Non-liver-related mortality in the DAA era: Insights from post-SVR patients with and without previous HCC history.

BACKGROUND AND AIMS: Mortality after sustained virological response (SVR) with interferon-free direct-acting antiviral (IFN-free DAA) therapy is crucial for optimizing post-SVR patient care, but it remains unclear, especially regarding non-liver-related mortality. METHODS: Consecutive post-SVR patients from 14 institutions were stratified into three cohorts: A (without advanced fibrosis and without prior HCC), B (with advanced fibrosis and without prior HCC), and C (curative HCC treatment). We assessed mortality (per 1000 person-years [/1000PY]) post-SVR. Mortality rates were compared between cohorts A and B and the general population using age- and sex-adjusted standardized mortality ratio (SMR). Comparison of survival between each cohort was performed using propensity-score (PS) matching with sex, age, and comorbidity. RESULTS: In cohort A (n = 762; median age, 65 years), 22 patients died (median follow-up, 36 months); all-cause mortality was 10.0/1000PY, with 86.4% non-liver-related deaths. In cohort B (n = 519; median age, 73 years), 27 patients died (median follow-up, 39 months); all-cause mortality was 16.7/1000PY, with 88.9% non-liver-related deaths. In both cohorts, malignant neoplasm was the most common cause of death; all-cause mortality was comparable to that of the general population (SMR: 0.96 and 0.92). In cohort C (n = 108; median age, 75 years), 15 patients died (median follow-up, 51 months); all-cause mortality was 36.0/1000PY, with 53.3% liver-related deaths. PS matching showed no significant survival differences between cohorts A and B, both of which had better survival than cohort C. CONCLUSIONS: Mortality varies based on HCC history in the DAA era; nevertheless, attention should be paid to non-liver-related deaths in all post-SVR patients.

Mouse Type-I Interferon-Mannosylated Albumin Fusion Protein for the Treatment of Chronic Hepatitis.

Although a lot of effort has been put into creating drugs and combination therapies against chronic hepatitis, no effective treatment has been established. Type-I interferon is a promising therapeutic for chronic hepatitis due to its excellent anti-inflammatory effects through interferon receptors on hepatic macrophages. To develop a type-I IFN equipped with the ability to target hepatic macrophages through the macrophage mannose receptor, the present study designed a mouse type-I interferon-mannosylated albumin fusion protein using site-specific mutagenesis and albumin fusion technology. This fusion protein exhibited the induction of anti-inflammatory molecules, such as IL-10, IL-1Ra, and PD-1, in RAW264.7 cells, or hepatoprotective effects on carbon tetrachloride-induced chronic hepatitis mice. As expected, such biological and hepatoprotective actions were significantly superior to those of human fusion proteins. Furthermore, the repeated administration of mouse fusion protein to carbon tetrachloride-induced chronic hepatitis mice clearly suppressed the area of liver fibrosis and hepatic hydroxyproline contents, not only with a reduction in the levels of inflammatory cytokine (TNF-α) and fibrosis-related genes (TGF-ß, Fibronectin, Snail, and Collagen 1α2), but also with a shift in the hepatic macrophage phenotype from inflammatory to anti-inflammatory. Therefore, type-I interferon-mannosylated albumin fusion protein has the potential as a new therapeutic agent for chronic hepatitis.

Cell-penetrating albumin enhances the sublingual delivery of antigens through macropinocytosis.

Innovations in oral immunotherapy have greatly advanced the therapeutic control of allergies. However, these therapeutic effects suffer from the fact that the amount of antigen delivered to antigen-presenting cells is limited given the formulations that are currently available. We recently designed a cell-penetrating albumin and found that this modified albumin enters cells via the induction of macropinocytosis. Herein, we report on a novel system for delivering antigens based on cell-penetrating albumin-inducible macropinocytosis that allows larger amounts of antigens to be delivered to antigen-presenting cells. A treatment with cell-penetrating albumin significantly increased the permeability of ovalbumin (45 kDa) or dextran (2000 kDa) on monolayers derived from human oral squamous carcinoma cells. Flow cytometric analyses showed that the cell-penetrating albumin treatment resulted in a significant elevation in the amount of dextran that was delivered to two types of antigen-presenting cells. Finally, mice that had been sensitized by Japanese cedar pollen extract (JCPE) and cell-penetrating albumin showed a decline in the frequency of nose-rubbing against a subsequent intranasal administration of JCPE. These findings suggest that the sublingual administration of cell-penetrating albumin efficiently delivers antigens to antigen-presenting cells via the induction of macropinocytosis, resulting in an enhancement in the therapeutic effect of sublingual immunotherapy.

