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

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

Genome-wide association studies identify PRKCB as a novel genetic susceptibility locus for primary biliary cholangitis in the Japanese population.

A previous genome-wide association study (GWAS) performed in 963 Japanese individuals (487 primary biliary cholangitis [PBC] cases and 476 healthy controls) identified TNFSF15 (rs4979462) and POU2AF1 (rs4938534) as strong susceptibility loci for PBC. In this study, we performed GWAS in additional 1,923 Japanese individuals (894 PBC cases and 1,029 healthy controls), and combined the results with the previous data. This GWAS, together with a subsequent replication study in an independent set of 7,024 Japanese individuals (512 PBC cases and 6,512 healthy controls), identified PRKCB (rs7404928) as a novel susceptibility locus for PBC (odds ratio [OR] = 1.26, P = 4.13 × 10-9). Furthermore, a primary functional variant of PRKCB (rs35015313) was identified by genotype imputation using a phased panel of 1,070 Japanese individuals from a prospective, general population cohort study and subsequent in vitro functional analyses. These results may lead to improved understanding of the disease pathways involved in PBC, forming a basis for prevention of PBC and development of novel therapeutics.

PNPLA3 as a liver steatosis risk factor following living-donor liver transplantation for hepatitis C.

AIM: Liver steatosis frequently occurs following liver transplantation (LT) and can affect patient outcome. Here, we aimed to clarify the steatosis and steatohepatitis risk factors that apply after living-donor LT for chronic hepatitis C. METHODS: We retrospectively examined 43 transplant recipients and donors, and tested for single nucleotide polymorphisms in the PNPLA3 gene. Liver biopsies taken 1 year after transplantation and yearly thereafter, or when abnormal liver enzyme levels were detected, were examined by histopathology. RESULTS: Liver steatosis (>5% steatotic hepatocytes) was evident in 13 of 43 cases (30%), and steatohepatitis in 3 (7.0%). The average time to steatosis after LT was 2.74 ± 1.55 years. The PNPLA3 rs738409 GG genotype, a steatosis risk factor, was identified in 13 recipients and 10 donors. Steatosis prevalence did not differ according to recipient genotype. However, this condition was significantly more common among patients who received tissue from donors carrying the rs738409 GG genotype compared to those with grafts from donors of the CC or CG genotype (60, 7, and 26%, respectively; P < 0.05). All 3 steatohepatitis cases were associated with the GG donor genotype. CONCLUSION: The PNPLA3 rs738409 GG donor genotype affects liver steatosis and steatohepatitis risk following living-donor LT.

Utilization and efficacy of elbasvir/grazoprevir for treating hepatitis C virus infection after liver transplantation.

AIM: Recently, elbasvir/grazoprevir combination therapy (EBR/GZR) was reported to have excellent antiviral effects for chronic genotype 1 hepatitis C virus (HCV) infection. However, it has not been recommended for patients with post-liver transplant (LT) HCV re-infections because of a lack of evidence for effectiveness and drug-drug interactions. METHODS: We report the usage of EBR/GZR in five post-LT HCV re-infected patients with the kinetics of renal function and tacrolimus trough levels during and after therapy. Furthermore, to evaluate the antiviral effects, we examined the HCV kinetics during and after therapy and compared this with other interferon-free therapy in post-LT patients (n = 19). RESULTS: All patients treated with EBR/GZR therapy obtained rapid virologic response and sustained at 12 weeks post-treatment. There was no evidence of worsening estimated glomerular filtration rates. Three patients were given tacrolimus as immunosuppressive therapy and its trough levels were controllable with dosage adjustments. One patient developed grade 1 diarrhea 3 days after therapy induction. To evaluate the antiviral effects of EBR/GZR therapy for these patients, we compared them to the effects of daclatasvir/asunaprevir combination therapy (n = 8) and sofosbuvir/ledipasvir combination therapy (n = 11). The EBR/GZR combination was not inferior to other therapies in its early phase and late-phase antiviral effects. CONCLUSIONS: Although further studies with a larger number of patients are required, we suggest that EBR/GZR therapy is an alternative therapy for patients with post-LT genotype 1 HCV re-infection.

