Association of Omega-3 Polyunsaturated Fatty Acids with Sarcopenia in Liver Cirrhosis Patients with Hepatocellular Carcinoma.

Background and Aims: Sarcopenia is associated with the prognosis of patients with liver cirrhosis and hepatocellular carcinoma (HCC). Given their diverse physiological activities, we hypothesized that plasma fatty acids might influence the progression of sarcopenia. This study aimed to clarify the association between fatty acids and sarcopenia in cirrhotic patients with HCC. Methods: In this single-center retrospective study, we registered 516 cases and analyzed 414 cases of liver cirrhosis and HCC. The skeletal muscle mass index was measured using a transverse computed tomography scan image at the third lumbar vertebra. The cutoff value for sarcopenia followed the criteria set by the Japan Society of Hepatology. Fatty acid concentrations were measured by gas chromatography. Results: Fatty acid levels, particularly omega-3 (n-3) polyunsaturated fatty acid (PUFA), were lower in patients with poor liver function (Child-Pugh grade B/C) and were negatively correlated with the albumin-bilirubin score (p<0.0001). The prognosis of HCC patients with low PUFA levels was significantly worse. Among the different fatty acid fractions, only n-3 PUFAs significantly correlated with skeletal muscle mass index (p=0.0026). In the multivariate analysis, the n-3 PUFA level was an independent variable associated with sarcopenia (p=0.0006). Conclusions: A low level of n-3 PUFAs was associated with sarcopenia in patients with liver cirrhosis and HCC.

Immunoglobulin-like transcript 2 as an impaired anti-tumor cytotoxicity marker of natural killer cells in patients with hepatocellular carcinoma.

Introduction: Natural killer (NK) cells play a pivotal role in immune surveillance in the liver. We aimed to identify potential targets for NK cell-mediated immune intervention by revealing the functional molecules on NK cells in HCC patients. Methods: To evaluate the impact of aging on NK cell phenotypes, we examined NK cells from healthy volunteers (HVs) of various ages. Because ILT2 expression on CD56dim NK cells increased with increasing age, we enrolled age-matched HCC patients and HVs. We determined the NK cell phenotypes in blood mononuclear cells (PBMCs) and intrahepatic lymphocytes (IHLs) from cancerous and non-cancerous tissues. We evaluated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of NK cells in vitro. Results: ILT2-positive CD56dim NK cells in PBMCs were increased in HCC patients compared with HVs. In HCC patients, ILT2-positive CD56dim NK cells were increased in cancerous IHLs compared with non-cancerous IHLs and PBMCs. We examined the impact of macrophage migration inhibitory factor (MIF) on ILT2 expression in co-cultures of HCC cells and NK cells. The enhanced expression of ILT2 on CD56dim NK cells from HCC patients was inhibited by masking antibodies against MIF and CXCR4. ILT2-positive CD56dim NK cells exhibited lower capacities for cytotoxicity and ADCC than ILT2-negative cells, which were partially restored by ILT2 blockade. Conclusions: In HCC patients, ILT2 is a signature molecule for cancerous CD56dim NK cells with impaired cytolytic capacity. The MIF-CXCR4 interaction is associated with ILT2 induction on CD56dim NK cells and ILT2 serves as a target for functional NK cell restoration.

Exercise changes the intrahepatic immune cell profile and inhibits the progression of nonalcoholic steatohepatitis in a mouse model.

