Primary Hepatoma Research Articles

Fangchinoline suppresses hepatocellular carcinoma by regulating ROS accumulation via the TRIM7/Nrf2 signaling pathway

BackgroundDysregulation of redox homeostasis is associated with developing hepatocellular carcinoma (HCC). Oxidative stress (OS) is distinguished by the accumulation of ROS, which plays a variety of roles in cancer pathology. Fangchinoline (FAN), a bis-benzylisoquinoline alkaloid, has anti-cancer pharmacological activity. However, the regulatory mechanism of FAN on OS and whether it can inhibit HCC by mediating OS are still unclear. Hypothesis/PurposeThis paper aims to explore the effectiveness of FAN in preventing HCC via regulating OS and identify the underlying molecular mechanisms. MethodsWe used the primary HCC mouse model and hepatoma cell line to explore the suppressive effect of FAN on hepatocarcinogenesis. To study the role of ROS in the anti-hepatocarcinoma effect of FAN in cell model and mouse model. The mechanism of FAN-induced nuclear factor erythroid 2-related factor 2 (Nrf2) pathway activation was studied through various techniques, including generation of Nrf2 and tripartite motif containing 7 (TRIM7) gene overexpressing or knockdown cell model, co-immunoprecipitation, immunohistochemistry and subcutaneous tumor xenograft models constructed by the stable TRIM7-overexpression HLE cells, etc. ResultsWe showed that FAN significantly inhibited cell proliferation and hepatocarcinogenesis in HCC cells and primary HCC mouse model. The FAN-induced mitochondrial dysfunction promoted ROS accumulation, and using N-Acetylcysteine to clear ROS reversed the anti-HCC effects of FAN. We observed that FAN is capable of activating the Nrf2 pathway. This effect was thought to be due to the fact that, in response to the FAN-induced OS, the cancer cells created a feedback loop to stable Nrf2 via depressing the K48-linkage ubiquitination of it, which was caused by reduced binding of kelch-like ECH-associated protein 1 (Keap1) and Nrf2 and elevated TRIM7 expression. Indeed, overexpression of TRIM7 suppressed the anti-hepatocarcinoma effect of FAN. ConclusionThe study determines the anti-liver cancer effect of FAN and first describes the positive regulatory effect of TRIM7 on Nrf2 signaling. We reveal that TRIM7/Nrf2 signaling served as a target of FAN-induced ROS accumulation in HCC, which helps to clarify the mechanism of action of FAN against HCC and provides a theoretical basis for FAN as an anti-cancer drug.

Hepatic and perihepatic PEComas: A study describing a series of five rare cases.

Perivascular epithelioid cell tumors (PEComas) encompass a group of rare mesenchymal neoplasms, with dual melanocytic and muscular differentiation. Hepatic PEComas are rare and difficult to diagnose, and their behavior is still unclear. Herein, we report a total of five cases of hepatic and perihepatic PEComas over a period of the last 5 years from our and collaborating center's archive. A detailed histological evaluation was done. A comprehensive panel of immunohistochemical stains was used and fluorescence in-situ hybridization analysis was performed for the TFE3 gene using break-apart probes. All these patients were women, with an average age of presentation of 44 years. The lesions were in the right hepatic lobe: three cases, the left hepatic lobe: one case, and gastrohepatic ligament: one case. The preoperative clinicoradiological diagnoses were hepatocellular carcinoma (HCC), focal nodular hyperplasia, hemangioma, metastasis, and gastrointestinal stromal tumor, respectively. Surgical excision was performed in four cases with no further adjuvant therapy. Histopathological examination and subsequent immunophenotyping revealed a diagnosis of PEComa. Fluorescence in-situ hybridization analysis was performed for TFE3 gene rearrangement in four cases. This series highlights the fact that accurate histological diagnosis of hepatic or perihepatic PEComas is important to prevent unnecessary aggressive treatment, unlike primary hepatocellular carcinomas or hepatoid/epithelioid metastatic tumors.

Qualitative and quantitative concentration-response modelling of gene co-expression networks to unlock hepatotoxic mechanisms for next generation chemical safety assessment.

