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Purpose: This study aims to determine the relationship between CpG island DNA hypermethylation and global genomic DNA hypomethylation and their prognostic implications in hepatocellular carcinoma. The association of DNA methylation changes with clinicopathologic factors and the chronological ordering of DNA methylation changes along multistep hepatocarcinogenesis were also assessed.Experimental Design: Hepatocellular carcinoma (n = 20) and nonneoplastic liver samples (n = 72) were analyzed for their methylation status at 41 CpG island loci and 3 repetitive DNA elements (LINE-1, ALU, and SAT2) using MethyLight or combined bisulfite restriction analysis. After selection of 19 CpG island loci showing cancer-specific DNA methylation, another set of 99 hepatocellular carcinoma samples was analyzed for these loci.Results: The number of methylated genes in hepatocellular carcinoma was significantly higher in hepatocellular carcinoma patients with a cirrhotic liver than in hepatocellular carcinoma patients with a noncirrhotic liver (9.9 versus 7.0, P = 0.001). Hepatocellular carcinoma from female patients showed a higher number of methylated genes than hepatocellular carcinoma from male patients (11.2 versus 8.4, P = 0.006). The genes CRABP1 and SYK showed significant association between CpG island hypermethylation and patients' poor survival. SAT2 hypomethylation occurred earlier than LINE-1 or ALU hypomethylation along the multistep hepatocarcinogenesis. Depending on the type of CpG island locus, a direct, inverse, or no relationship between CpG island hypermethylation and repetitive DNA hypomethylation was observed in hepatocellular carcinomas.Conclusion: The varying relationships between the hypermethylation of individual CpG island loci and the hypomethylation of repetitive elements suggests that they are not mechanically linked. SYK and CRABP1 hypermethylation may serve as useful tumor markers for prognostication of hepatocellular carcinoma patients.

Supplementary Data from Prognostic Implications of and Relationship Between CpG Island Hypermethylation and Repetitive DNA Hypomethylation in Hepatocellular Carcinoma

Supplementary Figures 1-5 from Identification of a Cholangiocarcinoma-Like Gene Expression Trait in Hepatocellular Carcinoma

Supplementary Tables 1-5 from Identification of a Cholangiocarcinoma-Like Gene Expression Trait in Hepatocellular Carcinoma

Acer tegmentosum Maxim., commonly known as Manchurian stripe maple, is a deciduous tree belonging to the family of Aceraceae and has been traditionally used in folk medicine for its remedial effects in liver diseases and traumatic bleedings. With a growing body of experimental evidence for its pharmacological efficacies, such as neuroprotective, hepatoprotective, antioxidant, and anti-inflammatory activities, A. tegmentosum has gradually gained popularity as a health supplement and functional food. However, the large part of essential toxicity information still remained lacking despite the possibility of mutagenic potentials as previously suggested, posing safety concerns for human consumption. In this study, we evaluated 90-day repeated oral toxicity of A. tegmentosum Maxim. water extract (ATWE) in SD rats with acute toxicity assessment in beagle dogs, and reevaluated genotoxicity using a combination of in vitro and in vivo assays. During the oral study period, ATWE did not cause toxicity-related clinical signs and mortality in rodents without adverse effects observed in the analysis of hematology, serum biochemistry, and histopathology, establishing >5,000 mg/kg BW as the NOAEL. In addition, doses up to 5,000 mg/kg BW did not cause acute toxicity in beagle dogs. When assessed for genotoxicity using bacterial reverse mutation, chromosome aberration, and micronucleus formation, ATWE showed lack of mutagenicity and clastogenicity. These results demonstrated that AWTE was safe in the present preclinical study for systemic toxicity and genotoxicity at the tested doses, providing a guideline for safe use in humans.

Cinnamomum cassia has been widely used as a natural product to treat diseases in Asia due to its diverse pharmacological functions including anti-inflammatory, anti-oxidant, anti-microbial, anti-diabetic, and anti-tumor effects. Despite its ethnomedicinal benefits, little information regarding its toxicity is currently available. The aim of this study was to evaluate its potential long-term toxicity and genotoxicity in compliance with test guidelines of the Organization for Economic Cooperation and Development. A 13-week repeat-dose oral toxicity study revealed that body weights of rats were normal after receiving cinnamon extract at up to 2000 mg/kg. High-dose intake of cinnamon extract (2000 mg/kg) showed potential nephrotoxicity and hepatotoxicity to both males and females as evidenced by obvious increases of kidney/liver weight along with a small but statistically elevation of total cholesterol level. Overall findings from genetic toxicity testing battery including Ames test, in vitro mammalian cell micronucleus assay, and in vivo bone marrow micronucleus assay indicated that cinnamon extract was not mutagenic or clastogenic. In conclusion, cinnamon extract may possess potential nephrotoxicity and hepatotoxicity at dose higher than its recommended daily safe dose. Further study is needed to clarify the mechanism involved in its induction of liver and kidney injury.

Intrahepatic cholangiocarcinoma (iCCA) is a devastating malignancy with no effective treatment. Nicotinamide (NA, the amide form of vitamin B3) has been shown to be effective in the treatment of various diseases. However, the effects of NA in iCCA have not been studied. In this study, four human iCCA cell lines (HuCCT1, JCK, OZ and Cho-CK) were used. We found that NA significantly inhibited cell viability and induced apoptosis in vitro. It arrested cell cycle in G1 phase, decreased Cyclin D1 and Cdk4 protein expression levels and increased p16 level. NA increased the levels of cleaved caspases 3 and 9, but had no effect on caspase 8. In HuCCT1 and OZ cell lines, NA treatment significantly impaired the invasion abilities and supressed epithelial-mesenchymal transition (EMT)- like changes. In conclusion, our findings provide the experimental basis for using NA as a potential anticancer agent against human iCCA in the future.

