Cultured Hepatoma Cells Research Articles

In Vitro Simulated Ketogenic Diet Inhibits the Proliferation and Migration of Liver Cancer Cells by Reducing Insulin Production and Down-regulating FOXC2 Expression.

Ketogenic diet (KD) may benefit patients with liver cancer, but the underlying mechanism of its anti-cancer effect remains an open issue. This work aimed to explore the influence of simulated KD on the proliferation and migration of cultured hepatoma cells. The low-glucose medium supplemented with β-hydroxybutyrate (BHB-Glow) was utilized to simulate clinical KD treatment. Western blot was utilized for detecting the expression of glycolysis-related proteins, Seahorse XF96 for oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), and ELISA for insulin content. Expression of FOXC2 in liver cancer cells was analyzed by bioinformatics and qPCR. Cell Count Kit-8 (CCK-8) testing kit was utilized for testing cell viability. KD treatment significantly reduced the expression of glycolysis-related proteins in Huh-7 cells, inhibited insulin production in β islet cells, reduced ECAR, and increased OCR. FOXC2 was significantly up-regulated in Huh-7 cell line, and sh-FOXC2 hindered the proliferation and migration of Huh-7 cells. The exogenous addition of insulin promoted the malignant progression of Huh-7 cells. Together, the medium simulating KD environment strengthened the protection of liver cancer cells by reducing insulin production and down-regulating FOXC2 expression. This study confirmed through in vitro cell experiments that KD could inhibit the proliferation and migration of liver cancer cells by targeting down regulation of insulin and FOXC2 expression, providing new theoretical basis for the treatment of liver cancer patients.

Abstract 3041: APOB Gene Deletion Increases The Expression Of Vitamin D Binding Protein In Human Hepatoma Cells

High plasma apolipoprotein B (apoB)-containing lipoproteins are both a biomarker and a causal mediator of many metabolic diseases. Inhibition of APOB mRNA decreases plasma lipid levels but at a risk of lipid accumulation in tissues. To understand the global consequences of reduced apoB synthesis, we generated a human hepatoma (Huh-7) cell culture model system deficient in apoB. ApoB deficient Huh-7 cells (Ako) were generated using the CRISPR/Cas9 system. Total RNA for transcriptomics and overnight conditioned media for proteomics and lipidomics was obtained from the Ako and Huh-7 cells (n=3). Statistical analysis was done using Student’s t-test while comparing two groups, p < 0.05 was considered significant. APOB gene deletion significantly reduced APOB mRNA and protein levels in the Ako cells compared to control cells. Ako cells supported apoB48 secretion when transfected with apoB48 expression plasmids. These cells did not accumulate significant amounts of triglyceride. KEGG pathway analysis of RNA-Seq data showed a significant increase in DNA replication/repair pathway and complement/coagulation cascade in the Ako cells. The most downregulated pathways included the PI3k-Akt signaling and MAPK signaling pathways. Differential gene expression analysis identified vitamin D binding protein (VDB, gene GC ) mRNA as the most highly upregulated transcript in Ako cells. The Ako media secretome contained significantly higher amounts of VDB compared to control Huh-7. Furthermore, targeted lipidomics of the secretome showed significant increases in media ergocalciferol. Our qPCR and western blot analysis confirmed increased expression of VDB mRNA and protein secretion in the Ako cells as compared to the Huh-7 cells. We established a cell culture model deficient in secreting apoB-containing lipoproteins. These cells, however, support the assembly and secretion of apoB48 when transfected with apoB48 expression plasmid. A major consequence of apoB deficiency was upregulation of the VDB expression. We interpret these data to suggest that human hepatoma cells have two differentially regulated pathways for the transport of vitamin D. In the absence of apoB-lipoprotein assembly, these cells upregulate VDB to augment secretion of vitamin D.