Next generation risk assessment of chemicals revolves around the use of mechanistic information without animal experimentation. In this regard, toxicogenomics has proven to be a useful tool to elucidate the underlying mechanisms of adverse effects of xenobiotics. In the present study, two widely used human in vitro hepatocyte culture systems, namely primary human hepatocytes (PHH) and human hepatoma HepaRG cells, were exposed to liver toxicants known to induce liver cholestasis, steatosis or necrosis. Benchmark concentration-response modelling was applied to transcriptomics gene co-expression networks (modules) to derive benchmark concentrations (BMCs) and to gain mechanistic insight into the hepatotoxic effects. BMCs derived by concentration-response modelling of gene co-expression modules recapitulated concentration-response modelling of individual genes. Although PHH and HepaRG cells showed overlap in deregulated genes and modules by the liver toxicants, PHH demonstrated a higher responsiveness, based on the lower BMCs of co-regulated gene modules. Such BMCs can be used as transcriptomics point of departure (tPOD) for assessing module-associated cellular (stress) pathways/processes. This approach identified clear tPODs of around maximum systemic concentration (Cmax) levels for the tested drugs, while for cosmetics ingredients the BMCs were 10-100-fold higher than the estimated plasma concentrations. This approach could serve next generation risk assessment practice to identify early responsive modules at low BMCs, that could be linked to key events in liver adverse outcome pathways. In turn, this can assist in delineating potential hazards of new test chemicals using in vitro systems and used in a risk assessment when BMCs are paired with chemical exposure assessment.

Human liver organoid models for assessment of drug toxicity at the preclinical stage.

The hepatotoxicity of drugs is one of the leading causes of drug withdrawal from the pharmaceutical market and high drug attrition rates. Currently, the commonly used hepatocyte models include conventional hepatic cell lines and animal models, which cannot mimic human drug-induced liver injury (DILI) due to poorly defined dose-response relationships and/or lack of human-specific mechanisms of toxicity. In comparison to 2D culture systems from different cell sources such as primary human hepatocytes and hepatomas,, 3D organoids derived from an inducible pluripotent stem cell (iPSC) or adult stem cells are promising accurate models to mimic organ behavior with a higher level of complexity and functionality owing to their ability to self-renewal. Meanwhile, the heterogeneous cell composition of the organoids enables metabolic and functional zonation of hepatic lobule important in drug detoxification and has the ability to mimic idiosyncratic DILI as well. Organoids having higher drug-metabolizing enzyme capacities can culture long-term and be combined with microfluidic-based technologies such as organ-on-chips for a more precise representation of human susceptibility to drug response in a high-throughput manner. However, there are numerous limitations to be considered about this technology, such as enough maturation, differences between protocols and high cost. Herein, we first reviewed the current preclinical DILI assessment tools and looked at the organoid technology with respect to in vitro detoxification capacities. Then we discussed the clinically applicable DILI assessment markers and the importance of liver zonation in the next generation organoid-based DILI models.

The efficacy of radical hepatectomy versus stereotactic body radiotherapy for primary hepatocellular carcinoma: A propensity score matching analysis.