BACKGROUND: NASH is an increasingly common cause of chronic liver disease and can progress to cirrhosis and HCC. Although exercise suppresses inflammation during acute hepatitis, its impact on the progression of chronic liver disease remains unclear. Here, we investigated the effects of exercise on disease progression and intrahepatic immune cell composition in a mouse model of NASH. METHOD: Mice were assigned to 4 groups: 2 control groups (normal diet) and 2 NASH groups (western diet and low-dose carbon tetrachloride injection). One of each group remained sedentary and one was exercised on a treadmill for 12 weeks (60 min/d, 5 times/wk). All mice were then analyzed for liver histomorphology, steatosis, inflammation, and fibrosis; liver, adipose tissue, and skeletal muscle expression of genes related to metabolism and inflammation; and intrahepatic immune cell composition. RESULT: Compared with the normal diet mice, NASH mice exhibited enhanced liver steatosis, inflammation, and fibrosis; upregulated expression of liver lipogenesis-related and inflammation-related genes; and increased frequencies of intrahepatic F4/80 int CD11b hi bone marrow-derived macrophages and programmed death receptor-1 (PD-1) + CD8 + T cells. Expression of inflammatory cytokines and the frequencies of bone marrow-derived macrophages and PD-1 + CD8 + T cells correlated positively with liver steatosis, inflammation, and fibrosis. Exercise was shown to reduce NASH-induced hepatic steatosis, liver inflammation, and fibrosis; induce alterations in metabolism-related genes and inflammatory cytokines in the liver; and suppress accumulation of liver bone marrow-derived macrophages and PD-1 + CD8 + T cells. In addition, we showed that exercise induced increased expression of IL-15 in muscle and its deficiency exacerbated the pathology of NASH. CONCLUSIONS: Exercise alters the intrahepatic immune cell profile and protects against disease progression in a mouse model of NASH.

Serum osteopontin predicts the response to atezolizumab plus bevacizumab in patients with hepatocellular carcinoma.

BACKGROUND: Combination therapy with anti-programmed death-ligand 1 and anti-vascular endothelial growth factor (VEGF) antibodies has become the standard treatment for un-resectable hepatocellular carcinoma (uHCC). We aimed to identify predictive circulating biomarkers for the outcome/response of the combination therapy in uHCC patients. METHODS: This prospective multicenter study enrolled 70 patients with uHCC who received atezolizumab and bevacizumab (Atez/Bev). We evaluated 47 circulating proteins in sera before and after 1 and 6 weeks of Atez/Bev therapy by multiplex bead-based immunoassay and ELISA. As controls, we analyzed the sera from 62 uHCC patients before treatment of lenvatinib (LEN) and healthy volunteers (HVs). RESULTS: The disease control rate was 77.1%. Median progression-free survival (PFS) was 5.7 months (95% confidence interval [CI] = 3.8-9.5). The pretreatment levels of osteopontin (OPN), angiopoietin-2, VEGF, S100-calcium-binding protein A8/S100-calcium-binding protein A9, soluble programmed cell death-1, soluble CD163, and 14 cytokines/chemokines were higher in patients with uHCC than in HVs. Among these, pretreatment OPN levels were higher in PD group than in non-PD group for Atez/Bev. The PD rate was higher in high OPN group than in low OPN group. Multivariate analysis identified high pretreatment OPN and high α-fetoprotein levels as independent predictors of PD. In the sub-analysis of Child-Pugh class A patients, PFS was also shorter in the high OPN group than in the low OPN group. Pretreatment OPN level was not associated with treatment response for LEN. CONCLUSION: High serum OPN levels were associated with poor response to Atez/Bev in patients with uHCC.

Plasma free amino acids are associated with sarcopenia in the course of hepatocellular carcinoma recurrence.

OBJECTIVE: Sarcopenia worsens the prognosis of patients with hepatocellular carcinoma (HCC). The aim of this study was to elucidate the plasma free amino acids (PFAAs) associated with sarcopenia or myosteatosis in the course of HCC recurrence. METHODS: In this cross-sectional study, 187 patients were enrolled retrospectively. All patients experienced more than one hospitalization (mean times, 2.65) owing to HCC recurrence. The skeletal muscle index (SMI) and muscle attenuation (MA) were measured by a transverse computed tomography (CT) scan image at the third lumbar vertebra (L3). The changes in the concentration of 24 PFAAs, SMI, and MA in the same patient between recurrences were defined as Δ. The associations between sarcopenia, myosteatosis, and PFAAs were evaluated by a logistic regression model. The ΔSMI and ΔMA were compared between the patients who received branched-chain amino acids (BCAAs) formulation and those who did not. RESULTS: Patients with sarcopenia showed lower survival rate; the 1-, 3-, and 5-y survival rates were 85%, 42%, and 9%, respectively. Multivariate analysis revealed that the level of total BCAAs was significantly associated with sarcopenia. The correlation coefficient value between the change of leucine (ΔLeu) and ΔSMI was highest (R = 0.256; P < 0.001) among the PFAAs. In the Child-Pugh grade B or C, the decrease of SMI was significantly more suppressed in the patients with the BCAAs formulation than in those without BCAAs formulation (ΔSMI: mean change -0.98 versus -3.45 cm²/m²; P = 0.038). CONCLUSION: Among the PFAAs, the level of BCAAs was associated with sarcopenia in the course of HCC recurrence.