Prevalence of Restless Legs Syndrome in Patients with Inflammatory Bowel Disease.

BACKGROUND AND AIM: There has been increased interest in sleep disorders in patients with inflammatory bowel disease (IBD). Studies in North America and Europe reported that the prevalence of restless legs syndrome (RLS) is much higher in patients with Crohn's disease (CD) than in the general population. The aim of this study was to reveal the prevalence and clinical features of RLS in Japanese patients with IBD and investigate the influence of RLS on sleep quality and quality of life (QOL). METHODS: The study included 80 outpatients with IBD who visited Nagasaki University Hospital between December 2012 and July 2014. All patients completed the international RLS study group rating scale, a validated measure of the presence of RLS. Sleep quality was assessed using the Japanese version of the Pittsburgh Sleep Quality Index (PSQI), and health-related QOL was assessed using the Japanese version of the 36-item short form healthy profile (SF-36) version 2. RESULTS: The prevalence of RLS in patients with IBD was 20%, including rates of 21.7% in patients with ulcerative colitis (UC) and 17.6% in patients with CD. Among patients with CD, the proportion of women and serum level of CRP were higher in the RLS group than in the non-RLS group. Among those with UC, there were no differences in clinical characteristics between the RLS and non-RLS groups. Patients in the RLS group slept significantly less well than those in the non-RLS group (PSQI > 5; 62.5 vs. 34.4%, P < 0.05). No significant relationships were observed between QOL indices and the presence of RLS (SF-36 physical score, 46.8 vs. 50.1; mental score, 43.8 vs. 45.7; role/social score, 48.1 vs. 49.2). CONCLUSIONS: RLS occurs frequently in Japanese patients with UC as well as CD. RLS affects sleep quality but not QOL, and it should be considered one of the causes of sleep disturbance in patients with IBD.

Hepatitis C Virus Infection Increases c-Jun N-Terminal Kinase (JNK) Phosphorylation and Accentuates Hepatocyte Lipoapoptosis.

BACKGROUND Hepatitis C virus (HCV) infection and metabolic diseases including nonalcoholic steatohepatitis (NASH) exhibit a complex interplay. Although free fatty acid-mediated apoptosis is a prominent feature of NASH, the impact of HCV infection on hepatocyte lipotoxicity has remained largely unexplored. The study aimed at identifying whether infection by HCV affected the apoptotic pathway in hepatocytes during fatty acid assault. MATERIAL AND METHODS OR6 cells, which are derived from human hepatocellular carcinoma Huh-7 cells and harbor a full-length HCV RNA genome replication system, were treated with palmitate. Apoptosis was examined by 4',6-diamidino-2-phenylindole staining. Activation and expression of JNK, Bim, cIAP-1, and Mcl-1 were examined by immunoblotting. mRNA expression of CHOP, a major player in endoplasmic reticulum stress-mediated apoptosis, was assessed by real-time PCR. RESULTS Palmitate-induced hepatocyte apoptosis was significantly enhanced in OR6 cells compared to cured cells, in which the HCV genome had been eradicated by treatment with interferon-α. Although basal expression of CHOP mRNA was enhanced in OR6 cells compared to cured cells, it was similarly upregulated in both cell lines following palmitate treatment. Notably, palmitate-induced JNK phosphorylation was accentuated in OR6 cells compared to cured cells. Inhibition of JNK with SP600125 attenuated palmitate-induced apoptosis. Palmitate-mediated upregulation of BH3-only protein Bim, which acts downstream of JNK, was also enhanced in OR6 cells compared to cured cells. In contrast, Mcl-1 and cIAP-1 were equally reduced in OR6 cells and cured cells following palmitate treatment. CONCLUSIONS These findings suggest that during lipoapoptosis, HCV infection may enhance hepatocyte toxicity by increasing JNK phosphorylation.

Elevated Fasting Plasma Glucose before Liver Transplantation is Associated with Lower Post-Transplant Survival.