4130 Background: Recent international guidelines have included stereotactic body radiotherapy (SBRT) which outstanding tumor control as a treatment option for hepatocellular carcinoma (HCC) in several settings. However, data evaluating surgery and SBRT are limited, and results published were controversial. The retrospective study aims to compare the efficacy of hepatectomy and SBRT for HCC. Methods: The study population started with patients in databases of primary HCC, treated with curative hepatectomy or SBRT, regardless of tumor number, tumor size, cirrhosis and portal vein thrombosis. Patients with prior cancer treatment other than transarterial chemoembolization were excluded. In order to reduce the potential confounding effect of treatment and selection bias, 3-to-1 propensity score models were constructed based on each patient’s estimated propensity score. The variables significant for overall survival were considered for the match, including age, sex, Child-Pugh score, ECOG performance status score, maximum diameter, BCLC stage, prothrombin time, total bilirubin, albumin, and white blood cell. Overall survival and progression-free survival were compared. Results: A total of 174 patients, 154 in hepatectomy group and 20 in SBRT group, were analyzed. 125 men and 29 women, mean 56.2 years of age, in hepatectomy group. 12 men and 8 women, mean 70.5 years of age, in SBRT group. ECOG was a significant favorable factor for overall survival. Maximum diameter, portal vein embolization, aspartate transferase and albumin was significant favorable factors for disease-free survival of patients with primary hepatocellular carcinomas. Before PSM, patients in the hepatectomy group tended to be younger, had better performance status, less cirrhosis, less tumor number and had better hepatic function than those in the SBRT group ( P < .05). The 3-year overall survival rate and median progression-free survival time for the surgical resection and SBRT groups were 77.9% vs 35.4 (P = .003) and 30 vs 20 month (P = .486), respectively. After PSM, 80 patients, 60 in hepatectomy group and 20 in SBRT group, were analyzed. The 3-year overall survival rate and median progression-free survival time for the surgical resection and SBRT groups were 71.9% vs 56.6 (P = .92) and 17 vs 26 month (P = .656), respectively. The patients of hepatectomy group had less cirrhosis, better liver function and less tumor number than those in 20 with SBRT. Conclusions: For primary HCC, regardless of tumor size and tumor number, stereotactic body radiotherapy can provide similar result on overall survival and tumor control compared to hepatectomy. Stereotactic body radiotherapy can also be less invasive and provide less inpatient time. So stereotactic body radiotherapy can be a effective treatment for patients with primary HCC.

Bioimaging of glutathione variation for early diagnosis of hepatocellular carcinoma using a liver-targeting ratiometric near-infrared fluorescent probe.

Reliable biomarkers are crucial for early diagnosis of diseases and precise therapy. Biological thiols (represented by glutathione, GSH) play vital roles in the antioxidant defense system for maintaining intracellular redox homeostasis in organisms. However, the aberrant variation in the cellular concentration of GSH correlates with diverse diseases including cancer. Here, a ratiometric near-infrared fluorescent probe CyO-Disu is constructed for the specific sensing of GSH variation in live cells and mice models of hepatic carcinoma (HCC). CyO-Disu features three key elements, a response moiety of bis(2-hydroxyethyl) disulfide, a near-infrared fluorescence signal transducer of heptamethine ketone cyanine, and a targeting moiety of D-galactose. By virtue of its liver-targeting capability, CyO-Disu was utilized for evaluating GSH fluctuations in primary and metastatic hepatoma living cells. To evaluate the efficacy of CyO-Disuin vivo, orthotopic HCC and pulmonary metastatic hepatoma mice models were employed for GSH imaging using two-dimensional and three-dimensional fluorescence molecular tomographic imaging systems. The bioimaging results offered direct evidence that GSH displayed varied concentrations during the progression of HCC. Therefore, the as-synthesized probe CyO-Disu could serve as a potential powerful tool for the early diagnosis and precise treatment of HCC using GSH as a reliable biomarker.

A Comparison of Primary Human Hepatocytes and Hepatoma Cell Lines to Model the Effects of Fatty Acids, Fructose and Glucose on Liver Cell Lipid Accumulation.

Non-alcoholic fatty liver disease (NAFLD) begins with lipid accumulation within hepatocytes, but the relative contributions of different macronutrients is still unclear. We investigated the impact of fatty acids, glucose and fructose on lipid accumulation in primary human hepatocytes (PHH) and three different cell lines: HepG2 (human hepatoblastoma-derived cell line), Huh7 (human hepatocellular carcinoma cell line) and McA-RH7777 (McA, rat hepatocellular carcinoma cell line). Cells were treated for 48 h with fatty acids (0 or 200 μM), glucose (5 mM or 11 mM) and fructose (0 mM, 2 mM or 8 mM). Lipid accumulation was measured via Nile Red staining. All cell types accumulated lipid in response to fatty acids (p < 0.001). PHH and McA, but not HepG2 or Huh7 cells, accumulated more lipid with 11 mM glucose plus fatty acids (p = 0.004, fatty acid × glucose interaction, for both), but only PHH increased lipid accumulation in response to fructose (p < 0.001). Considerable variation was observed between PHH cells from different individuals. Lipid accumulation in PHH was increased by insulin (p = 0.003) with inter-individual variability. Similarly, insulin increased lipid accumulation in both HepG2 and McA cells, with a bigger response in McA in the presence of fatty acids (p < 0.001 for fatty acid × insulin). McA were more insulin sensitive than either HepG2 or Huh7 cells in terms of AKT phosphorylation (p < 0.001 insulin × cell type interaction). Hence, glucose and fructose can contribute to the accumulation of lipid in PHH with considerable inter-individual variation, but hepatoma cell lines are not good models of PHH.

