Search

The proto-oncogene MYCN expression marked a cancer stem-like cell population in hepatocellular carcinoma (HCC) and served as a therapeutic target of acyclic retinoid (ACR), an orally administered vitamin A derivative that has demonstrated promising efficacy and safety in reducing HCC recurrence. This study investigated the role of MYCN as a predictive biomarker for therapeutic response to ACR and prognosis of HCC. MYCN gene expression in HCC was analyzed in the Cancer Genome Atlas and a Taiwanese cohort (N = 118). Serum MYCN protein levels were assessed in healthy controls (N = 15), patients with HCC (N = 116), pre- and post-surgical patients with HCC (N = 20), and a subset of patients from a phase 3 clinical trial of ACR (N = 68, NCT01640808). The results showed increased MYCN gene expression in HCC tumors, which positively correlated with HCC recurrence in non-cirrhotic or single-tumor patients. Serum MYCN protein levels were higher in patients with HCC, decreased after surgical resection of HCC, and were associated with liver functional reserve and fibrosis markers, as well as long-term HCC prognosis (>4 years). Subgroup analysis of a phase 3 clinical trial of ACR identified serum MYCN as the risk factor most strongly associated with HCC recurrence. Patients with HCC with higher serum MYCN levels after a 4-week treatment of ACR exhibited a significantly higher risk of recurrence (hazard ratio 3.27; p = .022). In conclusion, serum MYCN holds promise for biomarker-based precision medicine for the prevention of HCC, long-term prognosis of early-stage HCC, and identification of high-response subgroups for ACR-based treatment., (© 2024 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background and Aim: Immune checkpoint inhibitors pose the risk of immune-related adverse events (irAEs). Recent data suggest that irAEs may be associated with a favorable prognosis. This study aimed to investigate and analyze the association between these adverse events and the clinical benefits in patients with unresectable hepatocellular carcinoma., Methods: The study enrolled 130 patients with advanced hepatocellular carcinoma treated with atezolizumab plus bevacizumab between November 2020 and January 2023 at a single center. The relationship between irAEs and both response rate and post-treatment outcomes was investigated., Results: Out of the 130 patients, irAEs developed in 36 (27.7%) patients. The irAE group exhibited a significantly longer progression-free survival (PFS) than the non-irAE group, with a median PFS of 8.9 compared with 4.6 months (P < 0.01). No difference was found in the overall survival between the irAE and non-irAE groups. The irAE group demonstrated significantly higher disease control rate (DCR) than the non-irAE group (97.0% vs 65.5%, P < 0.01). The analysis by irAE severity revealed that the grade 1/2 group exhibited significantly longer PFS (7.9 vs 4.6 months, P = 0.007) and higher DCR (100% vs 65.5%, P < 0.01) than the non-irAE group. Furthermore, hypothyroidism correlated with a favorable PFS (8.9 vs 5.4 months, P = 0.02), DCR (100% vs 71.3%, P = 0.03), and overall response rate (58.3% vs 18.5%, P = 0.005)., Conclusion: The presence of irAEs is associated with prolonged PFS and higher DCR. Specifically, mild irAEs (grade 1/2) and hypothyroidism displayed prolonged PFS and higher DCR., (© 2024 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

A 69-year-old man was treated with lenvatinib after three sessions of proton beam therapy (PBT) for hepatocellular carcinoma. Five months after administration of lenvatinib, a dermatitis with huge skin ulcer formed in the site of PBT irradiation. Lenvatinib was immediately withdrawn, but the skin ulcer continued growing until about 2 weeks later. With topical and antibiotic treatment, the skin ulcer resolved after about 4 months. After administration of lenvatinib, potential skin damage due to PBT at the irradiated site may have become apparent. This is the first report describing skin ulcer by the combination of lenvatinib administration and PBT., (© 2023. Japanese Society of Gastroenterology.)

Transglutaminase 2 (TG2) is a multifunctional protein that promotes or suppresses tumorigenesis, depending on intracellular location and conformational structure. Acyclic retinoid (ACR) is an orally administered vitamin A derivative that prevents hepatocellular carcinoma (HCC) recurrence by targeting liver cancer stem cells (CSCs). In this study, we examined the subcellular location-dependent effects of ACR on TG2 activity at a structural level and characterized the functional role of TG2 and its downstream molecular mechanism in the selective depletion of liver CSCs. A binding assay with high-performance magnetic nanobeads and structural dynamic analysis with native gel electrophoresis and size-exclusion chromatography-coupled multi-angle light scattering or small-angle X-ray scattering showed that ACR binds directly to TG2, induces oligomer formation of TG2, and inhibits the transamidase activity of cytoplasmic TG2 in HCC cells. The loss-of-function of TG2 suppressed the expression of stemness-related genes, spheroid proliferation and selectively induced cell death in an EpCAM+ liver CSC subpopulation in HCC cells. Proteome analysis revealed that TG2 inhibition suppressed the gene and protein expression of exostosin glycosyltransferase 1 (EXT1) and heparan sulfate biosynthesis in HCC cells. In contrast, high levels of ACR increased intracellular Ca 2+ concentrations along with an increase in apoptotic cells, which probably contributed to the enhanced transamidase activity of nuclear TG2. This study demonstrates that ACR could act as a novel TG2 inhibitor; TG2-mediated EXT1 signaling is a promising therapeutic target in the prevention of HCC by disrupting liver CSCs., (© 2023. The Author(s).)

Aim: Using classification tree analysis, we evaluated the most useful magnetic resonance (MR) image type in the differentiation between early and progressed hepatocellular carcinoma (eHCC and pHCC)., Methods: We included pathologically proven 214 HCCs (28 eHCCs and 186 pHCCs) in 144 patients. The signal intensity of HCCs was assessed on in-phase (T1in) and opposed-phase T1-weighted images (T1op), ultrafast T2-weighted images (ufT2WI), fat-saturated T2-weighted images (fsT2WI), diffusion-weighted images (DWI), contrast enhanced T1-weighted images in the arterial phase (AP), portal venous phase (PVP), and the hepatobiliary phase. Fat content and washout were also evaluated. Fisher's exact test was performed to evaluate usefulness for the differentiation. Then, we chose MR images using binary logistic regression analysis and performed classification and regression tree analysis with them. Diagnostic performances of the classification tree were evaluated using a stratified 10-fold cross-validation method., Results: T1in, ufT2WI, fsT2WI, DWI, AP, PVP, fat content, and washout were all useful for the differentiation (p < 0.05), and AP and T1in were finally chosen for creating classification trees (p < 0.05). AP appeared in the first node in the tree. The area under the curve, sensitivity and specificity for eHCC, and balanced accuracy of the classification tree were 0.83 (95% CI 0.74-0.91), 0.64 (18/28, 95% CI 0.46-0.82), 0.94 (174/186, 95% CI 0.90-0.97), and 0.79 (95% CI 0.70-0.87), respectively., Conclusions: AP is the most useful MR image type and T1in the second in the differentiation between eHCC and pHCC., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: The incidence of non-alcoholic steatohepatitis (NASH) is dramatically increasing, but the effect of NASH on colon cancer liver metastasis (CLM) is controversial. The aim of this study was to investigate the impact and mechanism of action of NASH on CLM using a western diet (WD)-fed mouse model., Methods: Six-week-old male C57BL/6 J mice were used. They were divided into the WD group and control group with normal diet. MC38 colon cancer cells were injected into the spleen at 2, 6, 8 and 16 weeks, and mice were killed at 2 weeks after injection to evaluate hepatic steatosis, fibrosis, metastasis and mRNA/protein expression in the liver., Results: Only mice fed a WD for 16 weeks showed hepatic fibrosis. These mice showed significantly higher alanine aminotransferase and total cholesterol levels compared with the control group (p < 0.05). The WD group showed significantly lower tumor number and smaller tumor diameter (p < 0.05). In the WD group, expression of SAA1, IL6, STAT3 and MMP9 mRNA in the liver was significantly lower than in the control group (p < 0.05). Serum amyloid A1 protein expression was also lower in the WD group., Conclusions: The WD-fed NASH mouse model showed an inhibitory effect on CLM. Suppressed interleukin-6/signal transducer and activator of transcription 3 signaling and serum amyloid A/matrix metalloproteinase 9 expression may affect this phenomenon., Competing Interests: Declaration of competing interest The authors have no conflict of interest related to this study., (Copyright © 2022 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Background: Non-alcoholic steatohepatitis (NASH) is associated with a higher risk of hepatocellular carcinoma (HCC), and the importance of the gut?liver axis has been recognized in NASH-associated HCC. We investigated the effect of TU-100 on the intestinal microbiome and hepatocarcinogenesis in a NASH model., Methods: Seven-week-old Tsumura Suzuki obese diabetes mice, a model that shows the spontaneous onset of NASH and HCC, were used. They were divided into a TU-100 treated group and a control group. Mice were sacrificed at 24 and 48 weeks to evaluate hepatic steatosis, fibrosis, carcinogenesis, cytokine expression, and microbiome abundance., Results: At 24 weeks, the TU-100 group showed significantly lower expression of IL6, IL1B, and ACTA2 mRNA in the liver (P?