Introduction Pancreatic resection is technically difficult and has higher postoperative mortality and morbidity rates than those of other abdominal operations. As the incidence of postoperative morbidity and mortality has gradually decreased, some centers have reported remarkable achievements by attempting minimally invasive surgery. This study was performed to investigate the chronological trends of pancreatectomies by analyzing a large-scale database. Methods The medical records of 5,175 patients who underwent pancreatic resection between 1961 and 2019 at a single institution were collected and reviewed. The chronological trend of surgical outcome was investigated by dividing the period into 5-year intervals. To investigate the chronological change in survival outcomes of periampullary cancer, the survival data of 3,108 patients were analyzed. Results Patient age and the proportion of pancreatic cancer have increased over time. Pancreatic cancer was most common (35.9%) in 2015-2019, followed by pancreatic cyst (24.8%), common bile duct cancer (13.4%), and ampulla of Vater cancer (10.1%). The incidence of postoperative complications tended to decrease over time (26.0% in 2000-2004 and 20.8% in 2015-2019). A comparison of survival outcomes of periampullary malignancies by period revealed that pancreatic cancer significantly improved (5-year survival rate: 14.4% before 2000 vs. 15.2% in 2000-2009 vs. 29.0% after 2009, p < 0.001). Conclusions Postoperative complication rate and duration of recovery have improved in pancreatic resection over time. The proportion of minimally invasive pancreatectomies is gradually increasing. To improve outcomes in the future, active multidisciplinary approach and postoperative management are needed.

Artemisia capillaris (AC) has been used as an alternative therapy in obesity, atopic dermatitis, and liver diseases through several biological activity including anti-steatotic, antioxidant, and anti-inflammatory activities. Despite its ethnomedicinal benefits, no sufficient background information is available about the long-term safety and genotoxicity of the AC extract. Therefore, the present study was carried out to investigate the 13-week subchronic toxicity and genotoxicity of the AC extract according to the test guidelines published by the Organization for Economic Cooperation and Development. In the 13-week toxicity study using doses of 25, 74, 222, 667, and 2000 mg/kg body weight, oral administration of the AC extract in male and female rats did not result in any significant adverse effects in food/water consumption, body weight, mortality, hematology, serum biochemistry, organ weight and histopathology. Accordingly, the no-observed-adverse-effect level in rats of both genders was established for the AC extract at 2000 mg/kg/day, the highest dose level tested. In addition, the AC extract was not genotoxic in a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. In conclusion, we demonstrated that the AC extract is considered as a safe traditional medicine for human consumption.

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).

To evaluate the prognostic significance of phosphorylated Akt (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), and total phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressions in patients undergoing adjuvant chemoradiotherapy (CRT) for proximal extrahepatic bile duct (EHBD) cancer.Sixty-three patients with proximal EHBD cancer who underwent curative resection followed by adjuvant CRT were enrolled into this study. Postoperative radiotherapy was delivered to tumor bed and regional lymph nodes up to a median of 40 Gy (range, 40 to 54 Gy). Fifty-nine patients also received fluoropyrimidine chemotherapy as a radiosensitizer. p-Akt, p-mTOR, and PTEN expression were assessed with immunohistochemical staining on the tissue microarray.p-Akt, p-mTOR, and PTEN were expressed in 23 (36.5%), 17 (27.0%), and 24 patients (38.1%), respectively. p-Akt expression was associated with distant metastasis and overall survival (OS), but not with locoregional recurrence. The 5-year distant metastasis-free and OS rates were 25.8% versus 58.2% (P=0.007), and 27.5% versus 50.2% (P=0.0167) in patients with negative and positive expression, respectively. On multivariate analysis, nodal involvement was the only significant prognosticator predicting inferior distant metastasis-free survival (P=0.0105), whereas p-Akt expression had a borderline significance (P=0.0541). As for OS, p-Akt expression was a marginally significant prognosticator (P=0.0635), whereas other risk factors lost the statistical significance.p-Akt expression tended to be associated with a favorable prognosis in patients undergoing curative resection followed by adjuvant CRT for proximal EHBD cancer.

Alterations in mitochondrial respiration contribute to the development and progression of cancer via abnormal biogenesis, including generation of reactive oxygen species. Ubiquinol–cytochrome c reductase hinge protein (UQCRH) consists of the cytochrome bc1 complex serving respiration in mitochondria. In the present study, we analyzed UQCRH abnormalities in hepatocellular carcinoma (HCC) and its association with clinical outcomes of patients. UQCRH expression in HCC was determined via semiquantitative and quantitative real‐time reverse transcriptase polymerase chain reaction of 96 surgically resected HCC tissues positive for hepatitis B virus surface antigen. UQCRH was frequently overexpressed in HCC tissues (46.8%, based on 2.1‐fold cutoff). UQCRH overexpression was observed in HCCs with larger tumor size, poorer differentiation, or vascular invasion. Kaplan–Meier analysis revealed significantly shorter overall (P = 0.005) and recurrence‐free survival (P = 0.027) in patients with tumors overexpressing UQCRH. The prognostic impact of UQCRH was significant in subgroups of patients divided according to the α‐fetoprotein (AFP) level. The patient subgroup with higher AFP levels (≥20 ng/mL) exhibited significant differences in 5‐year overall (18.5% vs. 67.9%) and recurrence‐free survival rates (11.1% vs. 46.4%) between groups with and without UQCRH overexpression. In contrast, no marked survival differences were observed between subgroups with lower AFP levels (