Cell-penetrating mechanism of intracellular targeting albumin: Contribution of macropinocytosis induction and endosomal escape.

We recently developed a cell-penetrating drug carrier composed of albumin (HSA) combined with palmitoyl-cyclic-(D-Arg)12. While it is possible that the palmitoyl-cyclic-(D-Arg)12/HSA enters the cell mainly via macropinocytosis, the mechanism responsible for the induction of macropinocytosis and endosomal escape remain unknown. We report herein that palmitoyl-cyclic-(D-Arg)12/HSA might interact with heparan sulfate proteoglycan and the chemokine receptor CXCR4 followed by multiple activations of the PKC/PI3K/JNK/mTOR signaling pathways to induce macropinocytosis. This result was further confirmed by a co-treatment with 70 kDa dextran, a macropinocytosis marker. Using liposomes that mimic endosomes, the leakage of 5,6-carboxyfluorescein from liposome was observed in the presence of palmitoyl-cyclic-(D-Arg)12/HSA only in the case of the anionic late endosome-like liposomes but not the neutral early endosome-like liposomes. Heparin largely inhibited this leakage, suggesting the importance of electrostatic interactions between palmitoyl-cyclic-(D-Arg)12/HSA and the late-endosomal membrane. Immunofluorescence staining and Western blotting data indicated that the intact HSA could be transferred from endosomes to the cytosol. These collective data suggest that the palmitoyl-cyclic-(D-Arg)12/HSA is internalized via macropinocytosis and intact HSA is released from the late endosomes to the cytoplasm before the endosomes fuse with lysosomes. Palmitoyl-cyclic-(D-Arg)12/HSA not only functions as an intracellular drug delivery carrier but also as an inducer of macropinocytosis.

Development of Kupffer cell targeting type-I interferon for the treatment of hepatitis via inducing anti-inflammatory and immunomodulatory actions.

Because of its multifaceted anti-inflammatory and immunomodulatory effects, delivering type-I interferon to Kupffer cells has the potential to function as a novel type of therapy for the treatment of various types of hepatitis. We report herein on the preparation of a Kupffer cell targeting type-I interferon, an albumin-IFNα2b fusion protein that contains highly mannosylated N-linked oligosaccharide chains, Man-HSA(D494N)-IFNα2b, attached by combining albumin fusion technology and site-directed mutagenesis. The presence of this unique oligosaccharide permits the protein to be efficiently, rapidly and preferentially distributed to Kupffer cells. Likewise IFNα2b, Man-HSA(D494N)-IFNα2b caused a significant induction in the mRNA levels of IL-10, IL-1Ra, PD-L1 in RAW264.7 cells and mouse isolated Kupffer cells, and these inductions were largely inhibited by blocking the interferon receptor. These data indicate that Man-HSA(D494N)-IFNα2b retained the biological activities of type-I interferon. Man-HSA(D494N)-IFNα2b significantly inhibited liver injury in Concanavalin A (Con-A)-induced hepatitis model mice, and consequently improved their survival rate. Moreover, the post-administration of Man-HSA(D494N)-IFNα2b at 2 h after the Con-A challenge also exerted hepato-protective effects. In conclusion, this proof-of-concept study demonstrates the therapeutic effectiveness and utility of Kupffer cell targeting type-I interferon against hepatitis via its anti-inflammatory and immunomodulatory actions.

Design and tuning of a cell-penetrating albumin derivative as a versatile nanovehicle for intracellular drug delivery.