Male Tsumura-Suzuki Obese Diabetes (TSOD) mice, a spontaneous metabolic syndrome model, develop non-alcoholic steatohepatitis and liver tumors by feeding on a standard mouse diet. Nearly 70% of liver tumors express glutamine synthetase (GS), a marker of hepatocellular carcinoma. In contrast, approximately 30% are GS-negative without prominent nuclear or structural atypia. In this study, we examined the characteristics of the GS-negative tumors of TSOD mice. Twenty male TSOD mice were sacrificed at 40 weeks and a total of 21 tumors were analyzed by HE staining and immunostaining of GS, liver fatty acid-binding protein (L-FABP), serum amyloid A (SAA), and beta-catenin. With immunostaining for GS, six (29%) tumors were negative. Based on the histological and immunohistological characteristics, six GS-negative tumors were classified into several subtypes of human hepatocellular adenoma (HCA). One large tumor showed generally similar findings to inflammatory HCA, but contained small atypical foci with GS staining and partial nuclear beta-catenin expression suggesting malignant transformation. GS-negative tumors of TSOD mice contained features similar to various subtypes of HCA. Different HCA subtypes occurring in the same liver have been reported in humans; however, the diversity of patient backgrounds limits the ability to conduct a detailed, multifaceted analysis. TSOD mice may share similar mechanisms of HCA development as in humans. It is timely to review the pathogenesis of HCA from both genetic and environmental perspectives, and it is expected that TSOD mice will make further contributions in this regard.

Previous studies have shown that phosphorylation of the retinoid X receptor-α (RXRα) is associated with the development of hepatocellular carcinoma (HCC). However, these findings were revealed using HCC cell lines that express phosphorylated-RXRα (p-RXRα) proteins; therefore, it remains unclear whether p-RXRα affects hepatocarcinogenesis in vivo. Therefore, to investigate the biological function of p-RXRα in vivo, we developed a doxycycline-inducible ES cell line and transgenic mouse, both of which overexpress the phosphomimetic mutant form of RXRα, T82D/S260D, in a doxycycline-dependent manner. We found that the development of liver tumors, especially high-grade adenoma and HCC, was enhanced in diethylnitrosamine (DEN)-treated T82D/S260D-inducible mice. Moreover, the increased incidence of liver tumors in the transgenic mice was attributable to the promotion of cell cycle progression. Interestingly, the expression of β-catenin protein and its target gene cyclin D1 was elevated in the liver tumors of DEN-treated T82D/S260D-inducible mice, concurrent with increased cytoplasmic and nuclear β-catenin protein expression, indicating its stabilization and transcriptional activation. These results indicate that p-RXRα promotes DEN-induced hepatocarcinogenesis in mice through the activation of the β-catenin signaling pathway, suggesting that p-RXRα may serve as a possible therapeutic target for HCC., (© The Author(s) 2021. Published by Oxford University Press.)

Background: Direct-acting antiviral (DAA) therapy enables a high rate of sustained virologic response (SVR) in patients with hepatitis C virus associated cirrhosis. However, the impact of DAA therapy on liver-related events in patients with cirrhosis is unclear., Methods: A total of 350 patients with compensated and decompensated cirrhosis administered DAA therapy at 29 Japanese hospitals were enrolled (Child-Pugh class A [CP-A]: 195 patients, CP-B: 131 patients and CP-C: 24 patients)., Results: The SVR rates of patients with CP-A, CP-B and CP-C were 96.9%, 93.1% and 83.3%, respectively (p = 0.006). Seventy patients developed hepatocellular carcinoma (HCC), and male sex, previous HCC treatment, platelet counts < 10.0 × 10 4 /µl, alpha-fetoprotein levels ≥ 5.0 ng/ml and CP-C were identified as significant factors in the multivariate analysis. The cumulative HCC occurrence/recurrence rates at 1 year were 6.6%/45.2%. The cumulative rate of decompensated cirrhotic events requiring hospital admission at 1 year was 9.1%. In the multivariate analysis, CP-B and CP-C were identified as significant factors. During the median observation period of 14.9 months, 13 patients died and one patient received liver transplant. The overall survival rates at 1 year were 98.4% in patients with CP-A, 96.4% in those with CP-B and 85.6% in those with CP-C (CP-A vs. CP-B: p = 0.759, CP-A vs. CP-C: p = 0.001 and CP-B vs. CP-C: p = 0.005)., Conclusions: HCC development and mortality in patients with CP-B were not different from those with CP-A. On the other hand, in patients with CP-C, the development of HCC and decompensated cirrhotic events requiring hospital admission, and death were frequent., Trial Registration: University Hospital Medical Information Network (UMIN000036150)., (© 2022. Japanese Society of Gastroenterology.)

Metabolic syndrome (MS) is a risk factor for type 2 diabetes mellitus, vascular inflammation, atherosclerosis, and renal, liver, and heart diseases. Non-alcoholic steatohepatitis (NASH) is a progressive representative liver disease and may lead to the irreversible calamities of cirrhosis and hepatocellular carcinoma. Metabolic disorders such as hyperglycemia have been broadly reported to be related to hepatocarcinogenesis in NASH; however, direct evidence of a link between hyperglycemia and carcinogenesis is still lacking. Tsumura Suzuki Obese Diabetic (TSOD) mice spontaneously develop metabolic syndrome, including obesity, insulin resistance, and NASH-like liver phenotype, and eventually develop hepatocellular carcinomas. TSOD mice provide a spontaneous human MS-like model, even with significant individual variations. In this study, we monitored mice in terms of their changes in blood glucose levels, body weights, and pancreatic and liver lesions over time. As a result, liver carcinogenesis was delayed in non-hyperglycemic TSOD mice compared to hyperglycemic mice. Moreover, at the termination point of 40 weeks, liver tumors appeared in 18 of 24 (75%) hyperglycemic TSOD mice; in contrast, they only appeared in 5 of 24 (20.8%) non-hyperglycemic mice. Next, we investigated three kinds of oligosaccharide that could lower blood glucose levels in hyperglycemic TSOD mice. We monitored the levels of blood and urinary glucose and assessed pancreatic lesions among the experimental groups. As expected, significantly lower levels of blood and urinary glucose and smaller deletions of Langerhans cells were found in TSOD mice fed with milk-derived oligosaccharides (galactooligosaccharides and lactosucrose). At the age of 24 weeks, mild steatohepatitis was found in the liver but there was no evidence of liver carcinogenesis. Steatosis in the liver was alleviated in the milk-derived oligosaccharide-administered group. Taken together, suppressing the increase in blood glucose level from a young age prevented susceptible individuals from diabetes and the onset of NAFLD/NASH, as well as carcinogenesis. Milk-derived oligosaccharides showed a lowering effect on blood glucose levels, which may be expected to prevent liver carcinogenesis.

