Drug Screening for Hepatitis A Virus (HAV): Nicotinamide Inhibits c-Jun Expression and HAV Replication.

Hepatitis A virus (HAV) infection often causes acute hepatitis, which results in a case fatality rate of 0.2% and fulminant hepatitis in 0.5% of cases. However, no specific potent anti-HAV drug is available on the market to date. In the present study, we focused on inhibition of HAV internal ribosomal entry site (IRES)-mediated translation and investigated novel therapeutic drugs through drug repurposing by screening for inhibitors of HAV IRES-mediated translation and cell viability using a reporter assay and cell viability assay, respectively. The initial screening of 1,158 drugs resulted in 77 candidate drugs. Among them, nicotinamide significantly inhibited HAV HA11-1299 genotype IIIA replication in Huh7 cells. This promising drug also inhibited HAV HM175 genotype IB subgenomic replicon and HAV HA11-1299 genotype IIIA replication in a dose-dependent manner. In the present study, we found that nicotinamide inhibited the activation of activator protein 1 (AP-1) and that knockdown of c-Jun, which is one of the components of AP-1, inhibited HAV HM175 genotype IB IRES-mediated translation and HAV HA11-1299 genotype IIIA and HAV HM175 genotype IB replication. Taken together, the results showed that nicotinamide inhibited c-Jun, resulting in the suppression of HAV IRES-mediated translation and HAV replication, and therefore, it could be useful for the treatment of HAV infection. IMPORTANCE Drug screening methods targeting HAV IRES-mediated translation with reporter assays are attractive and useful for drug repurposing. Nicotinamide (vitamin B3, niacin) has been shown to effectively inhibit HAV replication. Transcription complex activator protein 1 (AP-1) plays an important role in the transcriptional regulation of cellular immunity or viral replication. The results of this study provide evidence that AP-1 is involved in HAV replication and plays a role in the HAV life cycle. In addition, nicotinamide was shown to suppress HAV replication partly by inhibiting AP-1 activity and HAV IRES-mediated translation. Nicotinamide may be useful for the control of acute HAV infection by inhibiting cellular AP-1 activity during HAV infection processes.

Molecular Mechanisms, Diagnosis and Treatments in Digestive Malignancy.

In this Special Issue, "Molecular Mechanisms, Diagnosis and Treatments in Digestive Malignancy", of the International Journal of Molecular Sciences, a total of 10 impactful articles have been published [...].

Gut-Microbiota Dysbiosis in Stroke-Prone Spontaneously Hypertensive Rats with Diet-Induced Steatohepatitis.

Metabolic-dysfunction-associated fatty-liver disease (MAFLD) is the principal worldwide cause of liver disease. Individuals with nonalcoholic steatohepatitis (NASH) have a higher prevalence of small-intestinal bacterial overgrowth (SIBO). We examined gut-microbiota isolated from 12-week-old stroke-prone spontaneously hypertensive-5 rats (SHRSP5) fed on a normal diet (ND) or a high-fat- and high-cholesterol-containing diet (HFCD) and clarified the differences between their gut-microbiota. We observed that the Firmicute/Bacteroidetes (F/B) ratio in both the small intestines and the feces of the SHRSP5 rats fed HFCD increased compared to that of the SHRSP5 rats fed ND. Notably, the quantities of the 16S rRNA genes in small intestines of the SHRSP5 rats fed HFCD were significantly lower than those of the SHRSP5 rats fed ND. As in SIBO syndrome, the SHRSP5 rats fed HFCD presented with diarrhea and body-weight loss with abnormal types of bacteria in the small intestine, although the number of bacteria in the small intestine did not increase. The microbiota of the feces in the SHRSP5 rats fed HFCD was different from those in the SHRP5 rats fed ND. In conclusion, there is an association between MAFLD and gut-microbiota alteration. Gut-microbiota alteration may be a therapeutic target for MAFLD.

Involvement of proliferation of atypical hepatocytes and CDT 1 in the liver cancer of rats administered the diethylnitrosamine.