Human serum albumin (HSA) is a superior carrier for delivering extracellular drugs. However, the development of a cell-penetrating HSA remains a great challenge due to its low membrane permeability. We report herein on the design of a series of palmitoyl-poly-arginine peptides (CPPs) and an evaluation of their cell-penetrating effects after forming a complex with HSA for use in intracellular drug delivery. The palmitoyl CPPs forms a stable complex with HSA by anchoring itself to the high affinity palmitate binding sites of HSA. Among the CPPs evaluated, a cyclic polypeptide composed of D-dodecaarginines, palmitoyl-cyclic-(D-Arg)12 was the most effective for facilitating the cellular uptake of HSA by HeLa cells. Such a superior cell-penetrating capability is primarily mediated by macropinocytosis. The effect of the CPP on pharmacological activity was examined using three drugs loaded in HSA via three different methods: a) an HSA-paclitaxel complex, b) an HSA-doxorubicin covalent conjugate and c) an HSA-thioredoxin fusion protein. The results showed that cell-penetrating efficiency was increased with a corresponding and significant enhancement in pharmacological activity. In conclusion, palmitoyl-cyclic-(D-Arg)12/HSA is a versatile cell-penetrating drug delivery system with great potential for use as a nano-carrier for a wide diversity of pharmaceutical applications.

Frequency of elevated biomarkers in patients with cryptogenic hepatocellular carcinoma.

BACKGROUND: The incidence of hepatocellular carcinoma (HCC) continues to increase in Japan, but the clinical characteristics of Japanese patients with HCC have not been well described. The aim of this study was to determine the frequencies and utilities of elevated a-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) levels as biomarkers in cryptogenic HCC. MATERIAL/METHODS: A total of 2638 patients with HCC diagnosed between 1999 and 2010 in the Nagasaki Association Study of Liver (NASLD) were recruited for this study. The cause of HCC was categorized into 4 groups; HCC-B, HCC-C, HCC-BC, and HCC-nonBC. The significance of factors was examined for HCC-nonBC using logistic regression analysis in all patients. RESULTS: Multivariate analysis identified age, sex, BMI, alcohol consumption, platelet count, AST, ALT, AFP, DCP, and TNM stage as independent and significant risk factors for HCC-nonBC. According to TNM stage, the median AFP levels in HCC-nonBC with TNM stages I, II, and III were significantly lower than in either HCC-B or HCC-C. In TNM stage IV, the median AFP level in HCC-nonBC was significantly lower than in either HCC-B or HCC-BC. The median DCP levels in HCC-nonBC with TNM stages I and II were significantly higher than those in either HCC-B or HCC-C. In TNM stage III, the median DCP level in HCC-nonBC was significantly higher than that in HCC-C. CONCLUSIONS: DCP was more sensitive than AFP for the diagnosis of early stage cryptogenic HCC. DCP should be used as the main serum test for cryptogenic HCC detection.

Long-acting human serum albumin-thioredoxin fusion protein suppresses bleomycin-induced pulmonary fibrosis progression.

Idiopathic pulmonary fibrosis (IPF) is thought to involve inflammatory cells and reactive oxygen species (ROS), such as superoxide anion radical (O2(·-)). There is currently no effective treatment of IPF. We previously developed a human serum albumin (HSA)-thioredoxin 1 (Trx) fusion protein (HSA-Trx) designed to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx, an antioxidative and anti-inflammatory protein. In this study, we examined the therapeutic effect of HSA-Trx on an IPF animal model of bleomycin (BLM)-induced pulmonary fibrosis. A pharmacokinetic study of HSA-Trx or Trx in BLM mice showed that the plasma retention and lung distribution of Trxc was markedly improved by fusion with HSA. A weekly intravenous administration of HSA-Trx, but not Trx, ameliorated BLM-induced fibrosis, as evidenced by a histopathological analysis and pulmonary hydroxyproline levels. HSA-Trx suppressed active-transforming growth factor (TGF)-ß levels in the lung and inhibited the increase of inflammatory cells in bronchoalveolar lavage fluid, pulmonary inflammatory cytokines, and oxidative stress markers. An in vitro EPR experiment using phosphate-buffered saline-stimulated neutrophils confirmed the O2(·-) scavenging ability of HSA-Trx. Furthermore, post-treatment of HSA-Trx had a suppressive effect against BLM-induced fibrosis. These results suggest that HSA-Trx has potential as a novel therapeutic agent for IPF, because of its long-acting antioxidative and anti-inflammatory modulation effects.