Although diabetes mellitus (DM) is a well-known risk factor for hepatocellular carcinoma (HCC), the underlying mechanisms have not yet to be defined. We previously reported that DIAR mice fed with standard murine diet developed type 1 diabetes and HCC at age of 16 weeks old with a neonatal streptozotocin treatment (n-STZ). Because DIAR mice did not manifest obesity nor develop steatohepatitis, hyperglycemia with streptozotocin trigger or streptozotocin alone might turn on the hepato-carcinogenesis. An insulin-recruitment to DIAR-nSTZ mice showed an increased frequency of HCC during the first 12 weeks of age, although the diabetic indications notably improved. To elucidate the role of hyperglycemia in hepato-carcinogenesis, we performed a head-to-head comparative study by using 4CS mice and DIAR mice with n-STZ treatment. Newborn 4CS mice and DIAR mice were divided into STZ treated group and control group. The blood glucose levels of DIAR-nSTZ mice increased at age of eight weeks, while that of 4CS-nSTZ mice were maintained in the normal range. At eight weeks old, three out of five DIAR-nSTZ mice (60%) and one out of ten 4CS-nSTZ mice (10%) developed multiple liver tumors. At age of 12 weeks old, all eight of DIAR-nSTZ mice (100%) and two of 10 4CS-nSTZ mice (20%) developed multiple liver tumors. At 16 weeks old, all animals of DIAR-nSTZ and 4CS-nSTZ mice occurred liver tumors. DIAR-nSTZ showed hyperglycemia and HCC, and 4CS-nSTZ developed HCC without hyperglycemia. These results were interpreted that the onset of HCC maybe not related to the presence or absence of hyperglycemia but nSTZ treatment. On the other hand, since the carcinogenesis of 4CS-nSTZ is delayed compared to DIAR-nSTZ, hyperglycemia may play a role in the progression of carcinogenesis. Histologically, the liver tumor appeared irregularly trabecular arrangements of hepatocytes with various degrees of nuclear atypia. By immunohistochemical analyses, all liver tumors showed positive staining of glutamine synthetase (GS), an established human HCC marker. The expression pattern of GS was divided into a strong diffuse pattern and weak patchy pattern, respectively. The liver tumor showing the weak GS-patchy pattern expressed biliary/stem markers, EpCAM, and SALL4, partially. Because 4CS-nSTZ mice did not show any metabolic complications such as gaining body weight or high blood glucose level, it is a unique animal model with a simple condition to investigate hepatic carcinogenesis by excluding other factors., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Background/aim: Tumor-derived exosomes play important roles in tumor metastases. In this report, we observed the fate of tumor-derived exosomes in pancreatic cancer metastatic nude-mouse models using color-coded imaging., Materials and Methods: Mia-PaCa-2 human pancreatic cancer cells expressing red fluorescent protein (RFP) were transduced by exosome-specific pCT-CD63-green fluorescent protein (GFP) and injected in the spleen of nude mice., Results: Four weeks after injection of these cells into the spleen, liver metastases developed and tumor-derived exosomes were observed within the metastatic cancer cells and in Kupffer cells. Furthermore, tumor-derived exosomes diffused to bone marrow and lung cells, especially macrophages, without any metastases present., Conclusion: In the present study, we visualized the distribution of cancer-derived exosomes for the first time at the cellular level, in a pancreatic-cancer metastatic model., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: We previously reported that the incidence of hepatocellular carcinoma (HCC) with non-viral etiologies increased rapidly between 1991 and 2010 in Japan., Methods: To update this investigation, we enrolled patients who were initially diagnosed as having non-B, non-C HCC at participating hospitals between 2011 and 2015. In addition to the patient characteristics investigated in the previous report, we also investigated the duration of alcohol consumption. The overall survival rate was analyzed using the Kaplan-Meier method, and the hazard function against the body mass index (BMI) was plotted using cubic splines., Results: A total of 2087 patients were enrolled. The proportion of patients with non-viral etiologies has continued to increase from 10.0% in 1991 to 32.5% in 2015. Patients were also older (median ages, 70-73 years) and more obese (median BMIs, 23.9-24.2 kg/m 2 ), and the proportions of patients with diabetes mellitus (46.1% to 51.6%), hypertension (42.7% to 58.6%), dyslipidemia (14.6% to 22.9%), and fatty liver (24.0% to 28.8%) had all increased significantly. There was a significant inverse relationship between the duration and the amount of daily alcohol consumption. The improvement in the overall survival was relatively small, with a decreased proportion of patients under surveillance (41.3% to 31.6%). A hazard function plot showed a curve similar to that in our previous report, with a lowest hazard of ~ 26 kg/m 2 ., Conclusions: The proportion of HCC patients with non-viral etiologies continues to increase in Japan. Lifetime total amount of alcohol consumption may be a risk factor.

Introduction: Radiofrequency ablation (RFA) has been accepted as safe and effective for treating early-stage hepatocellular carcinoma (HCC). However, it often causes severe pain. Therefore, in this study, we performed RFA under deep sedation and investigated its efficacy and safety., Methods: We conducted a retrospective study including 511 HCC patients who received approximately 886 RFA treatments between December 2014 and November 2016 at our institution. Respiratory depression was defined as oxygen saturation of below 90%; and severe body movement was defined as movement caused by pain, which was managed by lowering the power of the generator. Factors associated with respiratory depression and severe body movement were examined via univariate and multivariate regression analyses., Results: Respiratory depression occurred in 15.3% of the patients and severe body movement in 26.5% of the patients. In the multivariate analysis, BMI (≥ 25 kg/m 2 , odds ratio [OR] = 1.75, P = 0.035) and longer ablation (≥ 10 min, OR = 2.59, P = 0.002) were significant respiratory depression-related factors. Male sex (OR = 2.02, P = 0.005), Child-Pugh class A (odds ratio = 1.96, P = 0.018), and longer ablation (≥ 10 min, OR = 3.03, P < 0.001) were significant factors related to severe body movement., Conclusion: Deep sedation for RFA can be performed safely and effectively. Higher BMI and longer ablation were risk factors for respiratory depression and male sex, Child-Pugh class A, and longer ablation were independent predictors of severe body movement during RFA under deep sedation.

Diabetes mellitus (DM) is a risk factor for hepatocellular carcinoma (HCC). The purpose of this study was to investigate the impact of the disorder of glucose metabolism on the recurrence of HCC after curative treatment. Two hundred and eleven patients with HCC who received curative treatment in our hospital from 2006 to 2017 were enrolled in this study. Recurrence-free survival was estimated using the Kaplan⁻Meier method, and the differences between the groups partitioned by the presence or absence of DM and the values of hemoglobin A1c (HbA1c), fasting plasma glucose (FPG), fasting immunoreactive insulin (FIRI), and homeostasis model assessment-insulin resistance (HOMA-IR) were evaluated using the log-rank test. There were no significant differences in the recurrence-free survival rate between the patients with and without DM ( p = 0.144), higher and lower levels of HbA1c (≥6.5 and <6.5%, respectively; p = 0.509), FPG (≥126 and <126 mg/dL, respectively; p = 0.143), and FIRI (≥10 and <10 μU/mL, respectively; p = 0.248). However, the higher HOMA-IR group (≥2.3) had HCC recurrence significantly earlier than the lower HOMA-IR group (<2.3, p = 0.013). Moreover, there was a significant difference between the higher and lower HOMA-IR groups without DM ( p = 0.009), and there was no significant difference between those groups with DM ( p = 0.759). A higher HOMA-IR level, particularly in non-diabetic patients, was a significant predictor for HCC recurrence after curative treatment.