BACKGROUND Liver transplantation (LT) is the treatment of choice for hepatocellular carcinoma (HCC). The aim of this study was to investigate the recurrence rate of HCC after LT and prognostic factors for recurrence by comparing LT with non-transplanted resection. METHODS The participants were 338 patients who underwent LT between 1996 and 2012 at Seoul National University Hospital (LT group) and 520 HCC patients who underwent partial hepatectomy between 1995 and 2006 (control group, non-LT group). RESULTS In the LT group, 68 of 338 patients (19.8%) showed relapse, and the recurrence rate was lower than that in the non-LT group (64.9%, 357/520, p < .001). Stratification analysis by American Joint Committee on Cancer (AJCC) stage showed that the stage I-II LT group had a lower recurrence rate than the non-LT group. Univariate comparative analysis demonstrated that multiplicity of tumor, tumor size, gross type, Edmondson- Steiner (ES) nuclear grade, extent of tumor, angioinvasion, AJCC stage, Milan criteria, University of California at San Francisco criteria on explant pathology (all p < .001), positive expression of cytokeratin 19 (p = .002), and preoperative α-fetoprotein (AFP) (p < .001) were predictors of tumor recurrence. In multivariate analysis, LT, preoperative AFP, multiplicity of tumor, extent of tumor, size of tumor, and ES nuclear grade were independent prognostic factors. CONCLUSIONS LT might have a protective effect against the late recurrence of stage I-II HCC compared to non-LT, and the prognostic factors for recurrence were similar to previously well-known prognostic factors for HCC.

// Su-Hyung Lee 1 , Jun-Won Park 2 , Sang-Ho Woo 1 , Du-Min Go 1 , Hyo-Jung Kwon 3 , Ja-June Jang 4 , Dae-Yong Kim 1 1 Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea 2 Biomolecular Function Research Branch, National Cancer Center, Goyang, Gyeonggi 410-769, South Korea 3 Department of Veterinary Pathology, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, South Korea 4 Department of Pathology, College of Medicine, Seoul National University, Seoul 110-799, Korea Correspondence to: Dae-Yong Kim, email: daeyong@snu.ac.kr Keywords: osteopontin, EGFR, c-Jun, apoptosis, hepatocellular carcinoma Received: April 29, 2016 Accepted: November 01, 2016 Published: November 23, 2016 ABSTRACT Previous clinical reports have found elevated osteopontin (OPN) levels in tumor tissues to be indicative of greater malignancy in human hepatocellular carcinoma (HCC). However, the role of OPN on carcinogenesis and its underlying mechanism remain unclear. In the present study, we investigated the oncogenic role of OPN in diethylnitrosamine (DEN)-induced hepatic carcinogenesis in mice. The overall incidence of hepatic tumors at 36 weeks was significantly lower in OPN knockout (KO) mice than in wild-type (WT) mice. Apoptosis was significantly enhanced in OPN KO mice, and was accompanied by the downregulation of epidermal growth factor receptor (EGFR). In the in vitro study, OPN suppression also led to lower mRNA and protein levels of EGFR associated with the downregulation of c-Jun in Hep3B and Huh7 human HCC cells lines, which resulted in increased apoptotic cell death in both cell lines. Moreover, a positive correlation was clearly identified between the expression of OPN and EGFR in human HCC tissues. These data demonstrate that the OPN deficiency reduced the incidence of chemically induced HCC by suppressing EGFR-mediated anti-apoptotic signaling. An important implication of our findings is that OPN positively contributes to hepatic carcinogenesis.