Mechanism of PDZK1 in Hepatocellular Carcinoma Complicated with Hyperuricemia.

Hepatocellular carcinoma (HCC) is a kind of primary liver cancer that accounts for more than 90% of primary hepatocellular carcinomas. Hyperuricemia is closely related to the development, recurrence, metastasis, and prognosis of cancer. Previous studies have proved that the serum uric acid level can increase the incidence rate and mortality of malignant tumors. However, the specific pathogenesis remains unstudied. RT-qPCR analysis showed that the mRNA expression of PDZK1 and ABCG2 increased significantly after HCC cells were exposed to different concentrations of soluble uric acid (2.5, 5, 10, 20 mg/dl) for 24 hours. Then, in HCC shRNAs, PDZK1, or over expression PDZK1 were used. CCK8, wound healing, and Transwell assay showed that PDZK1 regulates cell proliferation, invasion, and migration. Flow cytometry results revealed that PDZK1 affects cell apoptosis. Western blot results show that PDZK1 affects the STAT3/C-myc pathway. Then, in vivo tumorigenesis, allopurinol maybe an effective drug to advance: the prognosis of HCC. In our study, RT-qPCR analysis showed that the mRNA expression of PDZK1 and ABCG2 increased significantly after different concentrations of soluble uric acid in HCC. Then, PDZK1 affects the proliferation, migration, and apoptosis of HCC through the STAT3/C-myc pathway. Hyperuricemia response affects the expression of PDZK1; PDZK1 affects the proliferation, migration, and apoptosis through the STAT3/C-myc pathway in hepatocellular carcinoma. It is suggested that PDZK1 maybe closely related to the occurrence, development, and prognosis of HCC and allopurinol maybe have potential anticancer effects.

Vitamin D Receptor Activation Targets ROS-Mediated Crosstalk Between Autophagy and Apoptosis in Hepatocytes in Cholestasic Mice

Observational epidemiologic studies have associated vitamin D deficiency with cholestasis. We reported previously that activation of the vitamin D/vitamin D receptor (VDR) axis in cholangiocytes mitigates cholestatic liver injury by remodeling the damaged bile duct. However, the function of VDR in hepatocytes during cholestasis remains unclear. Paricalcitol (VDR agonist, 200 ng/kg) was injected intraperitoneally into bile duct-ligated mice every other day for 5 days. Primary hepatocytes and HepG2 hepatoma cells were transfected with Vdr short hairpin RNA, control short hairpin RNA, Vdr plasmid, control vector, Atg5 small interfering RNA (siRNA), and control siRNA. Liver histology, cell proliferation, and autophagy were evaluated. Treatment with the VDR agonist paricalcitol improved liver injury in bile duct-ligated mice by up-regulating VDR expression in hepatocytes, which in turn reduced hepatocyte apoptosis by inhibiting reactive oxygen species (ROS) generation via suppressing the Ras-related C3 botulinum toxin substrate 1/reduced nicotinamide adenine dinucleotide phosphate oxidase 1 pathway. Mechanistically, upon exposure to an ROS-inducing compound, Vdr siRNA contributed to apoptosis, whereas the Vdr overexpression caused resistance to apoptosis. Interestingly, up-regulated VDR expression also increased the generation of autophagosomes and macroautophagic/autophagic flux, which was the underlying mechanism for reduced apoptosis following VDR activation. Autophagy depletion impaired the positive effects of VDR overexpression, whereas autophagy induction was synergystic with VDR overexpression. Importantly, up-regulation of VDR promoted autophagy activation by suppressing the activation of the extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (p38MAPK) pathway. Thus, a p38MAPK inhibitor abrogated the Vdr siRNA-induced decrease in autophagy and the Vdr siRNA-induced increase in apoptosis. In contrast, a Mitogen-activated protein kinase kinase (MEK)/ERK activator prevented the enhancement of autophagy and decreased apoptosis following Vdr overexpression. Moreover, the ROS inhibitor N-acetylcystein (NAC) blocked Vdr siRNA-enhanced activation of the ERK/p38MAPK pathway. VDR activation mitigated liver cholestatic injury by reducing autophagy-dependent hepatocyte apoptosis and suppressing the activation of the ROS-dependent ERK/p38MAPK pathway. Thus, VDR activation may be a potential target for the treatment of cholestatic liver disease.