A human serum albumin-thioredoxin fusion protein prevents experimental contrast-induced nephropathy.

Contrast-induced nephropathy (CIN), caused by a combination of the direct tubular toxicity of contrast media, a reduction in medullary blood flow, and the generation of reactive oxygen species, is a serious clinical problem. A need exists for effective strategies for its prevention. Thioredoxin-1 (Trx) is a low-molecular-weight endogenous redox-active protein with a short half-life in the blood due to renal excretion. We produced a long-acting form of Trx as a recombinant human albumin-Trx fusion protein (HSA-Trx) and examined its effectiveness in preventing renal injury in a rat model of ioversol-induced CIN. Compared with saline, a mixture of HSA and Trx, or Trx alone, intravenous HSA-Trx pretreatment significantly attenuated elevations in serum creatinine, blood urea nitrogen, and urinary N-acetyl-ß-D-glucosaminidase along with the decrease in creatinine clearance. HSA-Trx also caused a substantial reduction in the histological features of renal tubular injuries and in the number of apoptosis-positive tubular cells. Changes in the markers 8-hydroxy deoxyguanosine and malondialdehyde indicated that HSA-Trx significantly suppressed renal oxidative stress. In HK-2 cells, HSA-Trx decreased the level of reactive oxygen species induced by hydrogen peroxide, and subsequently improved cell viability. Thus, our results suggest that due to its long-acting properties, HSA-Trx has the potential to effectively prevent CIN.

Baseline serum cholesterol is associated with a response to pegylated interferon alfa-2b and ribavirin therapy for chronic hepatitis C genotype 2.

Background. HCV infection is associated with lipid disorders because this virus utilizes the host lipid metabolism to sustain its life cycle. Several studies have indicated that higher concentrations of serum cholesterol and LDL before treatment are important predictors of higher rates of sustained virological response (SVR). However, most of these studies involved patients infected with HCV genotype 1. Thus, we performed a multi-institutional clinical study to evaluate the impact of lipid profiles on SVR rates in patients with HCV genotype 2. Methods. A total of 100 chronic hepatitis C patients with HCV genotype 2 who received peg-IFN alfa-2b and ribavirin therapy were consecutively enrolled. The significance of age, sex, BMI, AST level, ALT level, WBC, hemoglobin, platelet count, gamma-glutamyltransferase, total cholesterol level (TC), LDL level, HCV RNA, and histological evaluation was examined for SVR using logistic regression analysis. Results. The 100 patients infected with HCV genotype 2 were divided into 2 groups, an SVR group and a non-SVR group. Characteristics of each group were subsequently compared. There was no significant difference in the level of HCV RNA, BMI, platelet, TG, or stage of fibrosis between the groups. However, there were significant differences in the levels of TC and LDL-C. In multivariate logistic regression analysis using baseline characteristics, high TC level was an independent and significant risk factor (relative risk 18.59, P = 0.015) for SVR. Conclusion. Baseline serum total cholesterol levels should be considered when assessing the likelihood of sustained treatment response following the course of peg-IFN and ribavirin therapy in patients with chronic HCV genotype 2 infection.

Tum-1, a tumstatin fragment, gene delivery into hepatocellular carcinoma suppresses tumor growth through inhibiting angiogenesis.