HDGF stimulates liver tumorigenesis by enhancing reactive oxygen species generation in mitochondria

Hepatoma-derived growth factor (HDGF) overexpression and uncontrolled reactive oxygen species (ROS) accumulation are involved in malignant transformation and poor prognosis in various types of cancer. However, the interplay between HDGF and ROS generation has not been elucidated in hepatocellular carcinoma. Here, we first analyzed the profile of HDGF expression and ROS production in newly generated orthotopic hepatomas by ultrasound-guided implantation. In situ superoxide detection showed that HDGF-overexpressing hepatomas had significantly elevated ROS levels compared with adjacent nontumor tissues. Consistently, liver tissues from HDGF-deficient mice exhibited lower ROS fluorescence than those from age- and sex-matched WT mice. ROS-detecting fluorescent dyes and flow cytometry revealed that recombinant HDGF (rHDGF) stimulated the production of superoxide anion, hydrogen peroxide, and mitochondrial ROS generation in cultured hepatoma cells in a dose-dependent manner. In contrast, the inactive Ser103Ala rHDGF mutant failed to promote ROS generation or oncogenic behaviors. Seahorse metabolic flux assays revealed that rHDGF dose dependently upregulated bioenergetics through enhanced basal and total oxygen consumption rate, extracellular acidification rate, and oxidative phosphorylation in hepatoma cells. Moreover, antioxidants of N-acetyl cysteine and MitoQ treatment significantly inhibited HDGF-mediated cell proliferation and invasive capacity. Genetic silencing of superoxide dismutase 2 augmented the HDGF-induced ROS generation and oncogenic behaviors of hepatoma cells. Finally, genetic knockdown nucleolin (NCL) and antibody neutralization of surface NCL, the HDGF receptor, abolished the HDGF-induced increase in ROS and mitochondrial energetics. In conclusion, this study has demonstrated for the first time that the HDGF/NCL signaling axis induces ROS generation by elevating ROS generation in mitochondria, thereby stimulating liver carcinogenesis.

LncRNA NEAT1 suppresses cellular senescence in hepatocellular carcinoma via KIF11-dependent repression of CDKN2A.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Therapeutic options for advanced HCC are limited, which is due to a lack of full understanding of pathogenesis. Cellular senescence is a state of cell cycle arrest, which plays important roles in the pathogenesis of HCC. Mechanisms underlying hepatocellular senescence are not fully understood. LncRNA NEAT1 acts as an oncogene and contributes to the development of HCC. Whether NEAT1 modulates hepatocellular senescence in HCC is unknown. The role of NEAT1 and KIF11 in cellular senescence and tumor growth in HCC was assessed both in vitro and in vivo. RNA pulldown, mass spectrometry, Chromatin immunoprecipitation (ChIP), luciferase reporter assays, RNA FISH and immunofluorescence (IF) staining were used to explore the detailed molecular mechanism of NEAT1 and KIF11 in cellular senescence of HCC. We found that NEAT1 was upregulated in tumor tissues and hepatoma cells, which negatively correlated with a senescence biomarker CDKN2A encoding p16INK4a and p14ARF proteins. NEAT1 was reduced in senescent hepatoma cells induced by doxorubicin (DOXO) or serum starvation. Furthermore, NEAT1 deficiency caused senescence in cultured hepatoma cells, and protected against the progression of HCC in a mouse model. During senescence, NEAT1 translocated into cytosol and interacted with a motor protein KIF11, resulting in KIF11 protein degradation and subsequent increased expression of CDKN2A in cultured hepatoma cells. Furthermore, KIF11 knockdown caused senescence in cultured hepatoma cells. Genetic deletion of Kif11 in hepatocytes inhibited the development of HCC in a mouse model. Conclusively, NEAT1 overexpression reduces senescence and promotes tumor progression in HCC tissues and hepatoma cells, whereas NEAT1 deficiency causes senescence and inhibits tumor progression in HCC. This is associated with KIF11-dependent repression of CDKN2A. These findings lay the foundation to develop potential therapies for HCC by inhibiting NEAT1 and KIF11 or inducing senescence.

SCD1 inhibits HBV replication by regulating autophagy under high lipid conditions.

Chronic hepatitis B virus (HBV) infection remains a significant public health concern worldwide. Several metabolic processes regulate HBV DNA replication, including autophagy and lipid metabolism. In this study, we clarified the effect of lipids on HBV replication and elucidated possible mechanisms. We discovered that lipid metabolic gene expression levels were negatively correlated with the HBV DNA in plasma. Our data showed that fatty acid stimulation significantly reduced HBV DNA, hepatitis B surface antigen (HBsAg), and hepatitis B e antigen (HBeAg) levels in HepG2.2.15 cells, which are human hepatoma cell cultures transfected with HBV DNA. The Stearoyl coenzyme A desaturase 1 (SCD1)-autophagy pathway has also been implicated in inhibiting HBV replication by fatty acids stimulation. SCD1 knockdown deregulates the inhibitory effect of fatty acids on HBV by enhancing autophagy. When 3 methyladenine (3MA) was added, the inhibitory effects of specific autophagy inhibitors eliminated the positive effects of SCD1 knockdown on HBV replication. Our results indicate that SCD1 participates in the regulation of inhibition of HBV replication by fatty acids stimulation through regulating autophagy.