Hepatitis B Virus Reactivation with Discontinuation of Nucleoside Analogue in Patients Who Received Allogeneic Hematopoietic Stem Cell Transplantation.

Reactivation of hepatitis B virus (HBV) is known to occur frequently after hematopoietic stem cell transplantation (HSCT). The reactivation can be prevented by nucleos(t)ide analogue (NA), but it is unclear how long NA should be continued. Here, we report 3 cases of HBV reactivation with discontinuation of NA following the discontinuation of immunosuppressive therapies after HSCT. Three male patients aged 34, 59, and 54 years received allogeneic HSCT (allo-HSCT) for chronic myeloid leukemia, mixed phenotype acute leukemia, and myelodysplastic syndrome, respectively. Before HSCT, 2 patients were positive for hepatitis B surface antigen (HBsAg) and 1 patient was negative for HBsAg and positive for antibodies to hepatitis B core antigen. NA (lamivudine or entecavir) was started at the same time as HSCT and stopped after the discontinuation of immunosuppressive therapies. In all patients, the serum HBV DNA levels were increased after the discontinuation of NAs. Two of the three patients developed severe hepatitis with high levels of HBV DNA (7.5 and 7.4 log IU/mL, respectively). A patient without hepatitis was re-administered NA soon after the HBV DNA started to increase (3.3 log IU/mL). Interestingly, the 2 patients who developed hepatitis cleared HBsAg promptly after the recovery from hepatitis and they could stop NAs without the reversion of HBsAg. It was speculated that transplanted immune cells, which were naïve for HBV, react strongly with HBV antigens that were increased after the NA discontinuation. The discontinuation of NA after allo-HSCT is not recommended generally because strong hepatitis might be induced even after several years.

Rapid enlargement of a hepatic hilar cyst leading to bile duct obstruction after acute exacerbation of chronic hepatitis B.

A hepatic cyst is usually asymptomatic but, in some cases, can be associated with various complications. Here we report a rare case with rapid enlargement of a hepatic hilar cyst that induced bile duct obstruction after an acute exacerbation of chronic hepatitis B. The case is a 60-year old female who discontinued entecavir by herself. Hyperbilirubinemia was prolonged along with bile duct obstruction due to an enlarged cyst. After the administration of entecavir and steroid pulse therapy, biliary drainage and punctuation of the cyst were performed. There was no evidence of malignancy in the cyst. The therapies were not effective enough, probably due to the prior liver damage, and she died of acute on chronic liver failure. This case suggests that a hepatic hilar cyst in a patient with acute hepatitis or an acute exacerbation of chronic hepatitis can become enlarged and may affect the clinical course of hepatitis. In such a case, the size of the cyst should be monitored frequently and bile duct obstruction should be treated early if it occurs.

Methotrexate-associated Lymphoproliferative Disorder in the Liver Resembling Hepatocellular Carcinoma Treated with Transarterial Chemoembolization.

Methotrexate-related lymphoproliferative disorder (MTX-LPD) is known to be a side effect of MTX, but its involvement in the liver has been rarely reported. We herein report a 70-year-old woman with autoimmune hepatitis and rheumatoid arthritis who developed multiple liver tumors. We initially considered that she had developed rapid-growing hepatocellular carcinoma (HCC) in the cirrhotic liver based on imaging tests. A tumor biopsy and transcatheter arterial chemoembolization were thus performed. The tumors were then diagnosed as diffuse large B-cell lymphoma pathologically and considered to be MTX-LPD. This case indicates that MTX-LPD should be considered even in cirrhotic patients with liver tumors resembling HCC.