We have reported that extent of proliferation of atypical hepatocytes (POAH) in non-cancerous liver in hepatocellular carcinoma and chromatin licensing and DNA replication factor 1 (CDT1) are associated with postoperative recurrence. Here, we investigated whether extent of POAH and expression of CDT1 in liver are also associated with chemically induced liver cancer in rats. Male Fisher strain rats were orally administered diethylnitrosamine (DEN) in their drinking water and sacrificed at 6, 8, 12, or 14 weeks after start of DEN administration. We serially monitored changes in extent of POAH, CDT1 expression by immunohistochemistry (IHC), and CDT1 mRNA expression in liver by real-time quantitative PCR. The extent of POAH in liver progressed in a time-dependent manner after start of DEN administration. CDT1 expression was higher at 8 weeks than at 6 weeks by IHC, suggesting that CDT1 expression may be a marker of POAH severity. CDT1 mRNA expression in liver was significantly higher at 12 weeks than at 6 weeks (p<0.0001). We found that extent of POAH and the expression of CDT1 are also important factors in the development of chemical carcinogen-induced hepatocarcinogenesis. Furthermore, the association with POAH and CDT1 expression in carcinogenic process is important regardless of the cause of hepatocarcinogenesis.

Muscle Cramps in Outpatients with Liver Diseases in Tokyo, Japan.

Background and Objectives: Muscle cramps are often observed in patients with liver diseases, especially advanced liver fibrosis. The exact prevalence of muscle cramps in outpatients with liver diseases in Japan is unknown. Patients and Methods: This study examined the prevalence of, and therapies for, muscle cramps in outpatients with liver diseases in Tokyo, Japan. A total of 238 outpatients with liver diseases were retrospectively examined. We investigated whether they had muscle cramps using a visual analog scale (VAS) (from 0, none, to 10, strongest), and also investigated their therapies. Results: Muscle cramps were observed in 34 outpatients with liver diseases (14.3%); their mean VAS score was 5.53. A multivariate analysis demonstrated that older age (equal to or older than 66 years) was the only significant factor as-sociated with muscle cramps. The prevalence of muscle cramps among patients with liver diseases seemed not to be higher. The problem was that only 11 (32.4%) of 34 outpatients received therapy for their muscle cramps. Conclusions: Only age is related to muscle cramps, which is rather weak, and it is possible that this common symptom may not be limited to liver disease patients.

Integrin ß1 Establishes Liver Microstructure and Modulates Transforming Growth Factor ß during Liver Development and Regeneration.

A unique and complex microstructure underlies the diverse functions of the liver. Breakdown of this organization, as occurs in fibrosis and cirrhosis, impairs liver function and leads to disease. The role of integrin ß1 was examined both in establishing liver microstructure and recreating it after injury. Embryonic deletion of integrin ß1 in the liver disrupts the normal development of hepatocyte polarity, specification of cell-cell junctions, and canalicular formation. This in turn leads to the expression of transforming growth factor ß (TGF-ß) and widespread fibrosis. Targeted deletion of integrin ß1 in adult hepatocytes prevents recreation of normal hepatocyte architecture after liver injury, with resultant fibrosis. In vitro, integrin ß1 is essential for canalicular formation and is needed to prevent stellate cell activation by modulating TGF-ß. Taken together, these findings identify integrin ß1 as a key determinant of liver architecture with a critical role as a regulator of TGF-ß secretion. These results suggest that disrupting the hepatocyte-extracellular matrix interaction is sufficient to drive fibrosis.

Quantitative Ultrasound Image Analysis Helps in the Differentiation of Hepatocellular Carcinoma (HCC) From Borderline Lesions and Predicting the Histologic Grade of HCC and Microvascular Invasion.