Image-guided percutaneous ablation is considered best in the treatment of early-stage hepatocellular carcinoma (HCC). Ablation is potentially curative, minimally invasive, and easily repeatable for recurrence. Ethanol injection used to be the standard in ablation. However, radiofrequency ablation has recently been the most prevailing ablation method for HCC. Many investigators have reported that radiofrequency ablation is superior to ethanol injection, from the viewpoints of treatment response, local tumor curativity, and overall survival. New-generation microwave ablation can create a larger ablation volume in a shorter time period. Further comparison studies are, however, mandatory between radiofrequency ablation and microwave ablation, especially in terms of complications and long-term survival. Irreversible electroporation, which is a non-thermal ablation method that delivers short electric pulses to induce cell death due to apoptosis, requires further studies, especially in terms of long-term outcomes. It is considerably difficult to compare outcomes in ablation with those in surgical resection. However, radiofrequency ablation seems to be a satisfactory alternative to resection for HCC 3 cm or smaller in Child-Pugh class A or B cirrhosis. Furthermore, radiofrequency ablation may be a first-line treatment in HCC 2 cm or smaller in Child-Pugh class A or B cirrhosis. Various innovations would further improve outcomes in ablation. Training programs may be effective in providing an excellent opportunity to understand basic concepts and learn cardinal skills for successful ablation. Sophisticated ablation would be more than an adequate alternative of surgery for small- and possibly middle-sized HCC.

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/β-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN + CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM + cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM - cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Background/aim: The lethal characteristic of pancreatic cancer is metastasis which is recalcitrant to currently-used chemotherapy. Our aim was to understand metastasis at the cellular level. We previously reported that multi-nucleate cells or spindle cells were more prominent in pancreatic cancer metastasis than in the primary tumor. In the present report, we investigated four representative human pancreatic cell lines for differences in cell morphology between the primary tumor and various metastatic organ targets for each cell line., Materials and Methods: Human pancreatic cancer cell lines AsPC-1, Panc-1, KP2 and KP3 were used. Pancreatic cancer cells were injected into spleen of nude mice resulting in experimental metastasis to various organs which were observed at the cellular level when the organs were placed into culture., Results: AsPC-1 and KP2 pancreatic cells formed many experimental liver metastases, in contrast to Panc-1 and KP3. Lung metastasis was only observed for AsPC-1. In the cultures established from the primary and metastatic tumors, multi-nucleate cells were found to be more prominent in the metastasis of the pancreatic cell lines with frequent metastasis, AsPC-1 and KP2. Spindle-like cells were observed prominently in AsPC-1 lung metastasis., Conclusion: Human pancreatic cancer cell lines have differential metastatic characteristics with regard to target organs and cell-morphology changes. Multi-nucleate and spindle cells may play an important role in pancreatic cancer metastasis to the liver and lung, respectively., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Sarcopenia impairs survival in patients with hepatocellular carcinoma (HCC). This study aimed to clarify the factors that contribute to decreased skeletal muscle volume in patients with HCC. The third lumbar vertebra skeletal muscle index (L3 SMI) in 351 consecutive patients with HCC was calculated to identify sarcopenia. Sarcopenia was defined as an L3 SMI value ≤ 29.0 cm²/m² for women and ≤ 36.0 cm²/m² for men. The factors affecting L3 SMI were analyzed by multiple linear regression analysis and tree-based models. Of the 351 HCC patients, 33 were diagnosed as having sarcopenia and showed poor prognosis compared with non-sarcopenia patients ( p = 0.007). However, this significant difference disappeared after the adjustments for age, sex, Child-Pugh score, maximum tumor size, tumor number, and the degree of portal vein invasion by propensity score matching analysis. Multiple linear regression analysis showed that age ( p = 0.015) and sex ( p < 0.0001) were significantly correlated with a decrease in L3 SMI. Tree-based models revealed that sex (female) is the most significant factor that affects L3 SMI. In male patients, L3 SMI was decreased by aging, increased Child-Pugh score (≥56 years), and enlarged tumor size (<56 years). Maintaining liver functional reserve and early diagnosis and therapy for HCC are vital to prevent skeletal muscle depletion and improve the prognosis of patients with HCC., Competing Interests: The authors declare no conflict of interest.

Cancer immunity is mediated through the effective priming and activation of tumour-specific class I MHC molecule-restricted CD8 + cytotoxic T lymphocytes (CTLs). DEC-205 + dendritic cells (DCs) can cross-present the epitope(s) of captured tumour antigens associated with class I MHC molecules alongside co-stimulatory molecules to prime and activate tumour-specific CD8 + CTLs. Immunosuppressive tolerogenic DCs with reduced co-stimulatory molecules may be a cause of impaired CTL induction. Hepa1-6-1 cells were established from the mouse hepatoma cell line Hepa1-6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 (B6) mice and do not prime CD8 + CTLs. In this study, we show that the growth of ongoing tumours was suppressed by activated CD8 + CTLs with tumour-specific cytotoxicity through the administration of the glycolipid α-galactosylceramide (α-GalCer), which is a compound known to stimulate invariant natural killer T (iNKT) cells and selectively activate DEC-205 + DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation with α-GalCer every 48 hr appeared to convert tolerogenic DEC-205 + DCs into immunogenic DCs with a higher expression of co-stimulatory molecules and a stronger cross-presentation capacity, which primed CTL precursors and induced tumour-specific CD8 + CTLs within the tumour environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC-205 + DCs within tumours into immunogenic DCs through the sequential administration of an immuno-potent lipid/glycolipid, and then activated immunogenic DCs with sufficient expression of co-stimulatory molecules prime and activate tumour-specific CD8 + CTLs within the tumour to control tumour growth., (© 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.)

Hepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5' ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers., Importance: Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5' ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication., (Copyright © 2016 Altinel et al.)

A73 -year-old man underwent a sigmoidectomy for sigmoid colon cancer with liver metastasis. After the operation, he received CapeOX combined with bevacizumab therapy. After 6 courses, the liver metastasis was undetectable on computed tomography scans. After 15 courses, computed tomography revealed ascites, and chemotherapy was discontinued. Two months later, computed tomography revealed portal vein thrombosis. Owing to the chronic nature of the thrombosis, thrombolytic therapy was not initiated. However, preservation therapy using antiplatelet drugs for 1 month resolved the ascites and the thrombosis. The risk of serious thrombosis must be considered when using bevacizumab.

Background: The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP., Materials and Methods: In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope., Results: EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture., Conclusion: Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Obesity and related metabolic abnormalities play a key role in liver carcinogenesis. Non-alcoholic steatohepatitis (NASH), which is often complicated with obesity and diabetes mellitus, is associated with the development of hepatocellular carcinoma (HCC). Sodium alginate (SA), which is extracted from brown seaweeds, is marketed as a weight loss supplement because of its high viscosity and gelling properties. In the present study, we examined the effects of SA on the progression of NASH and related liver carcinogenesis in monosodium glutamate (MSG)-treated mice, which show obesity, diabetes mellitus, and NASH-like histopathological changes. Male MSG-mice were intraperitoneally injected with diethylnitrosamine at 2 weeks of age, and, thereafter, they received a basal diet containing high- or low-molecular-weight SA throughout the experiment (16 weeks). At sacrifice, control MSG-treated mice fed the basal-diet showed significant obesity, hyperinsulinemia, steatosis and hepatic tumor development. SA administration suppressed body weight gain; improved insulin sensitivity, hyperinsulinemia, and hyperleptinemia; attenuated inflammation in the liver and white adipose tissue; and inhibited hepatic lipogenesis and progression of NASH. SA also reduced oxidative stress and increased anti-oxidant enzyme levels in the liver. Development of hepatic tumors, including liver cell adenoma and HCC, and hepatic pre-neoplastic lesions was significantly inhibited by SA supplementation. In conclusion, oral SA supplementation improves liver steatosis, insulin resistance, chronic inflammation, and oxidative stress, preventing the development of liver tumorigenesis in obese and diabetic mice. SA may have ability to suppress steatosis-related liver carcinogenesis in obese and diabetic subjects.