Intraductal papillary mucinous neoplasm (IPMN) is premalignant pancreatic lesion. International guidelines offer limited predictors of individual risk. A nomogram to predict individual IPMN malignancy risk was released, with good diagnostic performance based on a large cohort of Asian patients with IPMN. The present study validated a nomogram to predict malignancy risk and invasiveness of IPMN using both Eastern and Western cohorts.Clinicopathological and radiological data from patients who underwent pancreatic resection for IPMN at four centres each in Eastern and Western countries were collected. After excluding patients with missing data for at least one malignancy predictor in the nomogram (main pancreatic duct diameter, cyst size, presence of mural nodule, serum carcinoembryonic antigen and carbohydrate antigen (CA) 19-9 levels, and age).In total, data from 393 patients who fit the criteria were analysed, of whom 265 were from Eastern and 128 from Western institutions. Although mean age, sex, log value of serum CA19-9 level, tumour location, main duct diameter, cyst size and presence of mural nodule differed between the Korean/Japanese, Eastern and Western cohorts, rates of malignancy and invasive cancer did not differ significantly. Areas under the receiver operating characteristic (ROC) curve values for the nomogram predicting malignancy were 0·745 for Eastern, 0·856 for Western and 0·776 for combined cohorts; respective values for the nomogram predicting invasiveness were 0·736, 0·891 and 0·788.External validation of the nomogram showed good performance in predicting cancer in both Eastern and Western patients with IPMN lesions.La neoplasia mucinosa papilar intraductal (intraductal papillary mucinous neoplasm, IPMN) es una lesión pancreática premaligna. Las guías internacionales incluyen un número limitado de factores predictivos de riesgo individual. Para predecir el riesgo individual de malignidad del IPMN se ha propuesto un nomograma con un buen rendimiento diagnóstico, basado en una gran cohorte de pacientes asiáticos con IPMN. Este estudio validó el nomograma para predecir el riesgo de cáncer y de invasión de la IPMN utilizando cohortes tanto orientales como occidentales. MÉTODOS: Se recogieron datos clínico-patológicos y radiológicos de pacientes en los que se realizó una resección de páncreas por IPMN en 4 centros en países orientales y en 4 centros de países occidentales. Se excluyeron los pacientes en los que en el nomograma faltaba ≥ 1 factor(es) predictivo(s) de malignidad (diámetro del conducto pancreático principal, tamaño del quiste, presencia de nódulo mural, niveles séricos de CEA y CA19-9, y edad).En total, se analizaron datos de 393 pacientes que cumplían con los criterios de inclusión, de los cuales 265 eran de centros orientales y 128 de centros occidentales. Aunque la edad media, el sexo, el valor logarítmico del nivel sérico de CA19-9, la localización del tumor, el diámetro del conducto principal, el tamaño del quiste y la presencia de un nódulo mural difirieron entre las cohortes de Corea/Japón y las cohortes oriental y occidental, las tasas de malignidad y de cáncer invasivo no fueron significativamente diferentes. Las áreas bajo la curva operativa del receptor (area under the receiver operating curve, AUC) que mostró el nomograma para predecir la malignidad fueron: cohorte oriental: 0,745; cohorte occidental: 0,856 y cohortes combinadas: 0,776; y para predecir la invasión tumoral fueron: cohorte oriental: 0,736; cohorte occidental: 0,891, y cohortes combinadas: 0,788. CONCLUSIÓN: La validación externa del nomograma mostró un buen rendimiento en la predicción de cáncer, tanto en pacientes orientales como occidentales con lesiones IPMN.

The purpose of this study is to evaluate high-resolution ultrasound and magnetic resonance imaging (MRI) in monitoring of cholangiocarcinoma in the hamsters with C. sinensis infection and N-nitrosodimethylamine (NDMA). Twenty-four male Syrian golden hamsters of were divided into four groups composed of five hamsters as control, five hamsters receiving 30 metacercariae of C. sinensis per each hamster, five hamsters receiving NDMA in drinking water, and nine hamsters receiving both metacercariae and NDMA. Ultrasound was performed every other week from baseline to the 12th week of infection. MRI and histopathologic examination was done from the 4th week to 12th week. Cholangiocarcinomas appeared as early as the 6th week of infection. There were 12 cholangiocarcinomas, nine and ten of which were demonstrated by ultrasound and MRI, respectively. Ultrasound and MRI findings of cholangiocarcinomas in the hamsters were similar to those of the mass-forming intrahepatic cholangiocarcinomas in humans. Ultrasound and MRI also showed other findings of disease progression such as periductal increased echogenicity or signal intensity, ductal dilatation, complicated cysts, and sludges in the gallbladder. High-resolution ultrasound and MRI can monitor and detect the occurrence of cholangiocarcinoma in the hamsters non-invasively. • High-resolution ultrasound and MRI can monitor occurrence of cholangiocarcinoma in the hamsters. • Cholangiocarcinomas were detected as early as the 6th week after C. sinensis infection. • Axial T2-weighted MRI demonstrated cholangiocarcinomas and various inflammatory findings in the hamsters.

Clonorchis sinensis is a Group-I bio-carcinogen, associated with cholangiocarcinoma (CCA). The hamster is the only experimental model of C. sinensis-mediated CCA, but we oblige another animal model. The present study intended to develop a C. sinensis (Cs) mediated CCA model using C3H/He mice, co-stimulated with N-nitrosodimethyl-amine (NDMA) and dicyclanil (DC). The mice were divided into 8 groups with different combinations of Cs, NDMA, and DC. Six months later the mice were sacrificed and subjected to gross and histopathological examination. The body weights were significantly reduced among the groups treated with 2 or more agents (eg. Cs+NDMA, Cs+DC, NDMA+DC, and Cs+NDMA+DC). In contrast, liver weight percentages to body weight were increased in above groups by 4.1% to 4.7%. A Change of the spleen weight was observed only in Cs+NDMA group. Though C. sinensis infection is evident from hyperplastic changes, only 1 worm was recovered. T wo mice, 1 from Cs and the other from Cs+DC group, showed mass forming lesions; 1 (281.2 mm(3)) from the Cs group was a hepatocellular adenoma and the other (280.6 mm(3)) from the Cs+DC group was a cystic mass (peliosis). Higher prevalence of gray-white nodules was observed in Cs group (42.9%) followed by Cs+NDMA+DC group (21.4%). The mice of the Cs+NDMA+DC group showed hyper-proliferation of the bile duct with fibrotic changes. No characteristic change for CCA was recognized in any of the groups. In conclusion, C3H/He mice produce no CCA but extensive fibrosis when they are challenged by Cs, NDMA, and DC together.