BACKGROUND The risk of liver cirrhosis is higher among individuals with diabetes mellitus, and a cirrhotic patient with diabetes may have a poorer prognosis after liver transplantation compared to a patient without diabetes. Thus, we evaluated whether fasting plasma glucose prior to receiving a liver transplant was a prognostic factor for post-transplant survival. MATERIAL AND METHODS Ninety-one patients received a living donor liver transplant between November 2005 and December 2012. Patients were considered diabetic if they were prescribed diabetes medications or had impaired glucose tolerance as measured by an oral glucose tolerance test. Each patient was monitored through December 31, 2013, to evaluate prognosis. RESULTS Fasting plasma glucose of at least 100 mg/dL significantly decreased survival following transplant (52% in the high FPG group compared to 78% in the control group, p=0.04), while postprandial hyperglycemia had no effect on survival. Additionally, overall mortality and the incidence of vascular disease were significantly higher among patients with uncontrolled plasma glucose. Impaired fasting plasma glucose was significantly and inversely associated with overall survival in the univariate and multivariate analyses, while creatinine (at least 1 mg/dL) was inversely associated with survival in the univariate analysis. CONCLUSIONS Elevated fasting plasma glucose prior to liver transplantation was inversely associated with post-transplant survival. This effect may be due to underlying microangiopathy as a result of uncontrolled diabetes before transplantation. Our data demonstrated the importance of controlled blood glucose prior to liver transplantation.

Incidence of and risk factors for bile duct stones after living donor liver transplantation: An analysis of 100 patients.

AIM: Although bile duct stone (BDS) is one of the biliary complications of liver transplantation, analytical studies, particularly on living donor liver transplantation (LDLT) cases, are rare. This study aimed to clarify the incidence of and risk factors for BDS following LDLT. METHODS: We retrospectively reviewed the medical records of 100 patients who underwent LDLT at our institute from August 2000 to May 2012, and analyzed their clinical characteristics and risk factors for BDS. RESULTS: Of these, 10 patients (10.0%) developed BDS during the observation period. The median follow-up period to BDS diagnosis was 45.5 months (range, 5-84) after LDLT. Univariate analysis revealed male sex, right lobe graft and bile duct strictures as factors that significantly correlated with BDS formation. Multivariate analysis revealed bile duct strictures (odds ratio, 7.17; P = 0.011) and right lobe graft (odds ratio, 10.20; P = 0.040) to be independent risk factors for BDS formation. One patient with BDS and biliary strictures succumbed to sepsis from cholangitis. CONCLUSION: In the present study, right lobe graft and bile duct strictures are independent risk factors for BDS formation after LDLT. More careful observation and monitoring are required in the patients with high-risk factors.

Significance of miRNA-122 in chronic hepatitis C patients with serotype 1 on interferon therapy.

AIM: Peginterferon (PEG IFN) and ribavirin combination therapy is a curative treatment for chronic hepatitis C virus (HCV) infection, and virological response to IFN therapy has been strongly associated with genetic variation in IL28B single nucleotide polymorphisms (SNP). Recently, miRNA122 (miR-122), which is the most abundant miRNA in the liver, has been reported to be important for the replication of HCV RNA. Therefore, we investigated the correlation of miR-122 expression with virological response to IFN and other clinical data. METHODS: A total of 51 patients with HCV infection who were treated with IFN therapy at Nagasaki University Hospital from 2006 to 2011 were included in this study. We investigated the correlation of miR-122 expression in liver biopsy specimens with virological response to IFN therapy and other predictors of response, including IL28 SNP. RESULTS: miR-122 expression did not correlate with IL28 SNP. However, a significant difference was observed in miR-122 expression between patients who showed a sustained virological response (SVR) and those who did not (P < 0.05). Multivariate analysis indicated that miR-122 is an independent predictor of SVR. CONCLUSION: miR-122 expression could be a marker for predicting the outcome of IFN therapy. Therapies targeting miR-122 may have positive effects not only by directly inhibiting viral propagation but also by ameliorating cholesterol and lipid abnormalities.

Initiation of genome instability and preneoplastic processes through loss of Fhit expression.

Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.

[Bone disease in primary biliary cirrhosis].