Mechanism of action and side effects of colchicine based on biomechanical properties of cells.

Many diseases are related to changes in the biomechanical properties of cells; their study can provide a theoretical basis for drug screening and can explain the internal working of living cells. In this study, the biomechanical properties of nephrocytes (VERO cells), hepatocytes (HL-7702 cells), and hepatoma cells (SMCC-7721 cells) in culture were detected by atomic force microscopy (AFM) to analyze the side effects of colchicine at different concentrations (0.1 μg/mL (A) and 0.2 μg/mL (B)) at the nanoscale for 2, 4, and 6 h. Compared with the corresponding control cells, the damage to the treated cells increased in a dose-dependent manner. Among normal cells, the injury of nephrocytes (VERO cells) was markedly worse than that of hepatocytes (HL-7702 cells) in both colchicine solutions A and B. Based on the analyses of biomechanical properties, the colchicine solution reduced the rate of division and inhibited metastasis of SMCC-7721 cells. By comparing these two concentrations, we found that the anticancer effect of colchicine solution A was greater than that of solution B. Studying the mechanical properties of biological cells can help understand the mechanism of drug action at the molecular level and provide a theoretical basis for preventing the emergence and diagnosis of diseases at the nanoscale. This article is protected by copyright. All rights reserved.

Slow skeletal muscle troponin T acts as a potential prognostic biomarker and therapeutic target for hepatocellular carcinoma

Slow skeletal muscle troponin T (TNNT1) as a poor prognostic indicator is upregulated in colon and breast cancers. However, the role of TNNT1 in the disease prognosis and biological functions of hepatocellular carcinoma (HCC) is still unclear. The Cancer Genome Atlas (TCGA), real-time quantitative RT-PCR (qRT-PCR), immunoblot, and immunohistochemical analyses were applied to evaluate the TNNT1 expression of human HCC. The impact of TNNT1 levels on disease progression and survival outcome was studied using TCGA analysis. Moreover, the bioinformatics analysis and HCC cell culture were used to investigate the biological functions of TNNT1. Besides, the immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) were used to detect the extracellular TNNT1 of HCC cells and circulating TNNT1 of HCC patients, respectively. The effect of TNNT1 neutralization on oncogenic behaviors and signaling was further validated in the cultured hepatoma cells. In this study, tumoral and blood TNNT1 was upregulated in HCC patients based on the analyses using bioinformatics, fresh tissues, paraffin sections, and serum. From the multiple bioinformatics tools, the TNNT1 overexpression was associated with advanced stage, high grade, metastasis, vascular invasion, recurrence, and poor survival outcome in HCC patients. By the cell culture and TCGA analyses, TNNT1 expression and release were positively correlated with epithelial-mesenchymal transition (EMT) processes in HCC tissues and cells. Moreover, TNNT1 neutralization suppressed oncogenic behaviors and EMT in hepatoma cells. In conclusion, TNNT1 may serve as a non-invasive biomarker and drug target for HCC management. This research finding may provide a new insight for HCC diagnosis and treatment.

VEGF‑mediated tumour growth and EMT in 2D and 3D cell culture models of hepatocellular carcinoma