Spontaneous reactivation of hepatitis B virus with S gene mutations in an elderly patient with diabetic nephropathy.

Generally, reactivation of hepatitis B virus (HBV) infection is induced by the administration of immunosuppressants or anticancer agents, but reactivation without such drugs has rarely been reported. Here we report an elder case with spontaneous reactivation of HBV replication accompanied by hepatitis B surface antigen (HBsAg) mutations. A 69-year-old man with a history of diabetes mellitus and chronic kidney disease (CKD) was found to be positive for HBsAg (0.072 IU/ml) in June 2018. In May 2019, marked hepatic dysfunction and increased HBsAg (2533.2 IU/ml) were observed when he visited the hospital due to diarrhea and worsening of CKD. At that time, hepatitis B surface antibody (HBsAb) was positive (268.9 mIU/ml) and HBV DNA was 6.0 log IU/ml. After treatment with entecavir, HBV DNA and HBsAg rapidly decreased. Full-genome HBV sequence analysis revealed that the patient was infected with HBV of subgenotype B1 and it had an "a" determinant mutation M133L in the S gene coding HBsAg. Notably, both HBsAg and HBsAb were positive at the time of HBV reactivation, suggesting that the HBV with these mutations escaped from neutralization by HBsAb. This case suggests that immune escape mutations could play an important role in spontaneous HBV reactivation.

Effect of viral DNA methylation on expression of hepatitis B virus proteins depends on the virus genotype.

The frequency of HBV genomic methylation in the liver was reported to vary among patients, but the detailed mechanism is still unknown. In this study, the effects of HBV genome methylation on HBV replication were investigated in vitro. A total of 6 plasmids containing 1.24-fold the HBV genome of different genotypes (subgenotypes A1, A2, B1, and C2) were purified after in vitro methylation with CpG methyltransferase (M.SssI) and transfected into HepG2 cells. In genotype B and C strains, methylation markedly decreased the amount of hepatitis B e antigen (HBeAg) in the culture supernatant. A reduction of hepatitis B surface antigen (HBsAg) was found in some HBV strains but the reduction was smaller than that of HBeAg. There was no significant difference in particle-associated HBV DNA in the culture supernatant. These findings suggest that HBV genomic methylation might be involved in the HBeAg decline in genotype B and C, in part, and that the reduction of HBsAg was less than that of HBeAg. In conclusion, this study showed that the effect of HBV genomic methylation differs among HBV genotypes, suggesting a potential explanation for the different clinical outcomes of genotypes A, B, and C.

Comparison of hepatitis B virus genotypes B and C among chronically hepatitis B virus-infected patients who received nucleos(t)ide analogs: A multicenter retrospective study.

AIM: Hepatitis B virus genotype B (HBV/B) has been reported to have less risk of liver cirrhosis and hepatocellular carcinoma (HCC), but long-term observation has rarely been reported. We aimed to clarify the characteristics of HBV/B in nucleos(t)ide analog-treated patients in an area where HBV/B is more prevalent than in other areas of Japan. METHODS: A total of 498 chronically HBV-infected patients treated with nucleos(t)ide analog (lamivudine, entecavir, tenofovir disoproxil fumarate, or tenofovir alafenamide fumarate) for >6 months (mean 70.6 months) were included from nine hospitals in northeast Japan. The frequencies of hepatitis B surface antigen loss and HCC occurrence were analyzed. RESULTS: Among 427 patients whose genotype could be determined, 34.0% and 64.4% were infected with HBV/B and genotype C (HBV/C), respectively. The age of patients with HBV/B was significantly older than those with HBV/C (57.7 vs. 48.1). The cumulative rate of hepatitis B surface antigen loss was significantly higher in HBV/B than in HBV/C (3.6% vs. 0.7% at 10 years). Among 480 patients without HCC history, HCC occurrence was found in 40 patients (13.4% at 10 years). There was no cumulative rate difference of HCC occurrence among the genotypes, but after propensity score matching for age/sex, it was significantly lower in HBV/B than in HBV/C (5.3% vs. 18.5% at 10 years). CONCLUSIONS: Although a lower rate of HCC occurrence in HBV/B was shown by an age/sex-matched analysis than that in HBV/C, patients with HBV/B were significantly older and had a comparative risk of HCC occurrence in nucleos(t)ide analog-treated patients.

