Protein disulfide isomerase inhibition synergistically enhances the efficacy of sorafenib for hepatocellular carcinoma.

Sorafenib is the only approved targeted drug for hepatocellular carcinoma (HCC), but its effect on patients' survival gain is limited and varies over a wide range depending on pathogenetic conditions. Thus, enhancing the efficacy of sorafenib and finding a reliable predictive biomarker are crucial to achieve efficient control of HCCs. In this study, we utilized a systems approach by combining transcriptome analysis of the mRNA changes in HCC cell lines in response to sorafenib with network analysis to investigate the action and resistance mechanism of sorafenib. Gene list functional enrichment analysis and gene set enrichment analysis revealed that proteotoxic stress and apoptosis modules are activated in the presence of sorafenib. Further analysis of the endoplasmic reticulum stress network model, combined with in vitro experiments, showed that introducing an additional stress by treating the orally active protein disulfide isomerase (PDI) inhibitor (PACMA 31) can synergistically increase the efficacy of sorafenib in vitro and in vivo, which was confirmed using a mouse xenograft model. We also found that HCC patients with high PDI expression show resistance to sorafenib and poor clinical outcomes, compared to the low-PDI-expression group. CONCLUSION: These results suggest that PDI is a promising therapeutic target for enhancing the efficacy of sorafenib and can also be a biomarker for predicting sorafenib responsiveness. (Hepatology 2017;66:855-868).

Targeted Proteomics Predicts a Sustained Complete-Response after Transarterial Chemoembolization and Clinical Outcomes in Patients with Hepatocellular Carcinoma: A Prospective Cohort Study.

This study was aimed to identify blood-based biomarkers to predict a sustained complete response (CR) after transarterial chemoembolization (TACE) using targeted proteomics. Consecutive patients with HCC who had undergone TACE were prospectively enrolled (training (n = 100) and validation set (n = 80)). Serum samples were obtained before and 6 months after TACE. Treatment responses were evaluated using the modified Response Evaluation Criteria in Solid Tumors (mRECIST). In the training set, the MRM-MS assay identified five marker candidate proteins (LRG1, APCS, BCHE, C7, and FCN3). When this five-marker panel was combined with the best-performing clinical variables (tumor number, baseline PIVKA, and baseline AFP), the resulting ensemble model had the highest area under the receiver operating curve (AUROC) value in predicting a sustained CR after TACE in the training and validation sets (0.881 and 0.813, respectively). Furthermore, the ensemble model was an independent predictor of rapid progression (hazard ratio (HR), 2.889; 95% confidence interval (CI), 1.612-5.178; P value < 0.001) and overall an unfavorable survival rate (HR, 1.985; 95% CI, 1.024-3.848; P value = 0.042) in the entire population by multivariate analysis. Targeted proteomics-based ensemble model can predict clinical outcomes after TACE. Therefore, this model can aid in determining the best candidates for TACE and the need for adjuvant therapy.

Body Fat Distribution and Risk of Incident and Regressed Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Some studies have examined correlations between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) with nonalcoholic fatty liver disease (NAFLD) or between VAT and NAFLD. We investigated the longitudinal association between body fat distribution (VAT vs SAT) and incidence and regression of NAFLD, adjusting for risk factors, in a large population-based cohort. METHODS: We collected data from adults who underwent abdominal ultrasonography (to identify liver fat), abdominal fat computed tomography scan, and blood tests from March 2007 through December 2008. Each patient underwent an anthropometric assessment and completed a questionnaire about their medical history, physical activity, and diet. Our final analysis involved 2017 subjects from the initial cohort who participated in a voluntary follow-up health screen performed in 2011 and 2013. The median follow-up time was 4.43 years. RESULTS: We found 288 incident cases of NAFLD; 159 patients had NAFLD regression during the follow-up period. An increasing area of VAT was associated with higher incidence of NAFLD in the multivariable analysis (highest quintile vs lowest quintile of VAT hazard ratio [HR], 2.23; 95% confidence interval [CI], 1.28-3.89; P for trend = .002; HR, 1.36 [per 1 standard deviation]; 95% CI, 1.16-1.59). An increased area of SAT was significantly associated with regression of NAFLD (highest quintile vs lowest quintile of SAT HR, 2.30; 95% CI, 1.28-4.12; P for trend = .002; HR, 1.36 [per 1 standard deviation]; 95% CI, 1.08-1.72). CONCLUSIONS: In a large cohort study, larger areas of VAT were longitudinally associated with higher risk of incident NAFLD (during a period of approximately 4 years). In contrast, larger areas of SAT were longitudinally associated with regression of NAFLD. These data indicate that certain types of body fat are risk factors for NAFLD, whereas other types could reduce risk for NAFLD.