Background: Hepatitis B virus (HBV) plays well-known roles in tumorigenesis of hepatocellular carcinoma (HCC) in infected patients. However, HBV-associated protein status in tumor tissues and the relevance to tumor behavior has not been reported. Our study aimed to examine the expression of HBV-associated proteins in HCC and adjacent nontumorous tissue and their clinicopathologic implication in HCC patients. Methods: HBV surface antigen (HBsAg), HBV core antigen (HBcAg), and HBV X protein (HBx) were assessed in 328 HBV-associated HCCs and in 155 matched nontumorous tissues by immunohistochemistry staining. Results: The positive rates of HBsAg and cytoplasmic HBx staining in tumor tissue were lower than those in nontumorous tissue (7.3% vs. 57.4%, p < .001; 43.4% vs. 81.3%, p < .001). Conversely, nuclear HBx was detected more frequently in tumors than in nontumorous tissue (52.1% vs. 30.3%, p < .001). HCCs expressing HBsAg, HBcAg, or cytoplasmic HBx had smaller size; lower Edmondson-Steiner (ES) nuclear grade, pT stage, and serum alpha-fetoprotein, and less angioinvasion than HCCs not expressing HBV-associated proteins. Exceptionally, nuclear HBx-positive HCCs showed higher ES nuclear grade and more frequent large-vessel invasion than did nuclear HBx-negative HCCs. In survival analysis, only nuclear HBx-positive HCCs had shorter disease-free survival than nuclear HBx-negative HCCs in pT1 and ES nuclear grade 1–2 HCC subgroup (median, 126 months vs. 35 months; p = .015). Conclusions: Our data confirmed that expression of normal HBV-associated proteins generally decreases in tumor cells in comparison to nontumorous hepatocytes, with the exception of nuclear HBx, which suggests that nuclear HBx plays a role in recurrence of well-differentiated and early-stage HCCs.

Antitumor effects of metformin have recently emerged despite its original use for type II diabetes. In the present study, the effects of metformin on the development and recurrence of hepatocellular carcinoma (HCC) were investigated using the diethylnitrosamine (DEN)‑induced rat model of HCC. Tumor foci were characterized by gross examination and by histopathological characteristics, including proliferation, hepatic progenitor cell content and the expression of hepatocarcinoma‑specific molecular markers. Potential target molecules of metformin were investigated to determine the molecular mechanism underlying the inhibitory effects of metformin on chemically induced liver tumorigenesis. The antitumor effects of metformin were increased by the reduction of surface nodules and decreased the incidence of altered hepatocellular foci, hepatocellular adenoma and carcinoma. Also, decreased expression levels of glutathione S‑transferase placental form, proliferating cell nuclear antigen and cytokeratin 8 described the inhibitory effects of metformin on HCC. In the present study, Wistar rats receiving treatment with DEN were administered metformin for 16 weeks. In addition, metformin suppressed liver tumorigenesis via an AMPK‑dependent pathway. These results suggested that metformin has promising effects on the early stage of HCC in rats. Therefore, metformin may be used for the prevention of HCC recurrence following primary chemotherapy for HCC and/or for high‑risk patients, including chronic hepatitis and cirrhosis.

Background Seed of mature Croton tiglium Linne, also known as Tiglium seed (TS), has been widely used as a natural product due to its several health beneficial properties including anti-tumor and antifungal activities. Despite its ethnomedicinal beneficial properties, toxicological information regarding TS extract, especially its long-term toxicity, is currently limited. Therefore, the objective of the present study was to evaluate acute and subchronic toxicity of TS extract in rats after oral administration following test guidelines of the Organization for Economic Cooperation and Development (OECD). Methods Toxicological properties of TS extract were evaluated by toxicity assays to determine its single-dose acute toxicity (125, 250, 500, 1000, or 2000 mg/kg), 14-day repeated-dose toxicity (125, 250, 500, 1000, or 2000 mg/kg) and 13-week repeated-dose toxicity (31.25, 62.5, 125, 250, and 500 mg/kg) in Sprague-Dawley rats and F344 rats. Hematological, serum biochemical, and histopathological parameters were analyzed to determine its median lethal dose (LD50) and no-observed-adverse-effect-level (NOAEL). Results Oral single dose up to 2000 mg/kg of TS extract resulted in no mortalities or abnormal clinical signs. In 13-week toxicity study, TS extract exhibited no dose-related changes (mortality, body weight, food/water consumption, hematology, clinical biochemistry, organ weight, or histopathology) at dose up to 500 mg/kg, the highest dosage level suggested based on 14-day repeat-dose oral toxicity study. Conclusion Acute oral LD50 of TS extract in rats was estimated to be greater than 2000 mg/kg. NOAEL of TS extract administered orally was determined to be 500 mg/kg/day in both male and female rats. Results from these acute and subchronic toxicity assessments of TS extract under Good Laboratory Practice regulations indicate that TS extract appears to be safe for human consumption. Electronic supplementary material The online version of this article (10.1186/s12906-018-2315-5) contains supplementary material, which is available to authorized users.

Transmembrane protein 165 (TMEM165), a Golgi protein, functions in ion homeostasis and vesicular trafficking in the Golgi apparatus. While mutations in TMEM165 are known to cause human ‘congenital disorders of glycosylation’, a recessive autosomal metabolic disease, the potential association of this protein with human cancer development has not been explored to date. In the present study, we revealed that TMEM165 is overexpressed in HCC and its depletion weakens the invasive activity of cancer cells through suppression of matrix metalloproteinase-2 (MMP-2) expression. Levels of TMEM165 mRNA and protein were clearly increased in HCC patient tissues and cell cultures. Quantitative real-time RT-PCR analysis of fresh HCC tissues (n=88) revealed association of TMEM165 overexpression with more frequent macroscopic vascular invasion, microscopic serosal invasion and higher α-fetoprotein levels. Notably, depletion of TMEM165 led to a marked decrease in the invasive activity of two different HCC cell types, Huh7 and SNU475, accompanied by downregulation of MMP-2. Our collective findings clearly indicated that TMEM165 contributed to the progression of HCC by promoting invasive activity, supporting its utility as a novel biomarker and therapeutic target for cancer.