The purpose of the present study was to evaluate the effects of vascular endothelial growth factor (VEGF) on tumorigenic properties in two-dimensional (2D) and three-dimensional (3D) cultures of hepatoma cells. The proliferation and invasion of hepatoma cells was assessed using wound healing, chemotaxis Transwell, invasion, tube-forming and hanging drop assays in both 2D and 3D cultures. The expression levels of epithelial-mesenchymal transition (EMT) and stemness markers were analysed using reverse transcription-quantitative PCR (RT-qPCR) for mRNA expression and immunofluorescence assay for protein expression. To validate the role of VEGF in tumour growth, a VEGF receptor (VEGFR) inhibitor (sorafenib) was used. The results demonstrated that the hepatoma cells formed 3D spheroids that differed in size and density in the absence and presence of the growth factor, VEGF. In all spheroids, invasion and angiogenesis were more aggressive in 3D cultures in comparison to 2D conditions following treatment with VEGF. Mechanistically, the VEGF-mediated increase in the levels of EMT markers, including Vimentin, N-cadherin 2 (Cadherin 2) and Thy-1 Cell Surface Antigen was observed in the 2D and 3D cultures. Sorafenib treatment for 24 h culminated in a marked reduction in cell migration, cell-cell adhesion, spheroid compaction and EMT gene expression in 3D models as compared to the 2D models. On the whole, the findings of the present study suggested that as compared to the 2D cell cultures, 3D cell cultures model may be used as a more realistic model for the study of tumour growth and invasion in the presence of angiogenic factors, as well as for tumour inhibitor screening.

PPP2R5D promotes hepatitis C virus infection by binding to viral NS5B and enhancing viral RNA replication

BackgroundHepatitis C virus (HCV) infection increased the risk of hepatocellular carcinoma. Identification of host factors required for HCV infection will help to unveil the HCV pathogenesis. Adaptive mutations that enable the replication of HCV infectious clones could provide hints that the mutation-carrying viral protein may specifically interact with some cellular factors essential for the HCV life cycle. Previously, we identified D559G mutation in HCV NS5B (RNA dependent RNA polymerase) important for replication of different genotype clones. Here, we searched for the factors that potentially interacted with NS5B and investigated its roles in HCV infection.MethodsWild-type-NS5B and D559G-NS5B of HCV genotype 2a clone, J6cc, were ectopically expressed in hepatoma Huh7.5 cells, and NS5B-binding proteins were pulled down and identified by mass spectrometry. The necessity and mode of action of the selected cellular protein for HCV infection were explored by experiments including gene knockout or knockdown, complementation, co-immunoprecipitation (Co-IP), colocalization, virus infection and replication, and enzymatic activity, etc.ResultsMass spectrometry identified a number of cellular proteins, of which protein phosphatase 2 regulatory subunit B’delta (PPP2R5D, the PP2A regulatory B subunit) was one of D559G-NS5B-pulled down proteins and selected for further investigation. Co-IP confirmed that PPP2R5D specifically interacted with HCV NS5B but not HCV Core and NS3 proteins, and D559G slightly enhanced the interaction. NS5B also colocalized with PPP2R5D in the endoplasmic reticulum. Knockdown and knockout of PPP2R5D decreased and abrogated HCV infection in Huh7.5 cells, respectively, while transient and stable expression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that in wild-type Huh7.5 cells. Replicon assay revealed that PPP2R5D promoted HCV replication, but the phosphatase activity and catalytic subunit of PP2A were not affected by NS5B.ConclusionsPPP2R5D interactes with HCV NS5B and is required for HCV infection in cultured hepatoma cells through facilitating HCV replication.

Genetic Deficiency of Indoleamine 2,3-dioxygenase Aggravates Vascular but Not Liver Disease in a Nonalcoholic Steatohepatitis and Atherosclerosis Comorbidity Model

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that increases cardiovascular disease risk. Indoleamine 2,3-dioxygenase-1 (IDO1)-mediated tryptophan (Trp) metabolism has been proposed to play an immunomodulatory role in several diseases. The potential of IDO1 to be a link between NASH and cardiovascular disease has never been investigated. Using Apoe−/− and Apoe−/−Ido1−/− mice that were fed a high-fat, high-cholesterol diet (HFCD) to simultaneously induce NASH and atherosclerosis, we found that Ido1 deficiency significantly accelerated atherosclerosis after 7 weeks. Surprisingly, Apoe−/−Ido1−/− mice did not present a more aggressive NASH phenotype, including hepatic lipid deposition, release of liver enzymes, and histopathological parameters. As expected, a lower L-kynurenine/Trp (Kyn/Trp) ratio was found in the plasma and arteries of Apoe−/−Ido1−/− mice compared to controls. However, no difference in the hepatic Kyn/Trp ratio was found between the groups. Hepatic transcript analyses revealed that HFCD induced a temporal increase in tryptophan 2,3-dioxygenase (Tdo2) mRNA, indicating an alternative manner to maintain Trp degradation during NASH development in both Apoe−/− and Apoe−/−Ido1−/mice−. Using HepG2 hepatoma cell and THP1 macrophage cultures, we found that iron, TDO2, and Trp degradation may act as important mediators of cross-communication between hepatocytes and macrophages regulating liver inflammation. In conclusion, we show that Ido1 deficiency aggravates atherosclerosis, but not liver disease, in a newly established NASH and atherosclerosis comorbidity model. Our data indicate that the overexpression of TDO2 is an important mechanism that helps in balancing the kynurenine pathway and inflammation in the liver, but not in the artery wall, which likely determined disease outcome in these two target tissues.