Differential sensitivity of hepatocellular carcinoma cells to suppression of hepatocystin transcription under hypoxic conditions.

Mutations in the gene encoding hepatocystin/80 K-H (PRKCSH) cause autosomal dominant polycystic liver disease. Hepatocystin deficiency impairs glucosidase II activity, which is critical for processing and folding glycoproteins in the endoplasmic reticulum (ER). Hypoxia is known as a strong stimulus for generating survival signals in hepatocellular carcinoma (HCC) cells. However, hypoxia may induce cell apoptosis under conditions of severe ER stress. Thus, we hypothesized that suppression of hepatocystin transcription induces HCC cell death under hypoxic conditions due to excessive ER stress. A new human HCC cell line, SNU-3058, was established following primary culture of tumor cells harvested from a Korean patient with rapidly growing hypovascular HCC. In cell culture, human HCC cells (Huh-7, SNU-761, and SNU-3058) were treated with control siRNA or hepatocystin siRNA with or without doxorubicin under hypoxic conditions. Cell viability, ER stress, unfolded protein response (UPR), and apoptosis were assessed using the MTS assay, immunoblot assay, and RT-PCR. Suppression of hepatocystin transcription attenuated proliferation in Huh-7 and SNU-761 cells, while proliferation was amplified in SNU-3058 cells. Similar results were observed following treatment with doxorubicin. Hepatocystin siRNA transfection increased cell death in Huh-7 and decreased cell death in SNU-3058. In SNU-3058, hepatocystin siRNA amplified GRP78, known as a pro-survival and cyto-protective signal, and attenuated the pro-apoptotic signal CHOP. These findings suggest that suppression of hepatocystin transcription induce the UPR, which alleviates damage associated with ER stress in SNU-3058. UPR had a limited role in protecting SNU-761 cells, resulting in cell death through apoptosis. In addition, blocking of pro-survival UPR signal by bacitracin or GRP78 knockdown, attenuated hepatocystin siRNA-induced proliferation in SNU-3058 cells under hypoxia. In this study, we demonstrated that different sensitivities to hepatocystin siRNA among human HCC cell lines are dependent on appropriate UPRs to hypoxia-induced ER stress following hepatocystin siRNA transfection. Because UPR is the main evasive mechanism for apoptosis induced by suppression of hepatocystin, targeting hepatocystin via UPR suppression could be a strategy for treating HCC.

Hypoxia Enhances Tumor-Stroma Crosstalk that Drives the Progression of Hepatocellular Carcinoma.

BACKGROUND: Crosstalk between tumor cells and their microenvironment plays a crucial role in the progression of hepatocellular carcinoma (HCC). Hypoxia, a common feature of advanced HCC, has been shown to modulate the evolution of the tumor microenvironment. In this study, we investigated the effect of hypoxia on tumor-stroma crosstalk in HCC. METHODS: Human HCC cell lines (Huh-BAT, SNU-475) were cocultured with an activated human hepatic stellate cell line (HSCs; LX-2) under either normoxic or hypoxic conditions. Cell growth was evaluated with the MTS assay. Apoptotic signaling cascades were assessed by immunoblot analysis. Expression of CD31 and phosphorylated (p-) Akt in HCC tissues was detected by immunohistochemistry. RESULTS: Coculturing HCC cells with HSCs under hypoxic conditions enhanced their proliferation, migration, and resistance to bile acid (BA)-induced apoptosis compared to coculturing under normoxic conditions. Under hypoxia, of various HSC-derived growth factors, PDGF-BB was the most up-regulated, leading to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in HCC cells. Immunohistochemical study also revealed that p-Akt was highly expressed in hypoxic, hypovascular HCC as compared to hypervascular HCC. Neutralizing antisera to PDGF-BB or a PI3K inhibitor attenuated the proliferation of HCC cells cocultured with HSCs, and sensitized HCC cells to BA-induced apoptosis, especially under hypoxic conditions. CONCLUSIONS: In conclusion, hypoxic HSC-derived PDGF-BB stimulates the proliferation of HCC cells through activation of the PI3K/Akt pathway, while the inhibition of PDGF-BB or PI3K/Akt pathways enhances apoptotic cell death. Targeting tumor-stroma crosstalk might be a novel therapy in the management of human HCCs.