Hepatitis B virus promotes hepatocellular carcinoma development by activating GP73 to repress the innate immune response

BackgroundHepatitis B virus (HBV) causes acute and chronic infection in the clinic. Hepatocellular carcinoma (HCC) is closely linked to HBV infection. Serum Golgi protein 73 (GP73) increases during HBV infection. However, the role of GP73 during HBV infection and the occurrence of HBV-related HCC is still poorly understood.MethodsThe underlying role of HBV-induced GP73 in regulating HCC development was investigated in this study. GP73 expression in HBV-related clinical HCC tissues and in HBV-infected hepatoma cells and primary human hepatocytes was evaluated by immunohistochemistry, ELISAs, Western blotting and quantitative real-time PCR (qRT-PCR) analysis. Tumorigenicity of GP73 overexpressed cells was detected by flow cytometry, qRT-PCR, xenograft nude mouse analyses and sphere formation assays. The effects of GP73 and HBV infection on host innate immune responses in hepatocytes were further investigated by Western blotting and qRT-PCR analysis.ResultsInitially, we confirmed that HBV-positive HCC tissues had significantly higher expression of GP73. Ectopic expression of the HBV gene could induce GP73 expression in primary human hepatocytes and hepatoma cells in vitro. In addition, we discovered that GP73 promotes HCC in both normal liver cells and hepatoma cells. We also found that ectopic expression of HBV genes increases GP73 expression, suppressing the host's innate immune responses in hepatocytes.ConclusionsOur results demonstrate that HBV facilitates HCC development by activating GP73 to repress the host's innate immune response. This study adds to our understanding of the pathogenesis of HBV infection-induced HCC. The findings also provide preclinical support for GP73 as a potential HCC prevention or treatment target.

In Vitro Models for Studying Chronic Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a major clinical problem in terms of patient morbidity and mortality, cost to healthcare systems and failure of the development of new drugs. The need for consistent safety strategies capable of identifying a potential toxicity risk early in the drug discovery pipeline is key. Human DILI is poorly predicted in animals, probably due to the well-known interspecies differences in drug metabolism, pharmacokinetics, and toxicity targets. For this reason, distinct cellular models from primary human hepatocytes or hepatoma cell lines cultured as 2D monolayers to emerging 3D culture systems or the use of multi-cellular systems have been proposed for hepatotoxicity studies. In order to mimic long-term hepatotoxicity in vitro, cell models, which maintain hepatic phenotype for a suitably long period, should be used. On the other hand, repeated-dose administration is a more relevant scenario for therapeutics, providing information not only about toxicity, but also about cumulative effects and/or delayed responses. In this review, we evaluate the existing cell models for DILI prediction focusing on chronic hepatotoxicity, highlighting how better characterization and mechanistic studies could lead to advance DILI prediction.

An investigation of bacteriocin nisin anti-cancer effects and FZD7 protein interactions in liver cancer cells

The bacteriocin, nisin, produced by Lactococcus and Streptococcus species during fermentation, is widely used for bio preservatives in a wide variety of foods. Liver cancer has a high mortality rate and is the fourth leading cause of cancer-related deaths worldwide. Recently, researchers have shown the anti-cancer effects of nisin through in vitro and in vivo studies. This study aimed to investigate the effect of nisin on liver cancer cell lines, which represented two subgroups of the disease model. Nisin exhibited significant growth inhibition and apoptosis in both cell lines, HuH-7, and SNU182. Drug resistance is the main problem in liver cancer and the epithelial-to-mesenchymal transition has a role in the development of drug resistance in hepatocellular carcinoma. The expression of EMT transcription factors ZEB1, SNAI1, and TWIST1 were analyzed depending on nisin treatment, TWIST1 expression was down-regulated after nisin treatment compared to the untreated SNU182 and HuH-7 cell lines. Besides, due to the reported correlation between the overexpression of Frizzled (FZD) proteins, specifically FZD7, in primary hepatocellular carcinomas (HCCs), molecular docking was assessed for Nisin A in the binding site of FZD7. Results confirmed that Nisin A was able to form important hydrogen bonding with key residues. This research not only determined the role of nisin in different liver cancer cell models but it also provided the first result of FZD7 and nisin interaction.