Primary biliary cirrhosis(PBC)is a chronic autoimmune cholestatic liver disease. Metabolic bone disease is recognized in a complication of chronic liver disease, particularly in PBC. Bone disease in PBC includes osteoporosis and, osteomalacia which is more frequent in advanced liver disease. It is important that PBC occurs mainly in middle-aged women who are highest risk group in primary osteoporosis. In patients with PBC, the dysfunction in enterohepatic circulation of bile acids is associated with the impaired absorption of fats and fat soluble vitamins. Vitamin D and K deficiency leads to osteoporosis resulting in increased risk of bone fracture. This article describes the characteristic and molecular mechanism in bone disease of PBC.

Significance of serum and hepatic microRNA-122 levels in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is believed to be a type of metabolic syndrome. MicroRNA-122 (miR-122) is the most abundant microRNA in the liver and is an important factor for the metabolism of glucose and lipids. In the present study, we examined the correlation between the hepatic and serum miR-122 expression levels and the clinicopathological factors of patients with NAFLD. METHODS: We extracted the total RNA, along with preserved miRNAs, from liver biopsy samples of 67 patients with NAFLD. In 52 of these 67 patients, the total RNA was extracted from serum. The miR-122 that was obtained by quantitative reverse transcription-polymerase chain reaction was quantified using TaqMan MicroRNA assays. RESULTS: A significant correlation was detected between serum and hepatic miR-122 expression (correlation coefficient, 0.461; P=0.005). Patients with mild steatosis (<33%) showed significantly lower levels of hepatic miR-122 compared with patients with severe steatosis (>33%) (hepatic miR-122: mild/severe=2.158±1.786/4.836±7.506, P=0.0473; serum miR-122: mild/severe=0.002±0.005/0.007±0.001, P=0.0491). Moreover, hepatic and serum miR-122 levels were significantly higher in patients with mild fibrosis than in those with severe fibrosis (hepatic miR-122: mild/severe=5.201±7.275/2.394±1.547, P=0.0087; serum miR-122: mild/severe=0.008±0.011/0.002±0.004, P=0.0191). CONCLUSIONS: We found that the hepatic and serum miR-122 levels were associated with hepatic steatosis and fibrosis. The serum miR-122 level can be a useful predictive marker of liver fibrosis in patients with NAFLD.

Branched-chain amino acid deficiency stabilizes insulin-induced vascular endothelial growth factor mRNA in hepatocellular carcinoma cells.

Abnormal sugar metabolism is closely related to chronic liver diseases, including hepatocellular carcinoma (HCC). We previously reported that fasting hyperinsulinemia is a poor prognostic factor for HCC patients. A recent large-scale study has shown that long-term administration of branched chain amino acids (BCAA) reduces the risk of HCC development in obese cirrhotic patients who have been diagnosed with diabetes mellitus, although the mechanism by which it does so is unclear. In this study, we analyzed the expression of vascular endothelial growth factor (VEGF) in HepG2 cells under high-insulin culture conditions, and examined the effect of BCAA on VEGF expression. VEGF secretion was significantly increased by 200 nM of insulin under BCAA deficient conditions, but it was repressed by the addition of BCAA. BCAA activated the mTOR pathway and increase HIF-1α expression under high-insulin culture conditions, however quantitative PCR analysis showed that insulin-induced expression of VEGF mRNAs (VEGF121 and VEGF165) decreased 2 h after the addition of BCAA. The half-lives of both VEGF121 and 165 mRNAs were shortened in the presence of BCAA compared to the absence of BCAA. Therefore it is thought that BCAA regulate VEGF expression mainly at the post-transcriptional level. We also examined which of the Valine, Leucine, and Isoleucine components of BCAA were essential for VEGF mRNA degradation. All three BCAA components were required for acceleration of insulin-induced VEGF mRNA degradation. These results suggest that administration of BCAA may downregulate VEGF expression in patients who have hyperinsulinemia and are in the process of developing HCC.

Response of subtype-specific human breast cancer-derived cells to poly(ADP-ribose) polymerase and checkpoint kinase 1 inhibition.