Branched chain amino acids are associated with the heterogeneity of the area of lipid droplets in hepatocytes of patients with non-alcoholic fatty liver disease.

AIM: Macrovesicular steatosis around the central vein (zone 3) is one of the pathological features of non-alcoholic fatty liver disease or steatohepatitis (NAFLD/NASH). The aim of this study is to elucidate precisely the association between the area of lipid droplets (LDs) and the plasma metabolic parameters in patients with NAFLD/NASH. METHODS: Eighty patients with NAFLD/NASH diagnosed by needle biopsy were enrolled. The LDs around zone 3 were counted automatically by image processing software, the total area of LDs (TLDs), the maximum area of LDs (MAXLDs), the average area of LDs (AVELDs) and the heterogeneity by the coefficient of variation (CV [%]) were quantified. The correlations between these values and plasma metabolic parameters were analyzed. We evaluated the association between branched chain amino acids (BCAAs) and the heterogeneity of LDs in hepatocytes in vitro and in vivo. RESULTS: The MAXLDs was significantly correlated with more metabolic parameters than AVELDs and TLDs. The level of BCAAs was independently associated with the CV among the metabolic parameters. In early stage NAFLD, aspartate and alanine aminotransferase were significantly higher in the high CV group than in the low CV group. The high concentration of BCAAs increased the CV of LDs in hepatocytes accompanied by the expression of phosphor-p70 S6 kinase and sterol regulatory element-binding protein 1 in vitro. A high BCAA diet induced high heterogeneity of LDs around zone 3 in ob/ob mice. CONCLUSIONS: The levels of BCAAs were associated with the LD heterogeneity of hepatocytes around zone 3 in patients with NAFLD/NASH.

StAR-related lipid transfer domain 11 (STARD11)-mediated ceramide transport mediates extracellular vesicle biogenesis.

Extracellular vesicles are important carriers of cellular materials and have critical roles in cell-to-cell communication in both health and disease. Ceramides are implicated in extracellular vesicle biogenesis, yet the cellular machinery that mediates the formation of ceramide-enriched extracellular vesicles remains unknown. We demonstrate here that the ceramide transport protein StAR-related lipid transfer domain 11 (STARD11) mediates the release of palmitate-stimulated extracellular vesicles having features consistent with exosomes. Using palmitate as a model of lipotoxic diseases and as a substrate for ceramide biosynthesis in human and murine liver cell lines and primary mouse hepatocytes, we found that STARD11-deficient cells release fewer extracellular vesicles. Moreover, STARD11 reciprocally regulated exosome ceramide enrichment and cellular ceramide depletion. We further observed that in STARD11 knockout cells intracellular ceramide accumulates and that this apparent inability to transfer cellular ceramide into extracellular vesicles reduces cellular viability. Using endogenous markers, we uncovered structural and functional colocalization of the endoplasmic reticulum (ER), STARD11, and multivesicular bodies. This colocalization increased following palmitate treatment, suggesting a functional association that may mediate ceramide trafficking from the ER to the multivesicular body. However, the size and number of multivesicular bodies were comparable in WT and STARD11-knockout cells. In conclusion, we propose a model of how STARD11 mediates ceramide trafficking in palmitate-treated cells and stimulates exosome biogenesis.

Inhibitory effect of silibinin on hepatitis B virus entry.