Obesity and its related metabolic disorders are serious health problems worldwide, and lead to various health-related complications, including cancer. Among human cancers, hepatocellular carcinoma (HCC) is one of the most common malignancies affected by obesity. Therefore, obesity and its related disorders might be a key target for the prevention of HCC. Recently, new research indicates that the molecular abnormalities associated with obesity, including insulin resistance/hyperinsulinemia, chronic inflammation, adipokine imbalance, and oxidative stress, are possible molecular mechanisms underlying the pathogenesis of obesity-related hepatocarcinogenesis. Green tea catechins and branched-chain amino acids, both of which are classified as nutraceutical agents, have been reported to prevent obesity-related HCC development by improving metabolic abnormalities. The administration of acyclic retinoid, a pharmaceutical agent, reduced the incidence of HCC in obese and diabetic mice, and was also associated with improvements in insulin resistance and chronic inflammation. In this article, we review the detailed molecular mechanisms that link obesity to the development of HCC in obese individuals. We also summarize recent evidence from experimental and clinical studies using either nutraceutical or pharmaceutical agents, and suggest that nutraceutical and pharmaceutical approaches targeting metabolic abnormalities might be a promising strategy to prevent the development of obesity-related HCC.

We report here a color-coded imaging model in which metastatic niches in the lung and liver of breast cancer can be identified. The transgenic green fluorescent protein (GFP)-expressing nude mouse was used as the host. The GFP nude mouse expresses GFP in all organs. However, GFP expression is dim in the liver parenchymal cells. Mouse mammary tumor cells (MMT 060562) (MMT), expressing red fluorescent protein (RFP), were injected in the tail vein of GFP nude mice to produce experimental lung metastasis and in the spleen of GFP nude mice to establish a liver metastasis model. Niche formation in the lung and liver metastasis was observed using very high resolution imaging systems. In the lung, GFP host-mouse cells accumulated around as few as a single MMT-RFP cell. In addition, GFP host cells were observed to form circle-shaped niches in the lung even without RFP cancer cells, which was possibly a niche in which future metastasis could be formed. In the liver, as with the lung, GFP host cells could form circle-shaped niches. Liver and lung metastases were removed surgically and cultured in vitro. MMT-RFP cells and GFP host cells resembling cancer-associated fibroblasts (CAFs) were observed interacting, suggesting that CAFs could serve as a metastatic niche., (© 2015 Wiley Periodicals, Inc.)

Background/aim: The tumor microenvironment (TME) is critical for tumor growth and progression. We report here an imageable model of the TME of orthotopic liver cancer., Materials and Methods: The transgenic red fluorescent protein (RFP)-expressing nude mouse was used as the host. The RFP nude mouse expresses RFP in all organs. Non-colored Huh-7 human hepatoma cells were injected in the spleen of RFP nude mice to establish an orthotopic liver cancer model. TME formation resulting from the orthotopic liver tumor was observed using the Olympus OV100 small animal fluorescence imaging system., Results: Non-colored liver cancer cells formed tumor colonies in the liver 28 days after cell transplantation to the spleen. RFP-expressing host cells and blood vessels were recruited by the liver tumors as visualized by fluorescence imaging. A desmin- and sirus-red-positive area increased around and within the liver tumor over time., Conclusion: These results indicate cancer-associated fibroblasts (CAFs) were recruited by the liver tumors suggesting that CAFs, along with the angiogenic tumor blood vessels, were necessary for liver-tumor growth and could serve as visible therapeutic targets., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Aim: A syngeneic color-coded imageable lymphoma model has been developed to visualize recruitment of host stromal cells by malignant lymphoma during metastasis. The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene., Materials and Methods: EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were initially established. EL4-RFP cells were subsequently injected into the tail vein of C57/BL6-GFP transgenic mice., Results: EL4-RFP metastasis was observed in the lymph nodes of the upper mediastinum and in the liver 28 days after cell injection. Large EL4-RFP liver metastases in C57/BL6-GFP mice contained GFP-expressing stromal cells derived from the host. In addition, EL4-RFP lymphoma metastasis was formed in peri-gastric lymph nodes, which were also enriched in host GFP-expressing cells. Furthermore, EL4-RFP lymphoma cells were also observed in the peripheral blood and bone marrow of C57/BL6-GFP transgenic mice, where they were associated with GFP-expressing host cells., Conclusion: Lymph node, liver and bone marrow metastases were found approximately 4 weeks after transplantation and all RFP-expressing metastases were highly enriched in GFP-expressing host stromal cells. This model of malignant lymphoma can be used to study early tumor development, metastasis, and the role of the stroma, as well as for discovery and evaluation of novel therapeutics for this treatment-resistant disease., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Hepatitis B virus (HBV) infection is a global public health problem. HBV has been classified into eight genotypes (A to H) based on complete nucleotide sequencing. The prevalence of specific genotype varies geographically. The rationale for treatment in patients with chronic hepatitis B is to reduce the risk of progressive chronic liver disease, such as cirrhosis and hepatocellular carcinoma. Treatment strategies for chronic HBV infection include interferon and nucleotide analogues (lamivudine, adefovir dipivoxil, entecavir, and tenofovir disoproxil). HBV persists in the body even after serological recovery from acute hepatitis B. Thus, individuals who have been exposed to HBV are at risk of the reactivation of infection, which may result in an increase in serum aminotransferases or a flare when the immune response is suppressed. Patients requiring immunosuppressive therapy should undergo serologic testing for markers of HBV infection. This topic review summarizes these issues related to the management of hepatitis B.

The aim of this study was to determine whether skeletal muscle depletion predicts the prognosis of patients with hepatocellular carcinoma (HCC) that is being treated with sorafenib. We evaluated 40 consecutive HCC patients who received sorafenib treatment. The skeletal muscle cross-sectional area was measured by computed tomography at the third lumbar vertebra (L3), from which the L3 skeletal muscle index (L3 SMI) was obtained. The factors contributing to overall survival, sorafenib dose reduction, and discontinuation of sorafenib were analyzed using the Cox proportional hazards model. L3 SMI (p = 0.020) and log (α-fetoprotein (AFP)) (p = 0.010) were identified as independent prognostic factors in HCC patients treated with sorafenib. The initial dose of sorafenib (p = 0.008) was an independent risk factor for sorafenib dose reduction, and log (AFP) (p = 0.008) was the only significant risk factor for the discontinuation of this drug. L3 SMI was not a risk factor for either dose reduction (p = 0.423) or the discontinuation (p = 0.132) of sorafenib. A multiple linear regression analysis determined the following relationship between skeletal muscle mass (assessed as L3 SMI) and the explanatory factors: L3 SMI = -0.1896 × (Age) - 10.3441 × (Child-Pugh score) - 9.3922 × (log (AFP)) + 1.6139 × (log (AFP)) × (Child-Pugh score) + 112.9166. Skeletal muscle depletion is inversely associated with age, Child-Pugh score, and log (AFP). Moreover, it is an independent prognostic factor for HCC patients treated with sorafenib.

Obesity and related metabolic disorders, such as diabetes mellitus, raise the risk of liver carcinogenesis. Metformin, which is widely used in the treatment of diabetes, ameliorates insulin sensitivity. Metformin is also thought to have antineoplastic activities and to reduce cancer risk. The present study examined the preventive effect of metformin on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-+Leprdb/+Leprdb (db/db) obese and diabetic mice. The mice were given a single injection of DEN at 2 weeks of age and subsequently received drinking water containing metformin for 20 weeks. Metformin administration significantly reduced the multiplicity of hepatic premalignant lesions and inhibited liver cell neoplasms. Metformin also markedly decreased serum levels of insulin and reduced insulin resistance, and inhibited phosphorylation of Akt, mammalian target of rapamycin (mTOR), and p70S6 in the liver. Furthermore, serum levels of leptin were decreased, while those of adiponectin were increased by metformin. These findings suggest that metformin prevents liver tumorigenesis by ameliorating insulin sensitivity, inhibiting the activation of Akt/mTOR/p70S6 signaling, and improving adipokine imbalance. Therefore, metformin may be a potent candidate for chemoprevention of liver tumorigenesis in patients with obesity or diabetes.