OBJECTIVES: Quantitative image analysis is one of the methods to overcome the lack of objectivity of ultrasound (US). The aim of this study was to clarify the correlation between the features from a US image analysis and the histologic grade and microvascular invasion (MVI) of hepatocellular carcinoma (HCC) and differentiation of HCC smaller than 2 cm from borderline lesions. METHODS: We retrospectively analyzed grayscale US images with histopathologic evidence of HCC or a precancerous lesion using ImageJ version 1.47 software (National Institutes of Health, Bethesda, MD). RESULTS: A total of 148 nodules were included (borderline lesion, n = 31; early HCC [eHCC], n = 3; well-differentiated HCC [wHCC], n = 16; moderately differentiated HCC [mHCC], n = 79; and poorly differentiated HCC [pHCC], n = 19). A multivariate analysis selected lower minimum gray values (odds ratio [OR], 0.431; P = .003) and a higher standard deviation (OR, 1.880; P = .019) as predictors of HCC smaller than 2 cm. Median (range) minimum gray values of borderline lesions, eHCC, wHCC, mHCC, and pHCC were 29 (0-103), 7 (0-47), 6 (0-60), 10 (0-53), and 2 (0-38), respectively, and gradually decreased from borderline lesions to pHCC (P < 0.001). The multivariate analysis showed a higher aspect ratio (OR, 2.170; P = .001) and lower minimum gray value (OR, 0.475; P = .043) as predictors of MVI. An anechoic area diagnosed by a subjective evaluation was correlated with the minimum gray value (P < .0001). The proportion of the anechoic area gradually increased from eHCC to pHCC (P = .031). CONCLUSIONS: In a US image analysis, HCC smaller than 2 cm had features of greater heterogeneity and a lower minimum gray value than borderline lesions. Moderately differentiated HCC was smoother than borderline lesions, and the anechoic area correlated with histologic grading. Microvascular invasion was correlated with a slender shape and a lower minimum gray value.

Knockdown of Mitogen-Activated Protein Kinase Kinase 3 Negatively Regulates Hepatitis A Virus Replication.

Zinc chloride is known to be effective in combatting hepatitis A virus (HAV) infection, and zinc ions seem to be especially involved in Toll-like receptor (TLR) signaling pathways. In the present study, we examined this involvement in human hepatoma cell lines using a human TLR signaling target RT-PCR array. We also observed that zinc chloride inhibited mitogen-activated protein kinase kinase 3 (MAP2K3) expression, which could downregulate HAV replication in human hepatocytes. It is possible that zinc chloride may inhibit HAV replication in association with its inhibition of MAP2K3. In that regard, this study set out to determine whether MAP2K3 could be considered a modulating factor in the development of the HAV pathogen-associated molecular pattern (PAMP) and its triggering of interferon-ß production. Because MAP2K3 seems to play a role in antiviral immunity against HAV infection, it is a promising target for drug development. The inhibition of MAP2K3 may also prevent HAV patients from developing a severe hepatitis A infection.

Follow-Up of Patients Who Achieved Sustained Virologic Response after Interferon-Free Treatment against Hepatitis C Virus: Focus on Older Patients.

Background and Objectives: Direct-acting antiviral agents (DAAs) have improved sustained virologic response (SVR) rates in patients with chronic hepatitis C virus (HCV) infection. Our aim was to elucidate the occurrence of hepatocellular carcinoma (HCC) and to compare the outcomes of patients aged 75 years or older (older group) with those of patients younger than 75 years (younger group) after SVR. Materials and Methods: Among 441 patients treated with interferon-free DAA combinations, a total of 409 SVR patients were analyzed. We compared the two age groups in terms of HCC incidence and mortality rates. Results: Older and younger groups consisted of 68 and 341 patients, respectively. Occurrence of HCC after SVR did not differ between the two groups of patients with a history of HCC. Occurrence of HCC after SVR was observed more in younger patients without a history of HCC (p < 0.01). Although older patients without a history of HCC had a higher mortality rate (p < 0.01), their causes of death were not associated with liver diseases. Among younger patients without a history of HCC, none died. Conclusions: After SVR, liver disease may not be a prognostic factor in older HCV patients without a history of HCC.

Molecular Mechanisms: Connections between Nonalcoholic Fatty Liver Disease, Steatohepatitis and Hepatocellular Carcinoma.

Nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), causes hepatic fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The patatin-like phospholipase-3 (PNPLA3) I148M sequence variant is one of the strongest genetic determinants of NAFLD/NASH. PNPLA3 is an independent risk factor for HCC among patients with NASH. The obesity epidemic is closely associated with the rising prevalence and severity of NAFLD/NASH. Furthermore, metabolic syndrome exacerbates the course of NAFLD/NASH. These factors are able to induce apoptosis and activate immune and inflammatory pathways, resulting in the development of hepatic fibrosis and NASH, leading to progression toward HCC. Small intestinal bacterial overgrowth (SIBO), destruction of the intestinal mucosa barrier function and a high-fat diet all seem to exacerbate the development of hepatic fibrosis and NASH, leading to HCC in patients with NAFLD/NASH. Thus, the intestinal microbiota may play a role in the development of NAFLD/NASH. In this review, we describe recent advances in our knowledge of the molecular mechanisms contributing to the development of hepatic fibrosis and HCC in patients with NAFLD/NASH.

Co-Occurrence of Hepatitis A Infection and Chronic Liver Disease.

Hepatitis A virus (HAV) infection occasionally leads to a critical condition in patients with or without chronic liver diseases. Acute-on-chronic liver disease includes acute-on-chronic liver failure (ACLF) and non-ACLF. In this review, we searched the literature concerning the association between HAV infection and chronic liver diseases in PubMed. Chronic liver diseases, such as metabolic associated fatty liver disease and alcoholic liver disease, coinfection with other viruses, and host genetic factors may be associated with severe hepatitis A. It is important to understand these conditions and mechanisms. There may be no etiological correlation between liver failure and HAV infection, but there is an association between the level of chronic liver damage and the severity of acute-on-chronic liver disease. While the application of an HAV vaccination is important for preventing HAV infection, the development of antivirals against HAV may be important for preventing the development of ACLF with HAV infection as an acute insult. The latter is all the more urgent given that the lives of patients with HAV infection and a chronic liver disease of another etiology may be at immediate risk.

Noninvasive Assessment of Liver Fibrosis: Current and Future Clinical and Molecular Perspectives.

Liver fibrosis is one of the risk factors for hepatocellular carcinoma (HCC) development. The staging of liver fibrosis can be evaluated only via a liver biopsy, which is an invasive procedure. Noninvasive methods for the diagnosis of liver fibrosis can be divided into morphological tests such as elastography and serum biochemical tests. Transient elastography is reported to have excellent performance in the diagnosis of liver fibrosis and has been accepted as a useful tool for the prediction of HCC development and other clinical outcomes. Two-dimensional shear wave elastography is a new technique and provides a real-time stiffness image. Serum fibrosis markers have been studied based on the mechanism of fibrogenesis and fibrolysis. In the healthy liver, homeostasis of the extracellular matrix is maintained directly by enzymes called matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs). MMPs and TIMPs could be useful serum biomarkers for liver fibrosis and promising candidates for the treatment of liver fibrosis. Further studies are required to establish liver fibrosis-specific markers based on further clinical and molecular research. In this review, we summarize noninvasive fibrosis tests and molecular mechanism of liver fibrosis in current daily clinical practice.

Different Mechanisms of Action of Regorafenib and Lenvatinib on Toll-Like Receptor-Signaling Pathways in Human Hepatoma Cell Lines.

Multiple kinase inhibitors are available for patients with advanced hepatocellular carcinoma (HCC). It is largely unknown whether regorafenib or lenvatinib modulates innate immunity including Toll-like receptor (TLR)-signaling pathways in HCC. We performed real-time RT-PCR to investigate 84 TLR-associated gene expression levels and compared these gene expression levels in each hepatoma cells treated with or without regorafenib or lenvatinib. In response to regorafenib, nine and 10 genes were upregulated in Huh7 and HepG2 cells, respectively, and only C-X-C motif chemokine ligand 10 was upregulated in both cell lines. A total of 14 and 12 genes were downregulated in Huh7 and HepG2 cells, respectively, and two genes (Fos proto-oncogene, AP-1 transcription factor subunit, and ubiquitin conjugating enzyme E2 N) were downregulated in both cell lines. In response to lenvatinib, four and 16 genes were upregulated in Huh7 and HepG2 cells, respectively, and two genes (interleukin 1 alpha and TLR4) were upregulated in both cells. Six and one genes were downregulated in Huh7 and HepG2, respectively, and no genes were downregulated in both cell lines. In summary, regorafenib and lenvatinib affect TLR signaling pathways in human hepatoma cell lines. Modulation of TLR signaling pathway may improve the treatment of HCC patients with refractory disease.