A genome-wide association study identified new variants associated with the risk of chronic hepatitis B.

UNLABELLED: Hepatitis B virus (HBV) infection is the predominant risk factor for chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Recently, several genome-wide association studies (GWASs) of CHB identified human leukocyte antigen (HLA) loci, including HLA-DP and HLA-DQ in Asian populations, as being associated with the risk of CHB. To confirm and identify the host genetic factors related to CHB infection, we performed another GWAS using a higher-density chip in Korean CHB carriers. We analyzed 1400 samples from Korean population (400 CHB cases and 1000 population controls) using a higher-density GWAS chip [1 140 419 single nucleotide polymorphisms (SNPs)]. In subsequent replication analysis, we further analyzed in an independent study of a Korean CHB cohort consisting of 2909 Korean samples (971 cases and 1938 controls). Logistic regression methods were used for statistical analysis adjusting for age and sex as covariates. This study identified two new risk-associated loci for CHB on the HLA region of chromosome 6, e.g. rs652888 on euchromatic histone-lysine-methyltransferase 2 (EHMT2, P = 7.07 × 10(-13)) and rs1419881 on transcription factor 19 (TCF19, P = 1.26 × 10(-18)). Conditional analysis with nearby HLA CHB loci that were previously known, confirmed the independent genetic effects of these two loci on CHB. CONCLUSION: The GWAS and the subsequent validation study identified new variants associated with the risk of CHB. These findings may advance the understanding of genetic susceptibility to CHB.

Tenofovir monotherapy versus tenofovir plus lamivudine or telbivudine combination therapy in treatment of lamivudine-resistant chronic hepatitis B.

Tenofovir disoproxil fumarate (TDF) monotherapy is a therapeutic option for chronic hepatitis B (CHB) patients infected with hepatitis B virus (HBV) variants resistant to lamivudine (LAM). We evaluated the antiviral efficacy and safety of TDF alone compared to those of TDF plus LAM or telbivudine (LdT) combination in patients harboring HBV variants with genotypic resistance to LAM. This multicenter retrospective study included consecutive patients who had LAM-resistant HBV variants and were treated with TDF alone (monotherapy group) or TDF combined with LAM or LdT (combination therapy group) for at least 6 months. Inverse probability of treatment weighting (IPTW) for the entire cohort was applied to control for treatment selection bias. Overall, 153 patients (33 in the monotherapy group and 120 in the combination therapy group) were analyzed. The overall probability of achieving complete virologic suppression at month 12 was 91.6%: 88.6% in the monotherapy group and 92.6% in the combination therapy group. Combination therapy was not superior to monotherapy in viral suppression before and after IPTW (P=0.562 and P=0.194, respectively). Hepatitis B e antigen (HBeAg) loss, biochemical response, and virologic breakthrough did not differ between treatment groups. The probabilities of complete virologic suppression were comparable between treatment groups in the subsets according to HBeAg status and HBV DNA levels at baseline. No patient experienced any significant renal dysfunction during the treatment period. In conclusion, TDF monotherapy has antiviral efficacy comparable to that of TDF plus LAM or LdT combination therapy, with a favorable safety profile in CHB patients with LAM-resistant HBV variants.

Forns index predicts recurrence and death in patients with hepatitis B-related hepatocellular carcinoma after curative resection.