Since hepatocellular carcinoma (HCC) is a hypervascular cancer, anti-angiogenic therapy is a promising approach to treat HCC. In the present study, we investigated the antiangiogenic and antitumor effects of tum-1, a fragment of tumstatin, gene transduction into HCC in vitro and in vivo. Tum-1 gene was cloned into a pSecTag2B mammalian expression vehicle to construct pSecTag2B-tum-1. pSecTag2B-tum-1 or vehicle were transfected into human HCC cells, PLC/PRF/5 cells stably and Huh-7 cells tran-siently. pSecTag2B-tum-1 transfection slightly repressed the proliferation of both PLC/PRF/5 and Huh-7 cells in vitro. Addition of conditioned media (CM) from tum-1 expressing PLC/PRF/5 cells significantly inhibited the spontaneous and vascular endothelial growth factor (VEGF)-induced proliferation and migration of human umbilical vein endothelial cells (HUVEC) in vitro with diminishing the VEGF-induced phosphorylation of both Akt and extracellular signal-regulated kinase (ERK) that are known to mediate VEGF-induced proliferation and migration of endothelial cells. In in vivo experiments, intratumoral injection of pSecTag2B-tum-1 significantly repressed the growth of pre-established Huh-7 tumors in athymic mouse models accompanying the decreased density of CD34 positive vessels in tumors. In conclusion, our results suggest that antiangiogenic gene therapy using tum-1 gene may be an efficient strategy for the treatment of HCC.

Detection of HBV core promoter and precore mutations helps distinguish flares of chronic hepatitis from acute hepatitis B.

BACKGROUND AND AIM: Acute exacerbation of chronic hepatitis B has to be distinguished from acute hepatitis, because treatment strategies differ between them. METHODS: Mutations in the core promoter and precore region of hepatitis B virus (HBV) were determined in 36 patients with acute exacerbation of chronic hepatitis B, in whom alanine aminotransferase (ALT) increased above 500 IU/L, as well as the 36 patients with acute hepatitis. RESULTS: Mutations in the core promoter (A1762T/G1764A) and precore region (G1896A) were more frequent in patients with acute exacerbation of chronic hepatitis than acute hepatitis (81% vs 19%; P < 0.0001 and 58% vs 6%; P < 0.0001, respectively). Of the 19 patients with mutations in both the core promoter and precore region, 17 (89%) had acute exacerbation of chronic hepatitis. In contrast, among the 32 patients with the wild-type for both the core promoter and precore region, 29 (89%) developed acute hepatitis. By multivariate analysis, the double mutation in the core promoter was predictive of acute exacerbation in chronic hepatitis with the highest odds ratio at 26.4. CONCLUSIONS: In patients with hepatitis B having ALT levels >500 IU/L, mutations in the core promoter and precore region are useful in distinguishing acute exacerbation of chronic from acute HBV infection. Detection of these mutations would be useful for commencing prompt antiviral treatments on patients with acute exacerbation of chronic hepatitis for a better prognosis.

Clearance of hepatitis C virus after living-donor liver transplantation in spite of residual viremia on end date of interferon therapy before transplantation.

Interferon (IFN) therapy is the only treatment strategy for hepatitis C virus (HCV) infection after liver transplantation (LT), but prophylactic and treatable IFN therapy after LT has been shown to be insufficient due to the adverse effects of IFN and rivabirin. In this paper, we describe the disappearance of HCV after LT without IFN therapy in the presence of residual viremia on the day of LT. We herein report our findings since this is considered an important case for the anti-HCV strategy of post LT. A 60-year old woman with LC and HCC was referred to Nagasaki University Hospital in August 2004. After she underwent LT on February 18, 2005, we injected peg-IFN-alpha-2a the 11th time at 18 wk and HCV-RNA was still positive in the serum at LT. The serum HCV-RNA was negative one month after operation and subsequently dissolved 15 mo after operation without IFN therapy. As a result, we speculate that if HCV-RNA is positive while HCV core antigen is negative before LT, then it may lead to clearance of HCV after LT. Therefore long acting peg-IFN-alpha-2a is thus considered a potentially effective agent for the treatment of HCV-related cirrhosis before LT.

Abrogation of constitutive STAT3 activity sensitizes human hepatoma cells to TRAIL-mediated apoptosis.