Critical Investigation of the Usability of Hepatoma Cell Lines HepG2 and Huh7 as Models for the Metabolic Representation of Resectable Hepatocellular Carcinoma

Simple SummaryEnergy metabolism plays a central role in the liver. Therefore, metabolic alterations in liver cancer are fundamental for the development of diagnostic screening and therapeutic intervention. The aim of our experimental study was to investigate the extent to which commonly used hepatoma cell lines (HCLs) sufficiently represent tumor cells from hepatocellular carcinoma (HCC) from a metabolic point of view. To that end, we successfully established a method for the isolation of primary human hepatoma cells (PHCs). We present the unsurprising finding that cell lines are a poor substitute for primary cells. Surprisingly, our transcript data revealed that malign metabolic adaptions had already occurred in non-tumor-bearing liver tissue of HCC patients. In PHCs, we observed that downregulated metabolic key players showed a correlation with malign transformation and were predominantly pronounced in multilocular HCC. These findings should be taken into account for the future optimization of HCC models for in vitro research.Metabolic alterations in hepatocellular carcinoma (HCC) are fundamental for the development of diagnostic screening and therapeutic intervention since energy metabolism plays a central role in differentiated hepatocytes. In HCC research, hepatoma cell lines (HCLs) like HepG2 and Huh7 cells are still the gold standard. In this study, we characterized the metabolic profiles of primary human hepatoma cells (PHCs), HCLs and primary human hepatocytes (PHHs) to determine their differentiation states. PHCs and PHHs (HCC-PHHs) were isolated from surgical specimens of HCC patients and their energy metabolism was compared to PHHs from non-HCC patients and the HepG2 and Huh7 cells at different levels (transcript, protein, function). Our analyses showed successful isolation of PHCs with a purity of 50–73% (CK18+). The transcript data revealed that changes in mRNA expression levels had already occurred in HCC-PHHs. While many genes were overexpressed in PHCs and HCC-PHHs, the changes were mostly not translated to the protein level. Downregulated metabolic key players of PHCs revealed a correlation with malign transformation and were predominantly pronounced in multilocular HCC. Therefore, HCLs failed to reflect these expression patterns of PHCs at the transcript and protein levels. The metabolic characteristics of PHCs are closer to those of HCC-PHHs than to HCLs. This should be taken into account for future optimized tumor metabolism research.

Percutaneous ultrasound-guided microwave ablation for treatment of hepatocellular carcinomas in dogs: four cases (2019-2020).

To describe ultrasound-guided microwave ablation as a minimally invasive treatment option for primary or metastatic hepatocellular carcinomas. Four client-owned dogs underwent percutaneous ultrasound-guided microwave ablation of three primary and one metastatic hepatocellular carcinoma, diagnosed on cytology/histopathology. In each case, multiple ultrasound-guided thermal lesions were created in a 10 to 40 minutes ablation at 30 to 35 W. Real-time monitoring was performed using 10MHz diagnostic ultrasound transducers. The procedures were performed without complication and CT scans or abdominal ultrasounds were repeated immediately after. Patients were discharged on oral analgesia on the same day or 24 hours later. Hepatocellular carcinomas were successfully ablated, which in some cases resulted in an improvement in clinical signs and laboratory values. The patients were followed up for a time ranging between 39 and 649 days and no evidence of disease progression was found. Three out of four patients are still alive at the time of writing. In these four patients, minimally invasive ultrasound-guided microwave ablation was feasible and resulted in no immediate complications. Regular imaging follow-up is recommended after the procedure and further studies on microwave ablation are warranted to establish its effectiveness in dogs with hepatocellular carcinomas.