Hepatocellular carcinoma (HCC), which is a common malignancy worldwide, usually develops in a cirrhotic liver due to hepatitis virus infection. Metabolic syndrome, which is frequently complicated by obesity and diabetes mellitus, is also a critical risk factor for liver carcinogenesis. Green tea catechins (GTCs) may possess potent anticancer and chemopreventive properties for a number of different malignancies, including liver cancer. Antioxidant and anti-inflammatory activities are key mechanisms through which GTCs prevent the development of neoplasms, and they also exert cancer chemopreventive effects by modulating several signaling transduction and metabolic pathways. Furthermore, GTCs are considered to be useful for the prevention of obesity- and metabolic syndrome-related carcinogenesis by improving metabolic disorders. Several interventional trials in humans have shown that GTCs may ameliorate metabolic abnormalities and prevent the development of precancerous lesions. The purpose of this article is to review the key mechanisms by which GTCs exert chemopreventive effects in liver carcinogenesis, focusing especially on their ability to inhibit receptor tyrosine kinases and improve metabolic abnormalities. We also review the evidence for GTCs acting to prevent metabolic syndrome-associated liver carcinogenesis.

Background: Skeletal muscle depletion or sarcopenia has been identified as a poor prognostic factor for various diseases. The aim of this study is to determine whether muscle depletion is a prognostic factor for hepatocellular carcinoma (HCC)., Methods: We evaluated 217 consecutive patients with primary HCC. The skeletal muscle cross-sectional area was measured by computed tomography at the third lumbar vertebra (L3), from which the total body fat-free mass (FFM) and L3 skeletal muscle index (L3 SMI) were obtained. The factors contributing to overall survival (OS) were analyzed by univariate and multivariate Cox proportional hazards model., Results: In univariate analysis, FFM (P = 0.0422), Child-Pugh classification (P = 0.0058), serum albumin level (P < 0.0001), serum AFP level (P < 0.0001), serum proteins induced by vitamin K absence or antagonist-II level (P < 0.0001), cancer stage (P < 0.0001), and curability of the initial treatment (P < 0.0001) were significantly associated with the prognosis of HCC. Multivariate analysis indicated that FFM (P = 0.0499), albumin level (P = 0.0398), and curability of the initial treatment (P = 0.0008) were independent prognostic factors. Sarcopenia was defined as an L3 SMI value of ≤29.0 cm(2)/m(2) for women and ≤36.0 cm(2)/m(2) for men, and 24 patients (11.1%) have sarcopenia. Sarcopenic patients showed a significantly lower OS than those without sarcopenia (P = 0.0043). Sarcopenic patients who were overweight (BMI >22) died earlier (P = 0.0129)., Conclusions: Skeletal muscle depletion is an independent prognostic factor. Intervention to prevent muscle wasting might be an effective strategy for improving the outcome of HCC.

Obesity-related metabolic abnormalities, including adipokine imbalance and chronic inflammation, are involved in liver carcinogenesis. Chemerin, a novel adipokine, plays a critical role in adipogenesis, energy metabolism, and inflammation. We evaluated the impact of serum chemerin levels on liver functional reserves in hepatocellular carcinoma (HCC) patients and on the recurrence and prognosis of HCC. This study included 44 patients with any stage of HCC who underwent curative treatment at Gifu Municipal Hospital (Gifu, Japan) between 2006 and 2007. Recurrence-free survival and overall survival were estimated using the Kaplan-Meier method. Serum albumin levels (Pearson's correlation coefficient; r = 0.3110, p = 0.0399), platelet counts (r = 0.4159, p = 0.0050), and prothrombin times (r = 0.3775, p = 0.0115) were significantly correlated with serum chemerin levels in patients with HCC, and they were inversely correlated with Child-Pugh scores (r = -0.3732, p = 0.0126), serum alanine aminotransferase levels (r = -0.3864, p = 0.0105), and total bilirubin levels (r = -0.4023, p = 0.0068). Among these variables, a multiple comparison test identified that platelet counts and total bilirubin levels were associated with serum chemerin levels (p < 0.0083). No significant correlation was found between serum chemerin levels and recurrence-free survival (p = 0.3691) or overall survival (p = 0.7916). In HCC patients, serum chemerin concentrations were correlated with liver functional reserves and platelet counts, but not with recurrence or prognosis.

Non-alcoholic steatohepatitis (NASH), which involves hepatic inflammation and fibrosis, is associated with liver carcinogenesis. The activation of the renin-angiotensin system (RAS), which plays a key role in blood pressure regulation, promotes hepatic fibrogenesis. In this study, we investigated the effects of (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea catechins, on the development of glutathione S-transferase placental form (GST-P)-positive (GST-P(+)) foci, a hepatic preneoplastic lesion, in SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP-ZF) obese and hypertensive rats. Male 7-week-old SHRSP-ZF rats and control non-obese and normotensive WKY rats were fed a high fat diet and received intraperitoneal injections of carbon tetrachloride twice a week for 8weeks. The rats were also provided tap water containing 0.1% EGCG during the experiment. SHRSP-ZF rats presented with obesity, insulin resistance, dyslipidemia, an imbalance of adipokines in the serum, and hepatic steatosis. The development of GST-P(+) foci and liver fibrosis was markedly accelerated in SHRSP-ZF rats compared to that in control rats. Additionally, in SHRSP-ZF rats, RAS was activated and inflammation and oxidative stress were induced. Administration of EGCG, however, inhibited the development of hepatic premalignant lesions by improving liver fibrosis, inhibiting RAS activation, and attenuating inflammation and oxidative stress in SHRSP-ZF rats. In conclusion, obese and hypertensive SHRSP-ZF rats treated with a high fat diet and carbon tetrachloride displayed the histopathological and pathophysiological characteristics of NASH and developed GST-P(+) foci hepatic premalignant lesions, suggesting the model might be useful for the evaluation of NASH-related liver tumorigenesis. EGCG might also be able to prevent NASH-related liver fibrosis and tumorigenesis., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The poor prognosis for patients with hepatocellular carcinoma (HCC) is associated with its high rate of recurrence in the cirrhotic liver. Therefore, more effective strategies need to be urgently developed for the chemoprevention of this malignancy. The malfunction of retinoid X receptor α, a retinoid receptor, due to phosphorylation by Ras/mitogen-activated protein kinase is closely associated with liver carcinogenesis and may be a promising target for HCC chemoprevention. Acyclic retinoid (ACR), a synthetic retinoid, can prevent HCC development by inhibiting retinoid X receptor α phosphorylation and improve the prognosis for this malignancy. Supplementation with branched-chain amino acids (BCAA), which are used to improve protein malnutrition in patients with liver cirrhosis, can also reduce the risk of HCC in obese cirrhotic patients. In experimental studies, both ACR and BCAA exert suppressive effects on HCC development and the growth of HCC cells. In particular, combined treatment with ACR and BCAA cooperatively inhibits the growth of HCC cells. Furthermore, ACR and BCAA inhibit liver tumorigenesis associated with obesity and diabetes, both of which are critical risk factors for HCC development. These findings suggest that pharmaceutical and nutraceutical approaches using ACR and BCAA may be promising strategies for preventing HCC and improving the prognosis of this malignancy., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: A malfunction of RXRα due to phosphorylation is associated with liver carcinogenesis, and acyclic retinoid (ACR), which targets RXRα, can prevent the development of hepatocellular carcinoma (HCC). Activation of PI3K/Akt signaling plays a critical role in the proliferation and survival of HCC cells. The present study examined the possible combined effects of ACR and LY294002, a PI3K inhibitor, on the growth of human HCC cells., Methods: This study examined the effects of the combination of ACR plus LY294002 on the growth of HLF human HCC cells., Results: ACR and LY294002 preferentially inhibited the growth of HLF cells in comparison with Hc normal hepatocytes. The combination of 1 μM ACR and 5 μM LY294002, in which the concentrations used are less than the IC₅₀ values of these agents, synergistically inhibited the growth of HLF, Hep3B, and Huh7 human HCC cells. These agents when administered in combination acted cooperatively to induce apoptosis in HLF cells. The phosphorylation of RXRα, Akt, and ERK proteins in HLF cells were markedly inhibited by treatment with ACR plus LY294002. Moreover, this combination also increased RXRE promoter activity and the cellular levels of RARβ and p21(CIP1), while decreasing the levels of cyclin D1., Conclusion: ACR and LY294002 cooperatively increase the expression of RARβ, while inhibiting the phosphorylation of RXRα, and that these effects are associated with the induction of apoptosis and the inhibition of cell growth in human HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