Hepatitis B virus (HBV) infection is a worldwide health problem because of its potential to cause liver cirrhosis and hepatocellular carcinoma. Silibinin is a constituent of an extract of milk thistle, which is empirically used as a herbal medicine for the protection of liver, but its detailed effects on HBV are unknown. Because a previous study reported that silibinin hinders clathlin-mediated endocytosis (CME), we aimed to test whether silibinin inhibits the entry of HBV into hepatocytes. Using HepG2-NTCP-C4 cells, which overexpress sodium taurocholate cotransporting polypeptide (NTCP), it was shown that silibinin inhibited HBV infection dose-dependently. Similar effects were observed using human primary hepatocytes (PXB-cells). Additionally, a combination of silibinin and entecavir reduced HBV DNA in the culture supernatant more than either mono-treatment alone in HepG2-NTCP-C4 cells that had already been infected with HBV. Silibinin decreased transferrin uptake but did not affect the interaction between the HBV envelope and NTCP, suggesting that silibinin might inhibit HBV infection by hindering CME. In conclusion, this study showed that silibinin inhibits HBV entry in vitro.

Selective insulin resistance with differential expressions of IRS-1 and IRS-2 in human NAFLD livers.

BACKGROUND/OBJECTIVE: Insulin signals, via the regulation of key enzyme expression, both suppress gluconeogenesis and enhance lipid synthesis in the liver. Animal studies have revealed insulin signaling favoring gluconeogenesis suppression to be selectively impaired in steatotic livers. However, whether, and if so how, such selective insulin resistance occurs in human steatotic livers remains unknown. Our aim was to investigate selective insulin resistance in human livers with non-alcoholic fatty liver disease (NAFLD). SUBJECTS/METHODS: We examined mRNA expressions of key molecules for insulin signaling, gluconeogenesis and lipogenesis in human liver biopsy samples obtained from 51 non-diabetic subjects: 9 healthy controls and 42 NAFLD patients, and analyzed associations of these molecules with each other and with detailed pathological and clinical biochemistry data. RESULTS: In NAFLD patients, insulin receptor substrate (IRS)-2 expression was decreased, while those of key enzymes for gluconeogenesis were increased. These alterations of IRS-2 and gluconeogenesis enzymes were induced both in simple steatosis (SS) and non-alcoholic steatohepatitis (NASH), while these expression levels did not differ between SS and NASH. Furthermore, alterations in the expressions of IRS-2 and gluconeogenesis enzymes showed strong negative correlations and were concurrently induced in the early histological stage of NAFLD. In contrast, fatty acid synthase (FAS) expression was not decreased in NAFLD, despite IRS-2 downregulation, but correlated strongly with IRS-1 expression. Furthermore, no histological scores were associated with these molecules. Thus, IRS-1 signaling, which is not impaired in NAFLD, appears to modulate FAS expression. CONCLUSION: These analyses revealed that selective insulin resistance is present in human NAFLD livers and occurs in its early phases. The effect of insulin, during the IRS step, on gene expressions for lipogenesis and gluconeogenesis are apparently distinct and preferential downregulation of IRS-2 may contribute to selective resistance to the suppressive effects of insulin on gluconeogenesis.

Gastroesophageal Variceal Bleeding Successfully Controlled by Partial Splenic Embolization.

A 53-year-old male patient with a history of hepatocellular carcinoma developed gastroesophageal varices refractory to endoscopic injection sclerotherapy (EIS). He required EIS six times in 2 years for recurring variceal bleeding. After hepatic resection, he developed massive splenomegaly. Partial splenic embolization (PSE) was performed to reduce the portal pressure. Varices and variceal bleeding were not detected during 13-year follow up, until the patient died of hepatocellular carcinoma. This is a unique case of gastroesophageal varices controlled by PSE and improved portal hypertension.

Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner.

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions.

Expression of EpCAM increases in the hepatitis B related and the treatment-resistant hepatocellular carcinoma.