Organ weights of male and female F344 rats orally administered with Tiglium seed extract for 14Â days. (DOCX 30 kb)

Summary incidence of gross findings of F344 rats orally administered with Tiglium seed extract for 13Â weeks. (DOCX 18 kb)

Summary incidence of gross findings of F344 rats orally administered with Tiglium seed extract for 14Â days. (DOCX 17 kb)

Enhanced expression of the cancer stem cell (CSC) marker, CD133, is closely associated with a higher rate of tumor formation and poor prognosis in hepatocellular carcinoma (HCC) patients. Despite its clinical significance, the molecular mechanism underlying the deregulation of CD133 during tumor progression remains to be clarified. Here, we report on a novel mechanism by which interleukin‐6/signal transducer and activator of transcription 3 (IL‐6/STAT3) signaling up‐regulates expression of CD133 and promotes HCC progression. STAT3 activated by IL‐6 rapidly bound to CD133 promoter and increased protein levels of CD133 in HCC cells. Reversely, in hypoxic conditions, RNA interference silencing of STAT3 resulted in decrease of CD133 levels, even in the presence of IL‐6, with a concomitant decrease of hypoxia‐inducible factor 1 alpha (HIF‐1α) expression. Active STAT3 interacted with nuclear factor kappa B (NF‐κB) p65 subunit to positively regulate the transcription of HIF‐1α providing a mechanistic explanation on how those three oncogenes work together to increase the activity of CD133 in a hypoxic liver microenvironment. Activation of STAT3 and its consequent induction of HIF‐1α and CD133 expression were not observed in Toll‐like receptor 4/IL‐6 double‐knockout mice. Long‐term silencing of CD133 by a lentiviral‐based approach inhibited cancer cell‐cycle progression and suppressed in vivo tumorigenicity by down‐regulating expression of cytokinesis‐related genes, such as TACC1, ACF7, and CKAP5. We also found that sorafenib and STAT3 inhibitor nifuroxazide inhibit HCC xenograft formation by blocking activation of STAT3 and expression of CD133 and HIF‐1α proteins. Conclusion: IL‐6/STAT3 signaling induces expression of CD133 through functional cooperation with NF‐κB and HIF‐1α during liver carcinogenesis. Targeting STAT3‐mediated CD133 up‐regulation may represent a novel, effective treatment by eradicating the liver tumor microenvironment. (Hepatology 2015;62:1160‐1173)

The aim of this study was to evaluate silent mating type information regulation 2 homolog 1 (SIRT1) expression levels by subtype and evaluate its predictive power of axillary lymph node metastasis (LNM) and its association with clinical outcome. A total of 427 patients diagnosed with invasive ductal carcinoma were chosen, immunohistochemical staining for SIRT1 expression was performed on tissue microarrays, and in vitro experiments with each intrinsic subtype of human breast cancer cell line were carried out. Increased expression of SIRT1 in hormone receptor-positive breast cancer and HER2 breast cancer subtype significantly correlated with lower risks of LNM. On the contrary, in triple-negative breast cancer, increased SIRT1 expression was more frequently observed in LNM-positive subgroup than LNM-negative subgroup. Combination of statistically significant, independent parameters including SIRT1 revealed predictive performance for LNM with area under the curve of 0.602, 0.587, and 0.726 for hormone receptor-positive breast cancer, HER2 breast cancer, and triple-negative breast cancer subtype, respectively. Inhibition of SIRT1 expression with small interfering RNA suppressed tumor invasion in MDA-MB-231, specifically. This is the first study to examine SIRT1 expression in breast cancer by subtype, and we have observed the potentially different role of SIRT1 gene having tumor-suppressive or tumor-promoting influence depending on the subtype; thus, different associations between SIRT1 expression and prognosis by subtype should be considered in its target therapy.

Although silicon dioxide (SiO2), silver (Ag) and iron oxide (Fe2O3) nanoparticles are widely used in diverse applications from food to biomedicine, in vivo toxicities of these nanoparticles exposed via the oral route remain highly controversial. To examine the systemic toxicity of these nanoparticles, well-dispersed nanoparticles were orally administered to Sprague-Dawley rats daily over a 13-week period. Based on the results of an acute toxicity and a 14-day repeated toxicity study, 975.9, 1030.5 and 1000 mg kg(-1) were selected as the highest dose of the SiO2 , Ag and Fe2O3 nanoparticles, respectively, for the 13-week repeated oral toxicity study. The SiO2 and Fe2O3 nanoparticles did not induce dose-related changes in a number of parameters associated with the systemic toxicity up to 975.9 and 1000 mg kg(-1) , respectively, whereas the Ag nanoparticles resulted in increases in serum alkaline phosphatase and calcium as well as lymphocyte infiltration in liver and kidney, raising the possibility of liver and kidney toxicity induced by the Ag nanoparticles. Compared with the SiO2 and Fe2O3 nanoparticles showing no systemic distribution in all tissues tested, the Ag concentration in sampled blood and organs in the Ag nanoparticle-treated group significantly increased with a positive and/or dose-related trend, meaning that the systemic toxicity of the Ag nanoparticles, including liver and kidney toxicity, might be explained by extensive systemic distribution of Ag originating from the Ag nanoparticles. Our current results suggest that further study is required to identify that Ag detected outside the gastrointestinal tract were indeed a nanoparticle form or ionized form.