BACKGROUND & AIMS: Advanced liver fibrosis is associated with recurrence after curative resection of hepatocellular carcinoma (HCC). This study aimed to investigate whether noninvasive fibrosis indices could predict intrahepatic recurrence and death after curative resection of HCC. METHODS: Patients who underwent curative resection for hepatitis B virus (HBV)-related HCC between 2006 and 2010 at a single tertiary hospital were included. This study analysed the association of noninvasive fibrosis indices with recurrence and overall survival. RESULTS: A total of 303 patients were included. During a median follow-up period of 56.0 (interquartile range, 42.0-70.0) months, 151 (49.8%) patients experienced HCC recurrence and 54 (17.8%) died. Based on multivariate analysis, Forns index [hazard ratio (HR), 1.238; 95% confidence interval (CI), 1.097-1.398; P = 0.001] was independently associated with tumour recurrence after adjustment for anti-HBe positivity, histological cirrhosis, tumour size and number. Patients with Forns index <6.9 had a significantly longer recurrence-free survival rate than patients with Forns index ≥6.9 (P < 0.001 by log-rank test). Forns index (HR, 1.246; 95% CI, 1.034-1.501; P = 0.02) could also predict overall survival after adjustment for tumour size and number. Forns index detected cirrhosis with an AUROC of 0.700 (95% CI, 0.641-0.758). Aspartate aminotransferase-to-platelet ratio index, cirrhosis discriminant score, FIB-4 index, P2/MS and Lok index detected cirrhosis comparably to Forns index, but were not associated with tumour recurrence or death. CONCLUSIONS: Our data suggest that Forns index could be a useful predictor of recurrence and overall survival after curative resection of HBV-related HCC.

Efficacy of entecavir-tenofovir combination therapy for chronic hepatitis B patients with multidrug-resistant strains.

The emergence of multidrug-resistant (MDR) strains of hepatitis B virus (HBV) is a major concern. This study aimed to investigate the efficacy and safety of combination therapy with entecavir (ETV) plus tenofovir disoproxil fumarate (TDF) against MDR HBV. To adjust for differences in baseline characteristics, inverse probability weighting (IPW) using propensity scores for the entire cohort and weighted Cox proportional hazards models were applied. Ninety-three consecutive patients who were treated with ETV-TDF combination therapy for >6 months were included; at baseline, 45 were infected with HBV strains with genotypic resistance to lamivudine (LAM) and ETV (the LAM/ETV-R group), 28 with strains resistant to LAM and adefovir (ADV) (the LAM/ADV-R group), and 20 with strains resistant to LAM, ETV, and ADV (the LAM/ETV/ADV-R group). The median duration of rescue therapy was 13.0 (range, 6.7 to 31.7) months. Seventy-four of 93 patients (79.6%) achieved complete virologic suppression, after a median of 4.5 (95% confidence interval, 3.0 to 6.0) months. The cumulative probability of complete virologic suppression at month 6 was 63.6% (55.7%, 75.0%, and 65.0% in the LAM/ETV-R, LAM/ADV-R, and LAM/ETV/ADV-R groups, respectively). During the treatment period, these probabilities were not significantly different across the resistance profiles before and after IPW (P = 0.072 and P = 0.510, respectively). In multivariate analysis, a lower baseline HBV DNA level, but not resistance profiles, was an independent predictor of complete virologic suppression. Renal dysfunction was not observed during the treatment period. In conclusion, rescue therapy with ETV-TDF combination is efficient and safe in patients infected with MDR HBV strains regardless of the antiviral drug resistance profiles.

High-sensitivity C-reactive protein level is an independent predictor of poor prognosis in cirrhotic patients with spontaneous bacterial peritonitis.

BACKGROUND/AIMS: The production of high-sensitivity C-reactive protein (hs-CRP) may be affected by hepatic function, and the clinical importance of hs-CRP in patients with liver cirrhosis is still not clear. The aim of this study was to evaluate the clinical implications of hs-CRP in cirrhotic patients with spontaneous bacterial peritonitis (SBP). METHODS: We retrospectively investigated 336 consecutive patients treated for SBP from 2007 to 2012. The relationship between serum hs-CRP and the result of the treatment was assessed. RESULTS: A response to antibiotics was observed in 182 patients (54.2%), and 126 patients (37.5%) died of SBP. The initial hs-CRP (odds ratio=1.061, P=0.016), coexistent hepatocellular carcinoma, and Child-Pugh (CP) score were independent prognostic factors for high in-hospital mortality. Serum hs-CRP level was also an independent predictor of lower antibiotic response rate (odds ratio=0.916, P<0.001). However, hs-CRP was negatively correlated with the CP score (r=-0.199, P<0.001) and Model for End-Stage Liver Disease score (r=-0.182, P=0.001). CONCLUSIONS: This study found that serum hs-CRP level is related to a lower response rate to antibiotics, a higher mortality rate in patients with SBP. The hs-CRP level was negatively correlated with the CP and Model for End-Stage Liver Disease scores, which suggests that the prognostic function of hs-CRP was not a surrogate for hepatic dysfunction.