Increasing evidence supports the important role of cancer stem cells (CSCs). Many reports suggest that epithelial cell adhesion molecule (EpCAM) is a useful marker for cancer stem cells in hepatocellular carcinoma (HCC). To elucidate the mechanisms of cancer stem cells, the development of specific molecular targeted drugs has become very important. In the present study, we examined the EpCAM expression pattern and its characteristic expression in resected HCC. We studied the drug resistance of EpCAM expression cells. EpCAM expression was detected significantly more frequently with hepatitis B virus (HBV) than with other etiologies. In HCC resection patients who had received prior treatment (transcatheter arterial embolization or hepatic arterial infusion chemotherapy), EpCAM was strongly expressed. In particular, very strong expression was observed after hepatic arterial infusion chemotherapy. The PLC/PRF/5 human HCC cell line expressed bimodal EpCAM, and EpCAM-positive cells had CSC cell potency. The EpCAM expression in EpCAM-positive cells increased significantly by treatment with cisplatin. EpCAM-positive cells showed better viability than EpCAM-negative cells when treated with ciplatin. Collectively, our results suggest that cancer stem cells are highly expressed in hepatitis B and have potential anticancer drug resistance.

Involvement of miRNA-29a in epigenetic regulation of transforming growth factor-ß-induced epithelial-mesenchymal transition in hepatocellular carcinoma.

AIM: Epithelial-mesenchymal transition (EMT) is a crucial process during cancer invasion and metastasis, which is accompanied by the suppressed expression of E-cadherin initiated by stimuli such as transforming growth factor (TGF)-ß. Recent studies have shown that the epigenetic regulation of E-cadherin could be an alternate mechanism of EMT induction in hepatocellular carcinoma (HCC). miRNA-29a (miR-29a) is involved in the epigenetic regulation of genes by targeting DNA methyltransferases (DNMT), which methylate CpG islands to suppress the transcription of genes. We studied the involvement of miR-29a in TGF-ß-induced EMT in HCC cells. METHODS: We treated human HCC cell lines with TGF-ß to induce EMT. To investigate DNA methylation in EMT, cells were treated with a methylation inhibitor, 5-Aza-2'-deoxycytidine (5-Aza) and methylation status of CpG islands in the E-cadherin promoter was examined using methylation-specific PCR. Precursor miR-29a (pre-miR-29a) was electroporated to force the expression of miR-29a in HCC cells in order to study the role of miR-29a in EMT. RESULTS: TGF-ß transformed HCC cells into a spindle-shaped morphology accompanied by a decrease of E-cadherin with the induction of methylation of its promoter. Pretreatment of the cells with 5-Aza blocked this suppression of E-cadherin, indicating the involvement of DNA methylation. TGF-ß increased DNMT3B and DNMT1 and decreased miR-29a expression. The forced expression of miR-29a abrogated the suppression of E-cadherin induced by TGF-ß. CONCLUSION: miR-29a could regulate TGF-ß-induced EMT by affecting DNA methylation via the suppression of DNMT. These observations reveal the epigenetic regulation of genes by miRNA as a unique mechanism of EMT in HCC.

Plasma amino acids imbalance in cirrhotic patients disturbs the tricarboxylic acid cycle of dendritic cell.

An imbalance of plasma amino acids (AA) is observed cirrhotic patients. Here we report that the imbalance suppresses the maturation of dendritic cells (DCs) by reducing the intracellular ATP due to interference with the mitochondrial tricarboxylic acid (TCA) cycle. We used serum-free culture medium consistent with the average concentration of the plasma AA from a healthy volunteer (HCM) and that from patients with advanced cirrhosis (ACM). We compared the function of DCs and the metabolism of glucose-amino acids under each medium. The maturation and intracellular ATP of immature DCs were lower under ACM in spite of the enhancement of mitochondrial respiratory chain complex genes. Metabolomics revealed that the TCA cycle metabolite, fumarate and 2-oxoglutarate were increased in DCs generated under ACM. Consistent with in vitro, In CD1c(+) or CD14(+) cells from cirrhotic patients, the gene expression of 2-oxoglutarate-succinate-fumarate transition enzymes were significantly different from the cells of healthy controls.