Liver stiffness measurements in chronic hepatitis C: Treatment evaluation and risk assessment.

BACKGROUND AND AIM: Liver stiffness (LS), measured by transient elastography, has been validated as a non-invasive surrogate for liver fibrosis. METHODS: We investigated the long-term predictive ability of LS for hepatocellular carcinoma (HCC) development and overall survival in 1146 patients with chronic hepatitis C by using LS value at enrollment. We also investigated chronological changes in LS based on antiviral therapy and its outcome in 752 patients. RESULTS: During the mean follow-up period of 6.6 years, 190 patients developed HCC. Cumulative HCC incidence rates at 5 years were clearly stratified as 1.7% in the ≤ 5 kPa, 3.3% in 5.1-10 kPa, 16.7% in 10.1-15 kPa, 24.4% in 15.1-20 kPa, 36.3% in 20.1-25 kPa, and 43.7% in > 25 kPa subgroups (P < 0.001). Overall survival was also stratified: 10-year survival rates were 99.3% in the ≤ 5 kPa, 95.4% in 5.1-10 kPa, 81.4% in 10.1-15 kPa, 79.5% in 15.1-20 kPa, 66.1% in 20.1-25 kPa, and 49.1% in > 25 kPa subgroups (P < 0.001). LS decreased at a rate of 8.1% per year in those who achieved sustained virological responses, but increased at 0.1% per year in those who could not achieve sustained virological response instead of antiviral therapy, and increased at 3.7% per year in those who did not undergo antiviral therapy. CONCLUSIONS: Liver stiffness measurements can be useful in the prediction of HCC development and overall survival and in the evaluation of chronological changes in liver fibrosis grade during and after antiviral therapy.

SOCS2 Balances Metabolic and Restorative Requirements during Liver Regeneration.

After significant injury, the liver must maintain homeostasis during the regenerative process. We hypothesized the existence of mechanisms to limit hepatocyte proliferation after injury to maintain metabolic and synthetic function. A screen for candidates revealed suppressor of cytokine signaling 2 (SOCS2), an inhibitor of growth hormone (GH) signaling, was strongly induced after partial hepatectomy. Using genetic deletion and administration of various factors we investigated the role of SOCS2 during liver regeneration. SOCS2 preserves liver function by restraining the first round of hepatocyte proliferation after partial hepatectomy by preventing increases in growth hormone receptor (GHR) via ubiquitination, suppressing GH pathway activity. At later times, SOCS2 enhances hepatocyte proliferation by modulating a decrease in serum insulin-like growth factor 1 (IGF-1) that allows GH release from the pituitary. SOCS2, therefore, plays a dual role in modulating the rate of hepatocyte proliferation. In particular, this is the first demonstration of an endogenous mechanism to limit hepatocyte proliferation after injury.

Dicer-dependent production of microRNA221 in hepatocytes inhibits p27 and is required for liver regeneration in mice.

Dicer processes microRNAs (miRs) into active forms in a wide variety of tissues, including the liver. To determine the role of Dicer in liver regeneration, we performed a series of in vivo and in vitro studies in a murine 2/3 hepatectomy model. Dicer was downregulated after 2/3 hepatectomy, and loss of Dicer inhibited liver regeneration associated with decreased cyclin A2 and miR-221, as well as increased levels of the cell cycle inhibitor p27. In vitro, miR-221 inhibited p27 production in primary hepatocytes and increased hepatocyte proliferation. Specific reconstitution of miR-221 in hepatocyte-specific Dicer-null mice inhibited p27 and restored liver regeneration. In wild type mice, targeted inhibition of miR-221 using a cholesterol-conjugated miR-221 inhibited hepatocyte proliferation after 2/3 hepatectomy. These results identify Dicer production of miR-221 as an essential component of a miRNA-dependent mechanism for suppression of p27 that controls the rate of hepatocyte proliferation after partial hepatectomy.NEW & NOTEWORTHY Our findings demonstrate a direct role for microRNAs in controlling the rate of liver regeneration after injury. By deleting Dicer, an enzyme responsible for processing microRNAs into mature forms, we determined miR-221 is a critical microRNA in the physiological process of restoration of liver mass after injury. miR-221 suppresses p27, releasing its inhibitory effects on hepatocyte proliferation. Pharmaceuticals based on miR-221 may be useful to modulate hepatocyte proliferation in the setting of liver injury.

Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo.

Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue-DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications. A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis in the seemingly homogeneous enterocyte population. Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival. Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues.

Tumor markers are more useful in patients undergoing surveillance for hepatocellular carcinoma with unreliable results by ultrasonography.

AIM: The objectives of this study was to evaluate the utility of tumor markers in hepatocellular carcinoma (HCC) surveillance based on the reliability of ultrasonography. METHODS: We analyzed 313 patients with HCC detected through a surveillance program using ultrasonography combined with three tumor markers from February 2000 to December 2010. The patients were categorized into two groups based on the triggering event: the US group (n = 281) in which a tumor was first detected using ultrasonography and the TM group (n = 32) in which elevated tumor markers led to the diagnosis of a tumor that was undetected using ultrasonography. The reliability of ultrasonography was scored on a 4-point scale based on three items (coarseness of liver parenchyma, patient obesity and liver atrophy). Additionally, patient survival was assessed using the Kaplan-Meier method and log-rank test. RESULTS: The median tumor size was 20 mm (interquartile range, 15-24). The reliability of ultrasonography was evaluated as good in 208 (66.5%), satisfactory in 80 (8.0%), poor in 21 (6.7%) and unsatisfactory in four (1.2%) patients. The proportion of patients in the TM group increased significantly according to the score, from 7.2% to 25.0% (P = 0.01). The survival rates of patients at 3 and 5 years were 83.7% and 57.2% in the US group, and 79.3% and 59.4% in the TM group, respectively (P = 0.98). CONCLUSION: Tumor markers may play a diagnostic role in patients with unreliable ultrasonography results. The survival of patients diagnosed by elevated tumor markers was not significantly different from those diagnosed by ultrasonography.

Specific activin receptor-like kinase 3 inhibitors enhance liver regeneration.

Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor-like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease.

Sublethal heat treatment promotes epithelial-mesenchymal transition and enhances the malignant potential of hepatocellular carcinoma.

UNLABELLED: Radiofrequency ablation (RFA) is a potentially curative therapy for hepatocellular carcinoma (HCC). However, incomplete RFA can induce accelerated invasive growth at the periphery. The mechanisms underlying the RFA-induced tumor promotion remain largely unexplored. Three human HCC cell lines were exposed to 45°C-55°C for 10 minutes, simulating the marginal zone of RFA treatment. At 5-12 days post-treatment cell proliferation, parameters of epithelial-mesenchymal transition (EMT), and activation of mitogen-activated protein kinases were analyzed. Livers from patients with viral hepatitis without and with HCC (n = 114) were examined to confirm the relevance of altered kinase patterns. In vivo tumorigenic potential of heat-treated versus untreated HCC cells was studied in nude mice. Heating to 55°C killed all HCC cells, whereas 65%-85% of cells survived 48°C-50°C, developing spindle-like morphology and expressing CD133, cytokeratin (CK)7, CK19, procollagen-α1(I), and Snail at day 5 after heat exposure, which returned to baseline at day 12. Heat-exposed HCC cells showed enhanced proliferation and prominent activation of p46-Shc (Src homology and collagen) and downstream extracellular signal-related kinase (Erk)1/2. In patients, Shc expression correlated with malignant potential and overall survival. Blocking Erk1/2 reduced proliferation and EMT-like changes of heat-treated HCC cells. Implantation of heat-exposed HEPG2 cells into nude mice induced significantly larger, more aggressive tumors than untreated cells. CONCLUSIONS: Sublethal heat treatment skews HCC cells toward EMT and transforms them to a progenitor-like, highly proliferative cellular phenotype in vitro and in vivo, which is driven significantly by p46Shc-Erk1/2. Suboptimal RFA accelerates HCC growth and spread by transiently inducing an EMT-like, more aggressive cellular phenotype.