Tauroursodeoxycholic acid attenuates progression of steatohepatitis in mice fed a methionine-choline-deficient diet.

BACKGROUND AND AIM: Endoplasmic reticulum (ER) stress has been implicated in the development of nonalcoholic steatohepatitis. A methionine-choline-deficient (MCD) diet induces robust ER stress response and steatohepatitis, but the effects of ER stress modulation on the course of steatohepatitis remain uncertain. The present study evaluated whether reducing ER stress using the chemical chaperone tauroursodeoxycholic acid (TUDCA) could limit hepatocyte lipoapoptosis and progression of MCD diet-induced steatohepatitis. METHODS: HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH-Ntcp cells) and palmitate (PA) were used. Experimental steatohepatitis was induced in male C57BL/6 mice using an MCD diet, and three different doses of TUDCA (500, or 1,000 mg/kg, once daily; or 500 mg/kg twice daily) were administered by gavage from the start of the MCD diet regimen or after 4 weeks. RESULTS: TUDCA reduced PA-induced ER stress as manifested by decreased eIF2α phosphorylation, XBP1 splicing and expression of BiP, ATF4, and CHOP in HuH-Ntcp cells. TUDCA also decreased PA-induced JNK phosphorylation, Puma up-regulation and Bax activation, which in turn suppressed caspase-dependent hepatocyte lipoapoptosis. Mice given TUDCA did not show a significant decrease in the intrahepatic triglyceride contents and steatosis. However, TUDCA treatment significantly reduced hepatic damage compared to controls for both early and late treatment groups. TUDCA treatment reduced the expression of ER stress markers and pro-apoptotic proteins, leading to decreased apoptosis and oxidative stress. Finally, TUDCA reduced histological fibrosis along with the down-regulation of pro-fibrotic gene expression in both early and late treatment groups. CONCLUSIONS: These results show that TUDCA attenuates the progression of MCD diet-induced steatohepatitis by reducing ER stress.

Efficacy of adefovir-based combination therapy for patients with Lamivudine- and entecavir-resistant chronic hepatitis B virus infection.

Treatment strategies for entecavir (ETV)-resistant chronic hepatitis B (CHB) patients are not yet well established. The aim of this study was to evaluate overall antiviral efficacy and to compare the efficacy of combination therapy with adefovir (ADV) plus nucleoside analogues (lamivudine [LAM], telbivudine [LdT], or ETV) in patients infected with LAM- and ETV-resistant hepatitis B virus (HBV) variants. Virologic, biochemical, and serologic responses during combination therapy with ADV plus nucleoside analogues were assessed. Propensity score analysis was used to select a matched group of patients for the comparison of rescue therapy regimens. A total of 67 consecutive patients were analyzed. Complete virologic suppression was achieved in 27 patients. The overall cumulative incidence of complete virologic suppression at month 24 was 47.4%: 44.3% in the LAM or LdT plus ADV group and 51.4% in the group given ETV and ADV. There was no significant difference between these two groups (P = 0.234). The cumulative incidences of complete virologic suppression were still comparable between the two groups selected and matched using the propensity score model (P = 0.419). Virologic breakthrough was observed in 9 patients, and rtA181V substitution was newly detected in one patient. Hepatitis B e antigen (HBeAg) negativity and lower baseline HBV DNA level were associated with complete virologic suppression in univariate analysis. In multivariate analysis, lower baseline HBV DNA level remained an independent predictor. In conclusion, combination therapy with ADV plus nucleoside analogues fails to show sufficient antiviral efficacy in CHB patients with resistance to both LAM and ETV. Further study is warranted to evaluate the efficacy of a more potent tenofovir-based regimen in such patients.

Hepatocellular carcinoma: high hepatitis B viral load and mortality in patients treated with transarterial chemoembolization.