The human noncoding RNA gene RGM249 has been shown to regulate the degree of cancer cell differentiation. In this study, we investigated the effects of 3 microRNA-like molecules digested from RGM249 on the loss of malignant properties in cancer cells in immunodeficient KSN/Slc mice. We utilized small interfering RNAs (siRNAs) alone or in combination with a cationized drug delivery system (DDS) consisting of atelocollagen or gelatin hydrogel microspheres. The results demonstrated growth inhibition and apoptosis and the inhibition of both neovascularization and metastasis, indicating that the DDSs effectively infiltrated the majority of tumor cells in vivo. Systemic administration of the 3 siRNAs inhibited the metastatic ability of malignant cells. Cotransfection of these siRNAs exerted a regulatory effect upon the genes involved in differentiation, pluripotency, and proliferation in cancer cells. These results suggest that RGM249-derived oligonucleotides may be involved in the regulation of metastasis, proliferation, and differentiation in vivo, and that the tested siRNAs may therefore represent a new anticancer therapeutic approach.

Purpose: Oxidative stress plays an important role in liver carcinogenesis. To determine the impact of oxidative stress on the recurrence of stage I/II hepatocellular carcinoma (HCC) after curative treatment, we conducted a prospective case series analysis., Methods: This study included 45 consecutive patients with stage I/II HCC, who underwent curative treatment by surgical resection or radiofrequency ablation at Gifu Municipal Hospital from 2006 to 2007. In these 45 cases, recurrence-free survival was estimated using the Kaplan-Meier method. The factors contributing to HCC recurrence, including the serum levels of derivatives of reactive oxygen metabolites (d-ROM) as an index of oxidative stress, were subjected to univariate and multivariate analyses using the Cox proportional hazards model., Results: The serum levels of d-ROM (P = 0.0231), α-fetoprotein (AFP, P = 0.0274), and fasting plasma glucose (P = 0.0400) were significantly associated with HCC recurrence in the univariate analysis. Multivariate analysis showed that the serum levels of d-ROM (hazard ratio [HR] 1.0038, 95 % confidence interval [CI] 1.0002-1.0071, P = 0.0392) and AFP (HR 1.0002, 95 % CI 1.0000-1.0003, P = 0.0316) were independent predictors of HCC recurrence. Kaplan-Meier analysis showed that recurrence-free survival was low in patients with high serum d-ROM (≥570 Carr U, P = 0.0036) and serum AFP (≥40 ng/dL, P = 0.0185) levels., Conclusions: The serum levels of d-ROM and AFP can be used for screening patients with a high risk for HCC recurrence. Patients who show increased levels of these factors require careful surveillance.

Obesity and related metabolic abnormalities, including a state of chronic inflammation, increase the risk of hepatocellular carcinoma (HCC). Adipose tissue constitutively expresses the proinflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which are important tumor promoters in inflammation-related carcinogenesis. Dysregulation of TNF-α and IL-6 is associated with the development of steatosis and inflammation within the liver. These cytokines also lie at the core of the association between obesity and insulin resistance, which is a key factor in the development of obesity-related HCC. Here we present a detailed review of the relationship between metabolic abnormalities and the development of HCC, focusing on the role played by inflammation. Drawing from our basic and clinical research, the present report also reviews evidence that targeting metabolic abnormalities, such as attenuation of chronic inflammation and improvement of insulin resistance by either pharmaceutical or nutritional intervention, may be an effective strategy in preventing the development of HCC in obese individuals.

Obesity and its associated disorders, such as non-alcoholic steatohepatitis, increase the risk of hepatocellular carcinoma. Branched-chain amino acids (BCAA), which improve protein malnutrition in patients with liver cirrhosis, reduce the risk of hepatocellular carcinoma in these patients with obesity. In the present study, the effects of BCAA supplementation on the spontaneous development of hepatic premalignant lesions, foci of cellular alteration, in db/db obese mice were examined. Male db/db mice were given a basal diet containing 3.0% of either BCAA or casein, a nitrogen-content-matched control of BCAA, for 36 weeks. On killing the mice, supplementation with BCAA significantly inhibited the development of foci of cellular alteration when compared with casein supplementation by inhibiting cell proliferation, but inducing apoptosis. BCAA supplementation increased the expression levels of peroxisome proliferator-activated receptor-γ, p21(CIP1) and p27(KIP1) messenger RNA and decreased the levels of c-fos and cyclin D1 mRNA in the liver. BCAA supplementation also reduced both the amount of hepatic triglyceride accumulation and the expression of interleukin (IL)-6, IL-1β, IL-18 and tumor necrosis factor-α mRNA in the liver. Increased macrophage infiltration was inhibited and the expression of IL-6, TNF-α, and monocyte chemoattractant protein-1 mRNA in the white adipose tissue were each decreased by BCAA supplementation. BCAA supplementation also reduced adipocyte size while increasing the expression of peroxisome proliferator-activated receptor-α, peroxisome proliferator-activated receptor-γ and adiponectin mRNA in the white adipose tissue compared with casein supplementation. These findings indicate that BCAA supplementation inhibits the early phase of obesity-related liver tumorigenesis by attenuating chronic inflammation in both the liver and white adipose tissue. BCAA supplementation may be useful in the chemoprevention of liver tumorigenesis in obese individuals.

The poor prognosis for hepatocellular carcinoma (HCC) is associated with its high rate of recurrence in the cirrhotic liver. Therefore, development of effective strategies for preventing recurrence and secondary tumors will improve the clinical outcome of HCC patients. A malfunction of the retinoid X receptor-α (RXRα) due to phosphorylation by the Ras-MAPK signaling pathway is profoundly associated with liver carcinogenesis, and thus, may be a promising target for HCC chemoprevention. Acyclic retinoid (ACR), which inhibits Ras-MAPK activation and RXRα phosphorylation, successfully prevents HCC recurrence, thus improving patient survival. The fundamental concept of HCC chemoprevention by ACR is "clonal deletion," which is defined as the removal of latent malignant clones from the liver before they expand into clinically detectable HCC. "Combination chemoprevention" using ACR as a key drug holds great promise of a new effective strategy for the prevention of HCC because of its synergism. ACR is also expected to prevent the development of HCC in obese people, who are at an increased risk to HCC, because this agent significantly inhibits obesity-related liver tumorigenesis in the rodent model. Here, we review the detailed effects of ACR on preventing HCC development, especially based on the results of our basic and clinical research.