Deoxycholic and Ursodeoxycholic Acid Differentially Impact Cellular Steatosis and Lipid Peroxidation in Cultured Hepatoma Cells

Background: Bile acids (BAs) are the major lipid components of bile. They are synthesized from cholesterol in the liver and stored in the gallbladder. BAs have gained attention as drug candidates to control obesity and/or diabetic condition due to their role in lipid and glucose metabolism. Objective: This study aimed to evaluate the antisteatotic and antioxidant potential of deoxycholic ac-id (DCA) and ursodeoxycholic acid (UDCA), two BAs with opposite physico-chemical features. Methods: Different concentrations of DCA and UDCA in the micromolar range were tested on cul-tured hepatoma cells after loading with an excess of fatty acids to mimic non-alcoholic fatty liver disease (NAFLD) in vitro. Experimental analyses included cell viability, lipid accumulation and li-pid peroxidation in steatotic hepatocytes before and after exposure to either DCA or UDCA. Results: Both UDCA and DCA improved lipid dysmetabolism and oxidative stress conditions in the steatotic hepatocytes. However, while UDCA was more effective as lipid lowering agent, DCA showed a greater antioxidant effect. Conclusions: UDCA seems to have better protective and beneficial potential than DCA, as it is able to both alleviate lipid accumulation in the steatotic liver cells, but also to play antioxidant effect.

Zac1 and the Imprinted Gene Network program juvenile NAFLD in response to maternal metabolic syndrome.

Background and Aims:Within the next decade, NAFLD is predicted to become the most prevalent cause of childhood liver failure in developed countries. Predisposition to juvenile NAFLD can be programmed during early life in response to maternal metabolic syndrome (MetS), but the underlying mechanisms are poorly understood. We hypothesized that imprinted genes, defined by expression from a single parental allele, play a key role in maternal MetS-induced NAFLD, due to their susceptibility to environmental stressors and their functions in liver homeostasis. We aimed to test this hypothesis and determine the critical periods of susceptibility to maternal MetS.Approach and Results:We established a mouse model to compare the effects of MetS during prenatal and postnatal development on NAFLD. Postnatal but not prenatal MetS exposure is associated with histological, biochemical, and molecular signatures of hepatic steatosis and fibrosis in juvenile mice. Using RNA sequencing, we show that the Imprinted Gene Network (IGN), including its regulator Zac1, is up-regulated and overrepresented among differentially expressed genes, consistent with a role in maternal MetS-induced NAFLD. In support of this, activation of the IGN in cultured hepatoma cells by overexpressing Zac1 is sufficient to induce signatures of profibrogenic transformation. Using chromatin immunoprecipitation, we demonstrate that Zac1 binds the TGF-β1 and COL6A2 promoters, forming a direct pathway between imprinted genes and well-characterized pathophysiological mechanisms of NAFLD. Finally, we show that hepatocyte-specific overexpression of Zac1 is sufficient to drive fibrosis in vivo.Conclusions:Our findings identify a pathway linking maternal MetS exposure during postnatal development to the programming of juvenile NAFLD, and provide support for the hypothesis that imprinted genes play a central role in metabolic disease programming.

Canonical and Divergent N-Terminal HBx Isoform Proteins Unveiled: Characteristics and Roles during HBV Replication.