PURPOSE: To determine the relationship between hepatitis B virus (HBV) DNA level and the survival of patients with hepatocellular carcinoma treated by means of transarterial chemoembolization (TACE). MATERIALS AND METHODS: This study was approved by the institutional review board, and the requirement to obtain informed consent was waived. From January 2005 to March 2007, 183 patients with HBV-related hepatocellular carcinoma who underwent TACE but never received antiviral therapy were consecutively enrolled in our cohort. All patients were tested for pre-TACE serum level of HBV DNA, and overall survival was measured from date of enrollment until death from any cause. Radiologic progression was evaluated by using the modified response evaluation criteria in solid tumors by means of independent radiologic assessment. RESULTS: The median overall survival was 19 months (95% confidence interval: 13.7, 24.3) and median time to progression was 4 months (95% confidence interval: 3.03, 4.97). Multivariate analysis revealed that a high pre-TACE serum level of HBV DNA (> 2000 IU/L) was an independent risk factor for reduced overall survival (P = .021; hazard ratio [HR], 1.725), high cancer progression-related mortality (P = .014; HR, 1.936), and hepatic failure-related mortality associated with cancer progression (P = .005, HR, 3.908). Pre-TACE level of HBV DNA did not significantly affect hepatic failure-related mortality that was not caused by cancer progression. CONCLUSION: A high pre-TACE serum level of HBV DNA was associated with poor overall survival and rapid progression of hepatocellular carcinoma after TACE, and the cause of mortality was not hepatitis exacerbation but cancer progression.

Polymorphisms near interleukin 28B gene are not associated with hepatitis B virus clearance, hepatitis B e antigen clearance and hepatocellular carcinoma occurrence.

BACKGROUND: Polymorphisms near the IL28B gene have been proposed to be strongly associated with treatment response and the rate of spontaneous clearance of hepatitis C virus infection, and treatment response of hepatitis B virus (HBV) infection. In this study, we aimed to determine whether these polymorphisms could affect natural courses of HBV infection. METHODS: Genetic variations were identified through direct DNA sequencing using TaqMan assay in 1,439 patients with past or present HBV infection. Subjects included 404 spontaneously recovered patients, 313 chronic hepatitis B (CHB) patients, 305 liver cirrhosis (LC) patients and 417 hepatocellular carcinoma (HCC) patients. Three polymorphisms near the IL28B gene, rs8099917T>G, rs12979860C>T and rs12980275A>G, were identified. Associations between these polymorphisms and HBV clearance, hepatitis B e antigen (HBeAg) clearance as well as HCC occurrence among patients were analyzed using logistic regression analyses adjusted for age and gender. RESULTS: There were no significant associations between these polymorphisms and the HBV clearance both in CHB and LC groups. Similarly, these polymorphisms showed no significant associations with HBeAg clearance and the occurrence of HCC either. DISCUSSION: No significant association was identified between polymorphisms near the IL28B gene and the natural courses of chronic HBV infection, including the HBV clearance and HCC occurrence.

Guggulsterone attenuates activation and survival of hepatic stellate cell by inhibiting nuclear factor kappa B activation and inducing apoptosis.

BACKGROUND AND AIM: Liver fibrosis is associated with the deposition of the extracellular matrix, and hepatic stellate cells (HSCs) are the major source of these matrix proteins. Guggulsterone has recently been shown to induce apoptosis in several cell lines. Thus, the aim of this study was to evaluate whether guggulsterone has antifibrotic activities by reducing the activation and survival of HSCs. METHODS: Apoptotic and fibrosis-related signaling pathways and nuclear factor kappa B (NF-κB) activity were explored in LX-2 cells, an immortalized human HSC line, and in a mice model of liver fibrosis. RESULTS: Guggulsterone suppressed LX-2 cell growth in a dose- and activation-dependent manner. This growth suppression was due to the induction of HSC apoptosis, which was mediated by the activation of c-Jun N-terminal kinase and mitochondrial apoptotic signaling. Additionally, guggulsterone regulated phosphorylation of Akt and adenosine monophosphate-activated protein kinase, which were subsequently proven responsible for the guggulsterone-induced HSC growth suppression. Guggulsterone inhibited NF-κB activation in LX-2 cells, which is one of the major mediators in HSC activation. Indeed, guggulsterone decreased collagen α1 synthesis and α-smooth muscle actin expression in these cells. Compared with the control mice or mice treated with a low dose of guggulsterone, high dose of guggulsterone significantly decreased the extent of collagen deposition and the percentage of activated HSCs undergoing apoptosis. CONCLUSIONS: These results demonstrate that guggulsterone suppressed HSC activation and survival by inhibiting NF-κB activation and inducing apoptosis. Therefore, guggulsterone may be useful as an antifibrotic agent in chronic liver diseases.