Abnormalities in the expression and function of retinoid X receptor (RXR), a master regulator of the nuclear receptor superfamily, are associated with the development of hepatocellular carcinoma (HCC). Dysfunction of farnesoid X receptor (FXR), one of the nuclear receptors that forms a heterodimer with RXR, also plays a role in liver carcinogenesis. In the present study, we examined the effects of acyclic retinoid (ACR), a synthetic retinoid targeting RXRα, plus GW4064, a ligand for FXR, on the growth of human HCC cells. We found that ACR and GW4064 preferentially inhibited the growth of HLE, HLF, and Huh7 human HCC cells in comparison with Hc normal hepatocytes. The combination of 1μM ACR plus 1μM GW4064 synergistically inhibited the growth of HLE cells by inducing apoptosis. The combined treatment with these agents acted cooperatively to induce cell cycle arrest in the G(0)/G(1) phase and inhibit the phosphorylation of RXRα, which is regarded as a critical factor for liver carcinogenesis, through inhibition of ERK and Stat3 phosphorylation. This combination also increased the expression levels of p21(CIP1) and SHP mRNA, while decreasing the levels of c-myc and cyclin D1 mRNA in HLE cells. In addition, a reporter assay indicated that the FXRE promoter activity was significantly increased by treatment with ACR plus GW4064. Our results suggest that ACR and GW4064 cooperatively inhibit RXRα phosphorylation, modulate the expression of FXR-regulated genes, thus resulting in the induction of apoptosis and the inhibition of growth in HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Obesity and related metabolic abnormalities, including insulin resistance and a state of chronic inflammation, increase the risk of hepatocellular carcinoma. Abnormal activation of the insulin-like growth factor (IGF)/ IGF-1 receptor (IGF-1R) axis is also involved in obesity-related liver tumorigenesis. In the present study, we examined the effects of (-)-epigallocatechin gallate (EGCG), a major biologically active component of green tea, on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-db/db (db/db) obese mice. Male db/db mice were given tap water containing 40 ppm DEN for 2 weeks and then they received drinking water containing 0.1% EGCG for 34 weeks. At sacrifice, drinking water with EGCG significantly inhibited the development of liver cell adenomas in comparison with the control EGCG-untreated group. EGCG inhibited the phosphorylation of the IGF-1R, ERK (extracellular signal-regulated kinase), Akt, GSK-3β (glycogen synthase kinase-3β), Stat3, and JNK (c-Jun NH(2)-terminal kinase) proteins in the livers of experimental mice. The serum levels of insulin, IGF-1, IGF-2, free fatty acid, and TNF-α were all decreased by drinking EGCG, which also decreased the expression of TNF-α, interleukin (IL)-6, IL-1β, and IL-18 mRNAs in the livers. In addition, EGCG improved liver steatosis and activated the AMP-activated kinase protein in the liver. These findings suggest that EGCG prevents obesity-related liver tumorigenesis by inhibiting the IGF/IGF-1R axis, improving hyperinsulinemia, and attenuating chronic inflammation. EGCG, therefore, may be useful in the chemoprevention of liver tumorigenesis in obese individuals. Cancer Prev Res; 4(3); 396-403. ©2011 AACR.

The prognosis for patients with hepatocellular carcinoma (HCC) is poor and effective prevention strategies are urgently required. Here, we review abnormalities in the expression and function of retinoids and their receptors, and how they play a critical role in the development of HCC. In particular, a malfunction of RXRalpha due to phosphorylation by Ras-MAPK signaling pathway is profoundly associated with liver carcinogenesis and thus may be a promising target for HCC chemoprevention. Acyclic retinoid (ACR), a synthetic retinoid, inhibits Ras-MAPK activation and RXRalpha phosphorylation, thereby suppressing growth in HCC-derived cells. In clinical trials, ACR has been shown to improve patient survival by preventing viral HCC development, a possible manifestation of the concept of "clonal deletion" therapy. "Combination chemoprevention" with ACR as the key drug has great potential to become an effective strategy for the prevention of liver carcinogenesis. In summary, both basic and clinical research strongly suggest that ACR plays a critical role in preventing the development of HCC and that "clonal deletion" therapy is one of the most practical approaches for this purpose.

A malfunction of retinoid X receptor-alpha (RXRalpha) due to phosphorylation is associated with the development of hepatocellular carcinoma (HCC) and acyclic retinoid (ACR), which targets RXRalpha, can prevent the development of second primary HCC. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, induces apoptosis and cell cycle arrest in cancer cells. VPA can also enhance the sensitivity of cancer cells to retinoids. The present study examined the possible combined effects of ACR plus VPA in HepG2 human HCC cell line. The combination of 5muM ACR and 1mM VPA, about the IC(25) value for both compounds, synergistically inhibited the growth of HepG2 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VPA also acted synergistically to induce apoptosis and G(0)-G(1) cell cycle arrest in HepG2 cells. This combination further exerted a synergistic inhibition of the phosphorylation of RXRalpha, ERK, Akt and GSK-3beta proteins and caused an accumulation of acetylated histones H3 and H4 proteins. VPA enhanced the ability of ACR to raise the cellular levels of RARbeta and p21(CIP1). The combination of these agents markedly increased both the RARE and RXRE promoter activities in HepG2 cells. These results suggest that ACR and VPA cooperatively increase the expression of RARbeta and p21(CIP1), while inhibiting the phosphorylation of RXRalpha, and these effects were associated with induction of apoptosis and the inhibition of cell growth in HepG2 cells. This combination might therefore be an effective regimen for the chemoprevention and chemotherapy of HCC.

The receptor tyrosine kinase vascular endothelial growth factor (VEGF) receptor (VEGFR) plays an important role in tumor angiogenesis of hepatocellular carcinoma (HCC). (-)-Epigallocatechin gallate (EGCG), the major biologically active component of green tea, inhibits growth in a variety of human cancer cells by inhibiting the activation of several types of receptor tyrosine kinases. In this study, we examined the effects of EGCG on the activity of the VEGF-VEGFR axis in human HCC cells. The levels of total and phosphorylated (i.e. activated) form of VEGFR-2 protein (p-VEGFR-2) were observed to increase in a series of human HCC cell lines in comparison to the Hc normal human hepatocytes. EGCG preferentially inhibited the growth of HuH7 HCC cells, which express constitutive activation of the VEGF-VEGFR axis, in comparison to Hc cells. Treatment of HuH7 cells with EGCG caused a time- and dose-dependent decrease in the expression of VEGFR-2 and p-VEGFR-2 proteins. The production of VEGF from HuH7 cells was reduced by treatment with EGCG. Drinking of EGCG significantly inhibited the growth of HuH7 xenografts in nude mice and this was associated with inhibition of the activation of VEGFR-2 and its related downstream signaling molecules, including ERK and Akt. EGCG drinking also decreased the expression of Bcl-x(L) protein and VEGF mRNA in the xenografts. These findings suggest that EGCG can exert, at least in part, its growth-inhibitive effect on HCC cells by inhibiting the VEGF-VEGFR axis. EGCG might therefore be useful in the treatment of HCC.

Hepatocellular carcinoma (HCC) is a major health care problem worldwide. The prognosis of patients with HCC is poor because even in the early stages when surgical treatment might be expected to be curative, the incidence of recurrence in patients with underlying cirrhosis is very high due to multicentric carcinogenesis. Therefore, strategies to prevent recurrence and second primary HCC are required to improve the prognosis. One of the most practical approaches to prevent the multicentric development of HCC is 'clonal deletion' therapy, which is defined as the removal of latent (i.e. invisible) (pre)malignant clones from the liver in a hypercarcinogenic state. Retinoids, a group of structural and functional analogs of vitamin A, exert their biological function primarily through two distinct nuclear receptors, retinoic acid receptors and retinoid X receptors (RXR), and abnormalities in the expression and function of these receptors are highly associated with the development of various cancers, including HCC. In particular, a malfunction of RXRalpha due to phosphorylation by the Ras-mitogen-activated protein kinase signaling pathway is profoundly associated with the development of HCC and thus may be a critical target for HCC chemoprevention. Acyclic retinoid, which has been clinically shown to reduce the incidence of a post-therapeutic recurrence of HCC, can inhibit Ras activity and phosphorylation of the extracellular signal-regulated kinase and RXRalpha proteins. In conclusion, the inhibition of RXRalpha phosphorylation and the restoration of its physiological function as a master regulator for nuclear receptors may be a potentially effective strategy for HCC chemoprevention and clonal deletion. Acyclic retinoid, which targets phosphorylated RXRalpha, may thus play a critical role in preventing the development of multicentric HCC.