BACKGROUND/AIMS: Signal transducer and activator of transcription 3 (STAT3) is constitutively activated and regulates cell growth and survival of various cancer cells. We investigated the anti-tumor effect of AG490, a Janus kinase 2 specific inhibitor, inhuman hepatoma cells. METHODS: Effects of AG490 on STAT3 activation, on cell-growth and survival, and on the expression of cell-cycle- and apoptosis-related proteins were evaluated in Huh-1, Huh-7, HepG2 and Hep3B cells. Next, whether AG490 renders hepatoma cells susceptible to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was examined in vitro and in vivo. RESULTS: Constitutively activated STAT3 through tyrosine phosphorylation was detected in all hepatoma cells. AG490 inhibited the phosphorylation of STAT3 and its activity. AG490 induced cell cycle arrest in Huh-1, Huh-7 and HepG2 through cyclin D1 downregulation, and induced marked apoptosis in Hep3B. AG490 downregulated at least one of the anti-apoptotic proteins, Bcl-xL, survivin or XIAP in all hepatoma cells. AG490 sensitized Huh-1, Huh-7 and HepG2 to TRAIL-induced apoptosis in vitro. Intraperitoneal injection of AG490, the combination of AG490 and TRAIL more greatly, repressed the growth of subcutaneous Huh-7 tumors in athymic mice. CONCLUSIONS: Abrogation of constitutive activation of STAT3 by AG490 enhances the anti-tumor activity of TRAIL against human hepatoma cells.

Role of growth hormone, insulin-like growth factor 1 and insulin-like growth factor-binding protein 3 in development of non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Pituitary dysfunction including growth hormone (GH) deficiency may be associated with non-alcoholic fatty liver disease (NAFLD). Since the relationships among GH, IGF-1, IGFBP-3, and development of NAFLD without hypopituitarism are unclear, we examined the role of these hormones in the development of NAFLD based on clinical, laboratory and liver histology data. PATIENTS AND METHODS: A total of 55 consecutive patients (20 males and 35 females) with NAFLD. RESULTS: Aspartate amino transferase (AST), AST/ALT, platelet count and IGF-1, levels were significantly associated with differences in fibrosis, since these variables differed between stage 0-1 and stage 2-3 NAFLD. In multivariate analysis, platelet count (P = 0.0223, relative risk (RR), 5.899; 95% confidence interval (CI), 1.288-27.017), and IGF-1 (P = 0.0363, RR, 4.568; 95% CI, 1.101-18.945) showed significant associations with stage 2-3 NAFLD. Additionally, hyaluronic acid levels had a negative relationship with IGF-1 and the IGF-1/IGFBP-3 ratio. There was no relationship of fibrosis with GH level, but decreased GH (P = 0.0414, RR, 0.199; 95% CI, 0.042-0.989) was significantly associated with steatosis of stage 2-3. Low GH/IGF-1 and GH/IGFBP-3 ratios were found in advanced steatosis. CONCLUSION: GH, IGF-1 and IGFBP-3 are associated with hepatic fibrosis and steatosis in NAFLD. Low levels of IGF-1 might be associated with fibrosis while low level of GH with hepatic steatosis.

Association between liver fibrosis and insulin sensitivity in chronic hepatitis C patients.

BACKGROUND: Several clinical studies have suggested a possible link between chronic hepatitis caused by hepatitis C virus (HCV) and the development of diabetes mellitus. We investigated the association between liver fibrosis and glucose intolerance in HCV-infected patients by measuring insulin sensitivity and beta-cell function. METHOD: A total of 83 chronic HCV-infected patients were recruited into this study. We evaluated insulin sensitivity and beta-cell function of all patients in a fasting state (homeostasis model assessment of insulin resistance [HOMA-R] and homeostasis model assessment of beta-cell function [HOMA-beta]) and after an oral load of 75 g glucose (whole-body insulin sensitivity index [WBISI] and Delta-insulin/Delta-glucose 30). RESULTS: In a multivariate analysis, severe fibrosis was the only independent factor associated with insulin resistance. There were significant differences in both HOMA-R (P= 0.0063) and WBISI (P= 0.0159) between patients with mild fibrosis (N = 34) and those with severe fibrosis (N = 49). Although HOMA-beta was increased significantly in the subjects with severe fibrosis compared with those with mild fibrosis (P= 0.0169), Delta-insulin/Delta-glucose 30 showed no significant difference in stage of liver fibrosis, suggesting an uncertain association between liver fibrosis and beta-cell function. CONCLUSION: Our findings suggest that the development of liver fibrosis is associated with insulin resistance in HCV-infected patients.