The efficacy of adefovir plus entecavir combination therapy in patients with chronic hepatitis B refractory to both lamivudine and adefovir.

BACKGROUND: The efficacy of adefovir (ADV) plus entecavir (ETV) combination in patients with chronic hepatitis B (CHB) who developed multidrug refractoriness had not been fully evaluated. We aimed to evaluate the efficacy of ADV plus ETV as compared to that of lamivudine (LAM) plus ADV in the patients with antiviral refractoriness to sequential LAM monotherapy and then ADV monotherapy. METHODS: Twenty-seven patients were treated with a combination of ADV plus ETV and 63 patients were treated with a combination of LAM plus ADV. The virological and biochemical parameters were compared between the two groups, retrospectively. RESULTS: Treatment with a combination of ADV plus ETV produced significantly superior virological response than that of a combination of LAM plus ADV. At 12 months, the HBV DNA declined more in the ADV plus ETV group than in the LAM plus ADV (-4.52 ± 1.956 vs. -2.65 ± 1.723 log 10 IU/mL; p = 0.001). The rate of a complete response at 12 months was greater in the ADV plus ETV group than that in the LAM plus ADV group (63.16 vs. 14.81 %, p < 0.001). CONCLUSION: In the patients with CHB refractory to both LAM and ADV, the response to ADV plus ETV was significantly superior compared to that of the LAM plus ADV in suppressing HBV DNA. The result indicates that ADV plus ETV can be used as a bridging therapy in the patients with refractoriness to both LAM and ADV, especially in the areas where tenofovir is not yet available.

Enhancement of hexokinase II inhibitor-induced apoptosis in hepatocellular carcinoma cells via augmenting ER stress and anti-angiogenesis by protein disulfide isomerase inhibition.

3-bromopyruvate (3-BP), a hexokinase (HK) II inhibitor, promotes tumor cell death by inducing endoplasmic reticulum (ER) stress in human hepatocellular carcinoma (HCC) cell lines. Protein disulfide isomerase (PDI) is an essential folding catalyst and attenuates ER stress by folding the misfolded proteins. We examined if PDI is expressed in hypoxic HCC cells, and evaluated its inhibition potentiated HK II inhibitor-induced ER stress in hypoxic HCC cells. HCC apoptotic cell death was assessed by DAPI staining and apoptotic signaling pathways were explored by immunoblot analysis. An in vivo model of HCC was established in C3H mice intradermally with implanted MH134 cells. 3-BP with/without a PDI inhibitor (bacitracin) was subsequently administered. The anti-tumor efficacies were evaluated by measuring tumor volumes and quantifying apoptotic cells and microvessel densities (MVDs). HCC cells were found to express PDI in a hypoxia-inducible manner. The simultaneous treatment of bacitracin and 3-BP enhanced 3-BP-induced apoptosis. This enhancement was attributed to increased ER stress and JNK activation compared to the cells treated with just 3-BP. In an in vivo model of HCC, tumor growth was significantly suppressed in mice co-treated with bacitracin and 3-BP, and the percentages of apoptotic cells significantly increased and MVDs significantly decreased. These results demonstrated that PDI was induced in hypoxic HCC tissue and that PDI inhibition enhanced HK II inhibitor-induced anti-tumor efficacy synergistically via augmenting ER stress and anti-angiogenesis in vivo. Thus, blockage of PDI activity in combination with HK II inhibitor may be therapeutically useful in HCCs.

Simple scoring system predicting genotypic resistance during rescue therapy for Lamivudine-resistant chronic hepatitis B.