Hepatitis B virus (HBV) X protein (HBx) is a viral regulatory and multifunctional protein. It is well-known that the canonical HBx reading frame bears two phylogenetically conserved internal in-frame translational initiation codons at Met2 and Met3, thus possibly generating divergent N-terminal smaller isoforms during translation. Here, we demonstrate that the three distinct HBx isoforms are generated from the ectopically expressed HBV HBx gene, named XF (full-length), XM (medium-length), and XS (short-length); they display different subcellular localizations when expressed individually in cultured hepatoma cells. Particularly, the smallest HBx isoform, XS, displayed a predominantly cytoplasmic localization. To study HBx proteins during viral replication, we performed site-directed mutagenesis to target the individual or combinatorial expression of the HBx isoforms within the HBV viral backbone (full viral genome). Our results indicate that of all HBx isoforms, only the smallest HBx isoform, XS, can restore WT levels of HBV replication, and bind to the viral mini chromosome, thereby establishing an active chromatin state, highlighting its crucial activities during HBV replication. Intriguingly, we found that sequences of HBV HBx genotype H are devoid of the conserved Met3 position, and therefore HBV genotype H infection is naturally silent for the expression of the HBx XS isoform. Finally, we found that the HBx XM (medium-length) isoform shares significant sequence similarity with the N-terminus domain of the COMMD8 protein, a member of the copper metabolism MURR1 domain-containing (COMMD) protein family. This novel finding might facilitate studies on the phylogenetic origin of the HBV X protein. The identification and functional characterization of its isoforms will shift the paradigm by changing the concept of HBx from being a unique, canonical, and multifunctional protein toward the occurrence of different HBx isoforms, carrying out different overlapping functions at different subcellular localizations during HBV genome replication. Significantly, our current work unveils new crucial HBV targets to study for potential antiviral research, and human virus pathogenesis.

Sorafenib combined with dasatinib therapy inhibits cell viability, migration, and angiogenesis synergistically in hepatocellular carcinoma.

Sorafenib is a multikinase inhibitor used for treatment of advanced hepatocellular carcinoma. Sorafenib resistance may be related to Src-induced cell migration and angiogenesis, which are regulated by cancer stem cell activation and release of vascular endothelial growth factor. Dasatinib is a Src inhibitor that inhibits Src phosphorylation and suppresses Src-associated cell migration and angiogenesis. This study investigated whether combined treatment with dasatinib can overcome sorafenib resistance. Hepatoma cell lines were used for sorafenib and/or dasatinib treatment. Cell viability, cell migration, molecular expressions, and release of vascular endothelial growth factor by hepatoma cells were evaluated. Hepatoma cell culture medium was applied on human umbilical vein endothelial cells to monitor angiogenesis promoted by the hepatoma cells. Sorafenib and dasatinib combined therapy suppressed cell viability of hepatoma cells synergistically. Dasatinib suppressed sorafenib-induced cell migration via inhibiting sorafenib-induced Src/FAK phosphorylation, cell-to-cell contact and cancer stem cell activation. Culture medium from Chang liver and PLC/PRF/5 cells suppressed angiogenesis of human umbilical vein endothelial cells with any treatment, whereas sorafenib-treated medium of HepG2 cells induced angiogenesis. This sorafenib-induced angiogenesis was then suppressed by dasatinib. Vascular endothelial growth factor released from hepatoma cells was also inhibited by combined treatment. Src/FAK phosphorylation and cancer stem cell activation inducing cell migration and angiogenesis may be the key factors of sorafenib resistance. Sorafenib and dasatinib combined treatment suppresses cell migration and angiogenesis by inhibiting the Src/FAK phosphorylation, cell-to-cell contact, cancer stem cell activation, and release of vascular endothelial growth factor.

Testing in vitro tools for the prediction of cholestatic liver injury induced by non-pharmaceutical chemicals

Bile acid accumulation and subsequent liver damage is a frequent adverse effect induced by drugs. Considerable efforts have therefore been focused on the introduction and characterization of tools that allow reliable prediction of this type of drug-induced liver injury. Among those are the cholestatic index and transcriptomic profiling, which are typically assessed in in vitro settings. The present study was set up to test the applicability of both tools to non-pharmaceutical compounds with cholestatic potential, including the industrial compound bis(2-ethylhexyl)phthalate, the cosmetic ingredients triclosan and octynoic acid, the herbicides paraquat and quizalofop-para-ethyl, and the food additives sunset yellow and tartrazine, in a human hepatoma cell culture model of cholestatic liver injury. The cholestatic index method showed cholestatic liability of sunset yellow, tartrazine and triclosan. Of those, tartrazine induced transcriptional changes reminiscent of the transcriptional profile of cholestatic drugs. Furthermore, a number of genes were found to be uniquely modulated by tartrazine, in accordance with the cholestatic drugs atazanavir, cyclosporin A and nefazodone, which may have potential as novel transcriptomic biomarkers of chemical-induced cholestatic liver injury. In conclusion, unambiguous identification of the non-pharmaceutical compounds tested in this study as inducers of cholestasis could not be achieved.