Biliary atresia (BA) causes biliary obstruction in neonates. Although the Kasai operation can successfully treat certain BA cases, many patients exhibit recurrent jaundice and secondary biliary cirrhosis requiring liver transplantation. Consequently, studies of the prognostic factors of the Kasai operation are needed. Accordingly, sonic hedgehog (SHH) pathway expression at the extrahepatic bile duct (EHBD), an important bile duct repair mechanism, will be investigated via immunohistochemistry in patients with BA to examine the association with post-Kasai operation prognosis.Fifty-seven EHBD specimens were obtained during Kasai operations from 1992 to 2009. The SHH, patched (PTCH), and glioblastoma-2 (Gli-2) immunohistochemical staining results were analyzed quantitatively.Overall, 57.9% of patients had bile flow normalization after the Kasai operation; 43.1% did not. High preoperative serum total bilirubin, direct bilirubin, and aspartate aminotransferase levels were associated with sustained jaundice post-Kasai operation, as was an age ≥65days at the time of surgery (all p0.05). High Gli-2 and SHH expression rates were significantly associated with early post-Kasai operation jaundice relapse.Strong Gli-2 and SHH expression in the EHBD might be a poor prognostic factor in Kasai operation-treated patients with BA.

Gene therapy using RNA interference can be directed against tumors through various strategies, but has been hindered owing to the inefficiency of non-viral delivery. To evaluate the antitumor effects of adenine nucleotide translocase-2 (ANT2) short hairpin RNA (shRNA) by intraperitoneal injection using the polyethylenimine (PEI) and an ultrasound gene delivery method, human breast carcinoma MDA-MB-231 cells were injected subcutaneously into NOG (NOD/Shi-scid/IL-2Rγ(null)) mice. The results showed greater tumor regression (*P

Spleen tyrosine kinase (SYK) has predominantly been studied in hematopoietic cells, where it is involved in immunoreceptor-mediated signaling. However, SYK expression has been shown in numerous non-hematopoietic cells, and its downregulation has been shown to be involved in tumor formation and progression. SYK methylation has been demonstrated to identify a subset of hepatocellular carcinoma (HCC) cases with poor prognosis, but little is known regarding the biological role of SYK in HCC. We found that SYK methylation is a common event in HCC, and is inversely associated with its expression. We established stable HCC cell lines with inducible SYK expression vectors, and compared the differential RNA expression profiles of HCC cell lines with or without the induction of SYK. Gene ontology analysis revealed that the SYK-regulated genes were enriched for genes involved in cell adhesion. Accordingly, we found that the induction of SYK expression increased the adhesion of cells to fibronectin and decreased cell migration and invasion, and that cessation of SYK overexpression increased cell migration and invasion. Our findings suggest that SYK is involved in regulating cell to matrix adhesions, and that SYK loss affects the migration, and invasion of HCC cells.

Ecklonia cava (EC) is known to have antioxidant, anti-inflammatory, antidiabetic, and anticancer properties. Despite its wide use and beneficial properties, comprehensive toxicological information regarding EC extract is currently limited. Therefore, the purpose of this study was to investigate acute toxicity, subchronic toxicity, and genotoxicity of enzymatic EC extract according to test guidelines published by Organization for Economic Cooperation and Development. The acute oral LD50 values of this EC extract administered to rats and dogs were estimated to be more than 3000 mg/kg BW. In an oral 13-week toxicity study, changes in body weights of rats exposed to the EC extract up to 3000 mg/kg BW were found to be normal. In addition, repeated doses of EC extract failed to influence any systematic parameters of treatment-related toxic symptoms such as food/water consumption, mortality, urinalysis, hematology, serum biochemistry, organ weight, or histopathology. These results indicated that the no-observed-adverse-effect level for the EC extract was 3000 mg/kg/day for male and female rats. Data obtained from Ames test, chromosome aberration assay, and micronucleus assay indicated that EC extract was not mutagenic or clastogenic. Taken together, these results support the safety of enzymatic EC extract as a potential therapeutic for human consumption against various diseases.

Triple negative breast cancer (TNBC) is characterized by an aggressive biological behavior in the absence of a specific target agent. Nicotinamide has recently been proven to be a novel therapeutic agent for skin tumors in an ONTRAC trial. We performed combinatory transcriptomic and in-depth proteomic analyses to characterize the network of molecular interactions in TNBC cells treated with nicotinamide. The multi-omic profiles revealed that nicotinamide drives significant functional alterations related to major cellular pathways, including the cell cycle, DNA replication, apoptosis and DNA damage repair. We further elaborated the global interaction networks of molecular events via nicotinamide-inducible expression changes at the mRNA and functional protein levels. This approach indicated that nicotinamide treatment rewires interaction networks toward dysfunction in DNA damage repair and away from a pro-growth state in TNBC. To our knowledge, the high-resolution network interactions identified in the present study provide the first evidence to comprehensively support the hypothesis of nicotinamide as a novel therapeutic agent in TNBC.