Hepatocellular carcinomas in captive prosimians.

We performed a retrospective examination of spontaneous hepatocellular carcinomas (HCCs) (primary and metastatic tumors) in 14 captive prosimians brought to the Veterinary Medical Diagnostic Laboratory in North Carolina State University over a period of 11 years (2003 to 2014) to characterize the tumors. These animals are endangered primates; a better understanding of the main fatal neoplasms is crucial. In addition to the histologic evaluation, an immunohistochemical study was also performed, using a hepatocyte marker (hepatocyte paraffin 1 [HepPar-1]) and 2 cholangiocyte markers (keratin 7 [K7] and keratin 19 [K19]), in an attempt to identify a specific profile for HCCs with metastatic behavior. Six of the 14 HCCs had pulmonary metastases. The most frequent histopathological findings were a trabecular pattern (14/14, 100%), presence of multinucleated cells (12/14, 85.7%), and foci of extramedullary hematopoiesis (9/14, 64.3%). The mitotic count was significantly higher in the metastatic HCCs (P < .05). HepPar-1 was detected in all primary and metastatic HCCs, with a strong intensity of staining. Labeling for K7 and K19 was positive in 12 HCCs (85.7%) and 1 HCC (7.1%), respectively. Contrary to the less aggressive HCCs, most of the metastatic HCCs (5/6) expressed K7 in more than 15% of cells. The percentage of K7-positive neoplastic hepatocytes was significantly higher in metastatic HCCs. This study suggests that K7 might be a prognostically relevant marker in HCCs of captive prosimians.

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Hepatic CYP3A4 Enzyme Compensatively Maintains Endogenous Geranylgeranoic Acid Levels in MAOB-Knockout Human Hepatoma Cells.

Geranylgeranoic acid (GGA), developed as a preventive agent against second primary hepatoma, has been reported to be biosynthesized via the mevalonate pathway in human hepatoma-derived cells. Recently, we found that monoamine oxidase B (MAOB) catalyzed the oxidation of geranylgeraniol (GGOH) to produce geranylgeranial (GGal), a direct precursor of endogenous GGA in hepatoma cells, using tranylcypromine, an inhibitor of MAOs, and knockdown by MAOB siRNA. However, endogenous GGA level was unexpectedly unchanged in MAOB-knockout (KO) cells established using the CRISPR-Cas9 system, suggesting that some other latent metabolic pathways maintain endogenous GGA levels in the MAOB-KO cells. Here, we investigated the putative latent enzymes that oxidize GGOH in Hep3B/MAOB-KO cells. First, the broad-specific cytochrome P450 enzyme inhibitors decreased the amount of endogenous GGA in Hep3B/MAOB-KO cells in a dose-dependent manner. Second, among the eight members of cytochrome P450 superfamily that have been suggested to be involved in the oxidation of isoprenols and/or retinol in previous studies, only the CYP3A4 gene significantly upregulated its cellular mRNA level in Hep3B/MAOB-KO cells. Third, a commercially available recombinant human CYP3A4 enzyme was able to oxidize GGOH to GGal, and fourth, the knockdown of CYP3A4 by siRNA significantly reduced the amount of endogenous GGA in Hep3B/MAOB-KO cells. These results indicate that CYP3A4 can act as an alternative oxidase for GGOH when hepatic MAOB is deleted in the human hepatoma-derived cell line Hep3B, and that endogenous GGA levels are maintained by a multitude of enzymes.

Hepatic Leptin Signaling Improves Hyperglycemia by Stimulating MAPK Phosphatase-3 Protein Degradation via STAT3.