When DNA damage is detected, checkpoint signal networks are activated to stop the cell cycle, and DNA repair processes begin. Inhibitory compounds targeting components of DNA damage response pathways have been identified and are being used in clinical trials, in combination with chemotherapeutic agents, to enhance cancer therapy. Inhibitors of checkpoint kinases, Chk1 and Chk2, have been shown to sensitize tumor cells to DNA damaging agents, and treatment of BRCA1/2-deficient tumor cells, as well as triple negative breast cancers, with poly(ADP-ribose) polymerase (PARP) inhibitors has shown promise. But systematic studies to determine which tumor subtypes are likely to respond to these specific inhibitors have not been reported. The current study was designed to test sensitivity of specific breast cancer subtype-derived cells to two classes of these new inhibitory drugs, PARP and Chk1 inhibitors. Luminal, HER2 overexpressing, and triple negative breast cancer-derived cells were tested for sensitivity to killing by PARP inhibitors, ABT-888 and BSI-201, and Chk1 inhibitor, PF-00477736, alone or in combination with gemcitabine or carboplatin. Each of the triple negative breast cancer cell lines showed strong sensitivity to the Chk1 inhibitor, but only the BRCA1-deficient breast cancer cell lines showed sensitivity to the PARP inhibitors, suggesting that in vitro testing of cancer cell lines of specific subtypes, with panels of the different PARP and Chk1 inhibitors, will contribute to stratification of patients for clinical trials using these classes of inhibitors.

Liver Function in Older Patients With Unresectable Hepatocellular Carcinoma After Administration of Lenvatinib.

BACKGROUND: The age of patients with advanced hepatocellular carcinoma (HCC) eligible for molecular-targeted drug treatment is increasing. We assessed liver function after lenvatinib administration according to age in patients with advanced HCC. PATIENTS AND METHODS: In this retrospective, multicenter, observational study, we reviewed the records of patients with HCC who received lenvatinib treatment (March 2018-March 2020). Liver function was measured using the Albumin-Bilirubin Index (ALBI). RESULTS: Of 119 patients, with a median age of 72.0 years, median overall survival was 15.3 months. Overall survival was significantly better in the group which maintained liver function (p=0.02). Older age (≥72 years) was associated with liver-function deterioration within 8 weeks (odds ratio=2.47, 95% confidence interval=1.06-5.75, p=0.035). The ALBI score was significantly higher in the older group at 4 and 8 weeks after lenvatinib administration. CONCLUSION: Lenvatinib administration was more likely to adversely affect liver function in older patients; dose adjustment should be considered in such patients.

Pathology and biology associated with the fragile FHIT gene and gene product.

More than 12 years and >800 scientific publications after the discovery of the first gene at a chromosome fragile site, the FHIT gene at FRA3B, there are still questions to pursue concerning the selective advantage conferred to cells by loss of expression of FHIT, the most frequent target of allele deletion in precancerous lesions and cancers. These questions are considered in light of recent investigations of genetic and epigenetic alterations to the locus and in a retrospective consideration of biological roles of the Fhit protein discovered through functional studies.

Relationship between regulatory T cells and the combination of pegylated interferon and ribavirin for the treatment of chronic hepatitis type C.

BACKGROUND/AIM: The frequency of regulatory T cells (Tregs) may be related to persistent hepatitis C virus (HCV) infection. We studied the alteration of the Treg ratio in peripheral blood mononuclear cells (PBMCs) from chronic hepatitis C patients during combination therapy compared with the Treg ratio in liver-infiltrating lymphocytes (LILs) before therapy. METHOD: The study group consisted of 20 patients who were treatment-naive and had high virus titers of HCV genotype 1. Blood samples were collected prior to treatment and at several time points during treatment. All patients received a liver biopsy prior to treatment. Forkhead box P3 (Foxp3)+, CD3+, CD4+ and CD8+ cells in PBMCs and LILs were stained by specific antibodies. RESULTS: Ten patients had a sustained virological response (SVR), and 10 patients were non-responders. The SVR group had a significant increase in the Foxp3+/CD4+ ratio in PBMCs at 8 and 12 weeks as well as a significant decrease in the Foxp3+/CD4+ ratio and increase in the CD8+/Foxp3+ ratio in LILs. CONCLUSION: The evaluation of Tregs, a potentially significant factor for persistent HCV infection, in LILs prior to treatment and in PBMCs during treatment could predict the result of combination therapy.