Koji products have been considered as an effective fermented food consumed in East Asia with many health benefits. Particularly, rice koji with Aspergillus terreus (RAT) has been reported to be able to prevent hyperlipidemia and hepatic steatosis through regulating cholesterol synthesis. Despite its biological activities, there is a lack of comprehensive information to give an assurance of its safety. Therefore, the objective of this study was to perform a series of toxicological studies (repeated dose oral toxicity and genotoxicity) according to test guidelines published by the Organization for Economic Cooperation and Development. Along with acute toxicity study using rats and beagle dogs, a 13-week toxicity study revealed no clear RAT-related toxic changes, including body weight, mortality, hematology, serum biochemistry, organ weight, and histopathology after oral administration at doses of 500, 1000, and 2000 mg/kg BW. The no-observed-adverse-effect level of RAT was considered to be more than 2000 mg/kg BW/day in rats of both genders. In addition, potential genotoxicity was evaluated using a standard battery of tests (Ames test, chromosome aberration assay, and micronucleus assay) which revealed that RAT showed no genotoxicity. Accordingly, these results suggest that RAT is a safe and non-toxic functional food for human consumption at proper dose.

Background DNA methylation changes occurring in cancer cells are featured with both promoter CpG island hypermethylation and diffuse genomic hypomethylation. Long interspersed element-1 (LINE-1) is repeated in an interspersed manner with an estimated 500,000 copies per genome. LINE-1 has its CpG sites of the 5′ untranslated region methylated heavily in normal cells and undergoes demethylation in association with cancerization. However, little information is available regarding LINE-1 hypomethylation and its prognostic implication in intrahepatic cholangiocarcinomas. Methods A total of 172 cases of intrahepatic cholangiocarcinomas were analyzed for their methylation levels at four CpG sites of LINE-1 using bisulfite pyrosequencing. We examined the relation between tumoral LINE-1 methylation level and clinicopathological features, including survival. Results Tumor differentiation, lymphatic invasion, and T stage were associated with a low average methylation level of LINE-1 at the four CpG sites; LINE-1 methylation level tended to be lower in high-grade differentiation, lymphatic emboli, and higher T stage. LINE-1 hypomethylation was significantly linked with lower cancer-specific survival in patients with intrahepatic cholangiocarcinoma and was found to be an independent prognostic parameter. Conclusions Our findings suggest that tumoral LINE-1 hypomethylation could be a molecular biomarker heralding poor prognosis of patients with intrahepatic cholangiocarcinoma. Our findings need to be validated in further study. Electronic supplementary material The online version of this article (10.1186/s12885-017-3595-8) contains supplementary material, which is available to authorized users.

Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall’s corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS.

Clinicopathological features of intrahepatic cholangiocarcinomas with positive YAP1 expression (N = 239). Figure S1. Progress free survival curve with disease groups. Stem cell feature carcinomas including stem cell feature of cHC-CCA and cholangiolocellular IHCCA had better PFS than CK19(−) HCC (median, 103 vs. 23 month, p-value 0.123). However, CK19(+) HCC was included poor prognosis group, together with IHCCA and classical cHC-CCA (median, 18 vs. 11 vs. 12 month, log rank p-value 0.670) (DOCX 318 kb).

Background The Hippo pathway plays a vital role in liver regeneration and development by determining cellular lineage and regulating cell proliferation and apoptosis. In this study, we aimed to assess the role of the Hippo pathway in hepatic carcinogenesis and morphogenesis by examining Yes-associated protein 1 (YAP1) expression in the spectrum of hepatic carcinomas based on cellular lineage. Methods We examined 913 primary hepatic carcinomas, including hepatocellular carcinomas (HCCs), combined hepatocellular and cholangiocarcinomas (cHC-CCAs), intrahepatic cholangiocarcinomas (IHCCAs) and perihilar extrahepatic bile duct carcinomas (EHBCAs). Our study group was categorized into 8 disease groups, based on histological diagnosis and cytokeratin 19 (CK19) expression, and immunohistochemistry was used to detect and compare YAP1 expression levels between the groups. The eight disease groups we identified were: 1) CK19(−) HCC, 2) CK19(−) scirrhous HCC, 3) CK19(+) HCC, 4) stem cell feature of cHC-CCA, 5) classical cHC-CCA, 6) cholangiolocellular IHCCA, 7) non-cholangiolocellular IHCCA, and 8) EHBCA. Results Positive rates of YAP1 were the highest in the EHBCA group (21%). CK19(+) HCC and non-cholangiolocellular IHCCA groups also showed high expression levels (10% -11%), while the CK19 (−) HCC, CK19 (−) scirrhous HCC, cHC-CCA, and cholangiolocellular IHCCA groups showed low expression levels, ranging between 0% and 5%. Survival analysis, restricted to pT1 stage HCCs and IHCCAs, showed poor overall survival for YAP1(+) IHCCA patients (39 ± 17 vs. 109 ± 10 months, mean ± SD, log rank p-value 0.005). For HCCs, a trend of poor progression-free survival for YAP1(+) HCCs was observed (39 ± 18 vs. 81 ± 5 months, mean ± SD, log rank p-value 0.205) Conclusions YAP1 activation was more commonly found in CCAs than in pure HCCs. However, a differing pattern of YAP1 expression between cHC-CCAs and CK19(+) HCCs and the poor prognosis of YAP1 positive hepatic carcinomas suggests that YAP1 may have a preferential role in aggressive tumor behavior, rather than in the determination of cellular lineage in hepatic carcinomas. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3431-1) contains supplementary material, which is available to authorized users.