).

Suppression of signal transducers and activators of transcription 1 in hepatocellular carcinoma is associated with tumor progression.

Signal transducers and activators of transcription (STAT) 1 plays a pivotal role in cell-cycle and cell-fate determination, and vascular endothelial growth factor (VEGF) also contributes tumor growth. Recently, interferon (IFN) α has been reported to be effective for prevention of hepatocellular carcinomas (HCCs) recurrence, but the detailed mechanisms remain elusive. In vitro, cobalt chloride-treated VEGF induction and hypoxia responsive element (HRE) promoter activity were inhibited by IFNs and this abrogation was cancelled by introduction of small interfering RNA for STAT1. Immunoprecipitation/chromatin immunoprecipitation analyses showed STAT1 bound to hypoxia-inducible factor (HIF)-1α and dissociated HIF-complex from HRE promoter lesion. In a xenograft model using Balb/c nude mice, tumor growth was suppressed by IFNα through inhibition of VEGF expression and it was oppositely enhanced when STAT1-deleted cells were injected. This augmentation was due to upregulation of VEGF and hyaluronan synthase 2. In human samples, 29 HCCs were resected, divided into two groups based on STAT1 activation in tumor and the clinical features were investigated. Patients with suppressed STAT1 activity had a shorter recurrence-free survival. Histological and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses showed portal vein microinvasion and increased VEGF levels in tumors from suppressed STAT1 group. These human samples also showed a reverse correlation between VEGF and STAT1-regulated genes expression. These results in vitro and in vivo suggested that IFNα are potential candidates for prevention of vessel invasion acting through inhibition of VEGF expression and need to be properly used when STAT1 expression is suppressed.

Bak deficiency inhibits liver carcinogenesis: a causal link between apoptosis and carcinogenesis.

BACKGROUND & AIMS: Hepatocyte apoptosis is a key feature of chronic liver disease including viral hepatitis and steatohepatitis. A previous study demonstrated that absence of the Bcl-2 family protein Mcl-1 led to increased hepatocyte apoptosis and development of liver tumors in mice. Since Mcl-1 not only inhibits the mitochondrial pathway of apoptosis but can also inhibit cell cycle progression and promote DNA repair, it remains to be proven whether the tumor suppressive effects of Mcl-1 are mediated by prevention of apoptosis. METHODS: We examined liver tumor development, fibrogenesis, and oxidative stress in livers of hepatocyte-specific knockout (KO) of Mcl-1 or Bcl-xL, another key antagonist of apoptosis in hepatocytes. We also examined the impact of additional KO of Bak, a downstream molecule of Mcl-1 towards apoptosis but not the cell cycle or DNA damage pathway, on tumor development, hepatocyte apoptosis, and inflammation. RESULTS: Bcl-xL KO led to a high incidence of liver tumors in 1.5-year-old mice, similar to Mcl-1 KO. Bcl-xL- or Mcl-1-deficient livers showed higher levels of TNF-α production and oxidative stress than wild-type livers at as early as 6 weeks of age and oxidative DNA damage at 1.5 years. Deletion of Bak significantly inhibited hepatocyte apoptosis in Mcl-1 KO mice and reduced the incidence of liver cancer, coinciding with reduction of TNF-α production, oxidative stress, and oxidative DNA damage in non-cancerous livers. CONCLUSIONS: Our findings strongly suggest that chronically increased apoptosis in hepatocytes is carcinogenic and offer genetic evidence that inhibition of apoptosis may suppress liver carcinogenesis in chronic liver disease.

Interleukin-1ß enhances the production of soluble MICA in human hepatocellular carcinoma.

The production of soluble major histocompatibility complex class I-related chain A (MICA) is thought to antagonize NKG2D-mediated immunosurveillance. Interleukin-1ß (IL-1ß) is elevated in patients with chronic hepatitis C (CH), and this might contribute to the escape of hepatocellular carcinoma (HCC) cells from innate immunity. In this study, we investigated the immunoregulatory role of IL-1ß in the production of soluble MICA of HCC cells. First, we investigated the correlation between the serum IL-1ß levels and soluble MICA in CH patients. Serum IL-1ß levels were associated with soluble MICA levels in CH patients. The serum IL-1ß levels of CH patients with the HCC occurrence were significantly higher than those of CH patients without HCC. We next examined the MICA production of IL-1ß-treated HCC cells. Addition of IL-1ß resulted in significant increase in the production of soluble MICA in HepG2 and PLC/PRF/5 cells, human HCC cells. But soluble MICA was not detected in both non-treated and IL-1ß-treated normal hepatocytes. Addition of IL-1ß did not increase the expressions of membrane-bound MICA on HCC cells. These were observed similarly in various cancer cells including a gastric cancer (MKN1), two colon cancers (HCT116 and HT29) and a cervical cancer (HeLa). Addition of IL-1ß also increased the expression of a disintegrin and metalloproteinase (ADAM)9 in HCC cells, and the knockdown of ADAM9 in IL-1ß-treated HCC cells resulted in the decrease in the production of soluble MICA of HCC cells. These findings indicate that IL-1ß might enhance the production of soluble MICA by activating ADAM9 in human HCC.