Influence of Interferon-free Direct-acting Antiviral Therapy on Primary Hepatocellular Carcinoma Recurrence: A Landmark Time Analysis and Time-dependent Extended Cox Proportional Hazards Model Analysis.

Objective The influence of interferon (IFN)-free direct-acting antiviral (DAA) on hepatocellular carcinoma (HCC) recurrence remains unclear. Previous retrospective analyses revealed that the time interval between HCC curative treatment and IFN-free DAA induction is the critical factor affecting HCC recurrence. Thus, this study aimed to examine the influence of DAA therapy on HCC recurrence considering this interval. Methods Factors contributing to HCC recurrence were retrospectively analyzed using a landmark time analysis and time-dependent extended Cox proportional hazards model. Patients After screening 620 patients who were diagnosed with primary HCC from January 2001 to December 2016, 76 patients with early-stage (primary and solitary) disease who received curative treatment and were positive for serum hepatitis C virus RNA were included. Results HCC recurrence was observed in 8 of 17 (47.1%) patients who had received IFN-free DAA therapy and 45 of 59 (76.3%) who had not. No significant difference was seen between the IFN-free DAA (-) and IFN-free DAA (+) groups in the landmark time and time-dependent Cox proportional hazards model analyses. However, IFN-free DAA therapy tended to decrease the HCC recurrence rate after curative treatment for primary HCC in patients with chronic hepatitis. In addition, IFN-free DAA therapy tended to decrease the second HCC recurrence rate after treatment for the first HCC recurrence. Conclusion Our results, with a consideration of the time interval between HCC curative treatment and IFN-free DAA induction, showed that IFN-free DAA therapy was not associated with early-stage HCC recurrence after curative treatment.

Extracellular vesicles from senescent hepatic stellate cells promote cell viability of hepatoma cells through increasing EGF secretion from differentiated THP-1 cells.

Since the discovery of the senescence-associated secretory phenotype, the role of senescent hepatic stellate cells (HSCs) in hepatocellular carcinoma (HCC) development has gained increasing attention. Similar to cytokines, extracellular vesicles (EVs) are essential for intercellular communication. However, the function of EVs derived from senescent HSCs in HCC progression has not been extensively studied. The aims of the present study were to characterize the EVs derived from senescent HSCs and determine their role in the tumor microenvironment. Cellular senescence was induced in human hepatic stellate cells (HHSteCs) with various concentrations of etoposide. Induction was confirmed using EdU staining and 53BP1 and p21 immunostaining. EVs were isolated by ultracentrifugation and analyzed by nanoparticle tracking analysis. Multiplex immunoassays were used to compare the levels of growth factors secreted from hepatoma cell lines and macrophage cells pretreated with EVs derived from senescent HHSteCs (senescent EVs) with those pretreated with EVs derived from normal cultured HHSteCs (normal EVs). Treatment with 25 µM etoposide for 3 days was the most effective at inducing senescence in HHSteCs. This finding was confirmed by induction of irreversible cell-cycle arrest, upregulation of 53BP1 and p21 expression, and increased SA-ß-gal staining. Senescent HHSteCs released increased quantities of EV particles compared with normally cultured HHSteCs. Multiplex analysis revealed that there was no difference between hepatoma cell lines treated with normal EVs and those treated with senescent EVs in growth factor secretion. In contrast, the secretion of epidermal growth factor (EGF) was increased by macrophage cells treated with senescent EVs compared with those treated with normal EVs. Furthermore, senescent EVs did not affect the viability of hepatoma cells but increased the viability of hepatoma cells co-cultured with macrophage cells. In conclusion, the release of EVs from senescent HSCs was higher compared with normal HSCs. Furthermore, senescent EVs promoted HCC development by upregulating EGF secretion from macrophages.