GOAL: In this study, we aimed to devise a simple scoring system predicting the risk of genotypic resistance (GR) to current rescue therapies for patients with lamivudine (LAM)-resistant chronic hepatitis B. BACKGROUND: LAM and adefovir (ADV) combination therapy should be recommended for an initial rescue therapy against LAM-resistant hepatitis B virus (HBV). However, there still are many LAM-resistant patients being treated with entecavir (ETV) or ADV monotherapy. STUDY: This retrospective cohort study included consecutive patients treated for LAM-resistant chronic hepatitis B with ETV or ADV monotherapy, or LAM/ADV combination therapy. The cumulative probabilities of GR and virological responses and breakthrough according to clinical variables were analyzed by survival analyses and derived an index for future GR. RESULTS: A total of 224 patients were included (median treatment duration=117.9 wk). Using risk factors indentified on multivariable analyses, a simple index for future GR (Antiviral Resistance Prediction Index, ARPI) was developed with 3 clinical variables: the rescue therapy regimens (+0, ADV; +1, ETV; +2, LAM/ADV), HBV DNA reduction at 12 weeks (+0, <3 log10 copies/mL; +1, >3 log10 copies/mL), and the initial HBV DNA level (+0, >10 copies/mL; +1, <10 copies/mL). No patient with ARPI ≥2 exhibited GR, whereas 47% of the patients with an ARPI <2 developed GR by week 144 (P=0.005). CONCLUSIONS: The results of this study suggest that the ARPI is a simple and early index that can be used to determine the risk for subsequent GR during rescue therapy for LAM-resistant chronic hepatitis B.

Treatment and clinical outcome of needle-track seeding from hepatocellular carcinoma.

BACKGROUND/AIMS: Needle-track seeding is a rare but important complication of diagnostic and therapeutic ultrasound (US)-guided procedures in hepatocellular carcinoma (HCC). We examined the frequency of needle-track seeding after US-guided percutaneous ethanol injection (PEI), fine-needle aspiration biopsy (FNAB), and percutaneous transhepatic biliary drainage (PTBD) in order to determine the appropriate treatment for needle-track seeding and its clinical outcome. METHODS: We analyzed the clinical characteristics and treatment outcomes in eight patients who experienced needle-track seeding from HCC after an US-guided procedure (FNAB, PEI, or PTBD) between January 1990 and July 2004. RESULTS: Seven (0.14%) of 5,092 patients who experienced needle-track seeding (2 after PEI, 4 after FNAB, and 1 after PTBD) during the study period and 1 other patient who experienced needle-track seeding recently were recruited for this study. Two of the eight patients underwent mass excision and the other six patients underwent en-bloc wide excision for the needle-track seeding. Tumors recurred in the needle-tracks in both patients who underwent mass excision but not in the six patients who underwent en-bloc wide excision. Mortality occurred in three patients who experienced the recurrence and progression of intrahepatic HCC. CONCLUSIONS: The incidence of needle-track seeding after US-guided procedures in HCC was 0.14%. En-bloc wide excision seems to be the optimal treatment for minimizing the probability of tumor recurrence due to needle-track seeding.

Gene expression-based recurrence prediction of hepatitis B virus-related human hepatocellular carcinoma.

PURPOSE: The poor prognosis of hepatocellular carcinoma (HCC) is, in part, due to the high rate of recurrence even after "curative resection" of tumors. Therefore, it is axiomatic that the development of an effective prognostic prediction model for HCC recurrence after surgery would, at minimum, help to identify in advance those who would most benefit from the treatment, and at best, provide new therapeutic strategies for patients with a high risk of early recurrence. EXPERIMENTAL DESIGN: For the prediction of the recurrence time in patients with HCC, gene expression profiles were generated in 65 HCC patients with hepatitis B infections. RESULT: Recurrence-associated gene expression signatures successfully discriminated between patients at high-risk and low-risk of early recurrence (P=1.9 x 10(-6), log-rank test). To test the consistency and robustness of the recurrence signature, we validated its prognostic power in an independent HCC microarray data set. CD24 was identified as a putative biomarker for the prediction of early recurrence. Genetic network analysis suggested that SP1 and peroxisome proliferator-activated receptor-alpha might have regulatory roles for the early recurrence of HCC. CONCLUSION: We have identified a gene expression signature that effectively predicted early recurrence of HCC independent of microarray platforms and cohorts, and provided novel biological insights into the mechanisms of tumor recurrence.