Aging of patients with hepatitis C virus-associated hepatocellular carcinoma: long-term trends in Japan.

The incidence of hepatocellular carcinoma (HCC) in Japan has been increasing. The aim of the present study was to analyze epidemiological changes in Japanese HCC patients. A total of 463 patients with HCC diagnosed at our hospital between 1982 and 2001 were recruited for this study. Cohorts of patients with HCC were categorized into intervals of five years. The number of HBV- and HCV-associated HCC cases had decreased and increased in 1987-1991, respectively, and thereafter reached a plateau. The mean age of patients at diagnosis of HCV-associated HCC showed a steady significant increase from 60 to 68 years of age during the period, suggesting that these findings were associated with a shift toward an older-age group that had the highest rate of HCV infection. The mean age of patients with other types of HCC did not significantly change during the period. Since it is known that the prevalence of HCV infection in young Japanese persons is low and that the incidence of HCV infection is very low at present, our findings may indicate that the prevalence of HCC will decline in Japan, an advanced country with regard to HCV-associated HCC, in the near future.

The impact of newer treatment modalities on survival in patients with hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. However, although the therapeutic approaches for HCC have progressed rapidly, it remains unknown whether the current management of patients with HCC has reduced its mortality. We analyzed changes of survival rate in patients with HCC over a 20-year period. METHODS: Between 1982 and 2001, 463 patients were diagnosed with HCC at our hospital. Subjects were enrolled in the current cohort according to the following inclusion criteria: HCC lesion measuring less than 3 cm in diameter, no evidence of extrahepatic metastasis, and no evidence of main portal vein infiltration/thrombosis. A total of 257 patients with HCC were recruited for this study, and categorized into 5-year intervals. RESULTS: The survival rates improved significantly during the study period. When the patients were stratified according to Child-Pugh score, only patients with Child's B showed improved survival rates. Furthermore, patients with surgical resection or transarterial chemoembolization during the latter period had a better prognosis than those during the early period. CONCLUSIONS: Our findings suggest that the development of therapeutic interventions for HCC have led to improvements in the prognosis for HCC patients.

DHMEQ, a novel NF-kappaB inhibitor, induces apoptosis and cell-cycle arrest in human hepatoma cells.

Several reports have indicated that nuclear factor-kappaB (NF-kappaB) is constitutively activated in a variety of cancer cells including hepatoma cells and plays a key role in their growth and survival. Dehydroxymethylepoxyquinomicin (DHMEQ) derived from the structure of an antibiotic epoxyquinomicin C is a novel NF-kappaB inhibitor. In the present study, we evaluated the effect of DHMEQ on the NF-kappaB activity in human hepatoma cells, Huh-7, HepG2 and Hep3B, and the anti-tumor effect of DHMEQ on these cells in vitro and in vivo. DHMEQ inhibited the steady-state transcriptional activity of NF-kappaB in all hepatoma cells. DHMEQ blocked the constitutive DNA-binding activity and TNF-alpha-mediated nuclear translocation of NF-kappaB in Huh-7 cells. DHMEQ (5-20 microg/ml) dose-dependently reduced the viable cell number of all hepatoma cells. DHMEQ (20 microg/ml) induced apoptosis in all hepatoma cells, especially in Hep3B cells, and cell-cycle arrest in Huh-7 and HepG2 cells. These effects were accompanied by downregulation of proteins involved in anti-apoptosis (Bcl-xL, XIAP or c-IAP2) and cell-cycle progression (cyclin D1), and induction of proteins involved in pro-apoptosis (Bax) and cell-cycle retardation (p21Waf1/Cip1), although the degree of changes by DHMEQ was different in each hepatoma cell type. Moreover, intraperitoneal administration of DHMEQ (8 mg/kg) significantly repressed the growth of Huh-7 tumor subcutaneously transplanted into BALB/c nu/nu athymic mice. Our results suggest that DHMEQ could qualify as a candidate for a new chemotherapeutic agent against human hepatoma.