Background & AimsObesity-related hyperglycemia, with hepatic insulin resistance, has become an epidemic disease. Central neural leptin signaling was reported to improve hyperglycemia. The aim of this study was to investigate the effect of hepatic leptin signaling on controlling hyperglycemia.MethodsFirst, the effect of leptin signaling on gluconeogenesis was investigated in primary mouse hepatocytes and hepatoma cells. Second, glucose tolerance, insulin tolerance, blood glucose levels, and hepatic gluconeogenic gene expression were analyzed in obese mice overexpressing hepatic OBRb. Third, expression of mitogen-activated protein kinase phosphatase (MKP)-3, phosphorylation level of signal transducer and activator of transcription (STAT) 3, and extracellular regulated protein kinase (ERK) were analyzed in hepatocytes and mouse liver. Fourth, the role of MKP-3 in hepatic leptin signaling regulating gluconeogenesis was analyzed. Lastly, the role of ERK and STAT3 in the regulation of MKP-3 protein by leptin signaling was analyzed.ResultsActivation of hepatic leptin signaling suppressed gluconeogenesis in both hepatocytes and obese mouse liver, and improved hyperglycemia, insulin tolerance, and glucose tolerance in obese mice. The protein level of MKP-3, which can promote gluconeogenesis, was decreased by leptin signaling in both hepatocytes and mouse liver. Mkp-3 deficiency abolished the effect of hepatic leptin signaling on suppressing gluconeogenesis in hepatocytes. STAT3 decreased the MKP-3 protein level, while inactivation of STAT3 abolished the effect of leptin signaling on reducing the MKP-3 protein level in hepatocytes. Moreover, STAT3 could combine with MKP-3 and phospho-ERK1/2, which induced the degradation of MKP-3, and leptin signaling enhanced the combination.ConclusionsHepatic leptin signaling could suppress gluconeogenesis at least partially by decreasing the MKP-3 protein level via STAT3-enhanced MKP-3 and ERK1/2 combination.

MiR1908-5p regulates energy homeostasis in hepatocyte models

We previously identified genomic variants that are quantitative trait loci for circulating miR-1908-5p and then showed this microRNA to causally associate with plasma levels of LDL-C, fasting blood glucose and HbA1c. The link to LDL-C was subsequently validated and clarified by the identification of a miR1908-5p-TGFB-LDLR regulatory axis. Here, we continue our investigations on miR1908-5p function by leveraging human primary hepatocytes and HuH-7 hepatoma models. Expression of miR1908-5p was shown to be sensitive to glucose and agents affecting glucose metabolism. Transcriptome-wide changes in primary hepatocytes and HuH-7 cells treated with a miR1908-5p mimic were investigated by enrichment approaches to identify targeted transcripts and cognate pathways. Significant pathways included autophagy and increased mitochondrial function. Reduced activation and/or levels of several key energy and metabolic regulators (AKT, mTOR, ME1, G6PD, AMPK and LKB) were subsequently confirmed in mimic treated HuH-7 cells. These effects were associated with reduced NADPH to NADP+ ratio in HuH-7 cells. LKB1 was validated as a direct target of miR1908-5p, the reintroduction of which was however insufficient to compensate for the impact of the miR1908-5p mimic on AMPK and ACC1. These findings implicate miR1908-5p in metabolic and energy regulation in hepatocyte models via multiple, independent, pathways.

A rapid increase in lysophospholipids after geranylgeranoic acid treatment in human hepatoma-derived HuH-7 cells revealed by metabolomics analysis

Geranylgeranoic acid (GGA) was developed as a preventative agent against second primary hepatoma, and was reported to induce cell death in human hepatoma cells via Toll-like receptor 4 (TLR4)-mediated pyroptosis. We recently reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived HuH-7 cells and that endogenous GGA is found in most rat organs including the liver. An unbiased metabolomics analysis of ice-cold 50% acetonitrile extracts from control and GGA-treated cells was performed in this study to characterize the intracellular metabolic changes in GGA-induced pyroptosis and to analyze their relationship with the mechanism of GGA-induced cell death. The total positive ion chromatograms of the cellular extracts in ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were apparently unchanged after GGA treatment, but an orthogonal partial least squares-discriminant analysis score plot clearly discriminated the intracellular metabolite profiles of GGA-treated cells from that of control cells. S-plot analysis revealed 15 potential biomarkers up-regulated by 24-h GGA treatment according to their variable importance in the projection value of more than 1, and the subsequent metabolomics analysis identified nine of these metabolites as a group of lysophospholipids containing lysophosphatidylcholine with C16:0, C20:4, or C20:3 fatty acids. The possible roles of these lysophospholipids in GGA-induced pyroptosis are discussed.