In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide

An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.

Inhibition of histone methyltransferase G9a attenuates liver cancer initiation by sensitizing DNA-damaged hepatocytes to p53-induced apoptosis

While the significance of acquired genetic abnormalities in the initiation of hepatocellular carcinoma (HCC) has been established, the role of epigenetic modification remains unknown. Here we identified the pivotal role of histone methyltransferase G9a in the DNA damage-triggered initiation of HCC. Using liver-specific G9a-deficient (G9aΔHep) mice, we revealed that loss of G9a significantly attenuated liver tumor initiation caused by diethylnitrosamine (DEN). In addition, pharmacological inhibition of G9a attenuated the DEN-induced initiation of HCC. After treatment with DEN, while the induction of γH2AX and p53 were comparable in the G9aΔHep and wild-type livers, more apoptotic hepatocytes were detected in the G9aΔHep liver. Transcriptome analysis identified Bcl-G, a pro-apoptotic Bcl-2 family member, to be markedly upregulated in the G9aΔHep liver. In human cultured hepatoma cells, a G9a inhibitor, UNC0638, upregulated BCL-G expression and enhanced the apoptotic response after treatment with hydrogen peroxide or irradiation, suggesting an essential role of the G9a-Bcl-G axis in DNA damage response in hepatocytes. The proposed mechanism was that DNA damage stimuli recruited G9a to the p53-responsive element of the Bcl-G gene, resulting in the impaired enrichment of p53 to the region and the attenuation of Bcl-G expression. G9a deletion allowed the recruitment of p53 and upregulated Bcl-G expression. These results demonstrate that G9a allows DNA-damaged hepatocytes to escape p53-induced apoptosis by silencing Bcl-G, which may contribute to the tumor initiation. Therefore, G9a inhibition can be a novel preventive strategy for HCC.

Monitoring metabolism of synthetic cannabinoid 4F-MDMB-BINACA via high-resolution mass spectrometry assessed in cultured hepatoma cell line, fungus, liver microsomes and confirmed using urine samples

PurposeA tert-leucinate derivative synthetic cannabinoid, methyl (2S)-2-([1-(4-fluorobutyl)-1H-indazole-3-carbonyl]amino)-3,3-dimethylbutanoate (4F-MDMB-BINACA, 4F-MDMB-BUTINACA or 4F-ADB) is known to adversely impact health. This study aimed to evaluate the suitability of three different modes of monitoring metabolism: HepG2 liver cells, fungus Cunninghamella elegans (C. elegans) and pooled human liver microsomes (HLM) for comparison with human in-vivo metabolism in identifying suitable urinary marker(s) for 4F-MDMB-BINACA intake.MethodsTentative structure elucidation of in-vitro metabolites was performed on HepG2, C. elegans and HLM using liquid chromatography–tandem mass spectrometry and high-resolution mass spectrometry analysis. In-vivo metabolites obtained from twenty authentic human urine samples were analysed using liquid chromatography–Orbitrap mass spectrometry.ResultsIncubation with HepG2, C. elegans and HLM yielded nine, twenty-three and seventeen metabolites of 4F-MDMB-BINACA, respectively, formed via ester hydrolysis, hydroxylation, carboxylation, dehydrogenation, oxidative defluorination, carbonylation or reaction combinations. Phase II metabolites of glucosidation and sulfation were also exclusively identified using C. elegans model. Eight in-vivo metabolites tentatively identified were mainly products of ester hydrolysis with or without additional dehydrogenation, N-dealkylation, monohydroxylation and oxidative defluorination with further oxidation to butanoic acid. Metabolites with intact terminal methyl ester moiety, i.e., oxidative defluorination with further oxidation to butanoic acid, were also tentatively identified.ConclusionsThe in-vitro models presented proved useful in the exhaustive metabolism studies. Despite limitations, HepG2 identified the major 4F-MDMB-BINACA ester hydrolysis metabolite, and C. elegans demonstrated the capacity to produce a wide variety of metabolites. Both C. elegans and HLM produced all the in-vivo metabolites. Ester hydrolysis and ester hydrolysis plus dehydrogenation 4F-MDMB-BINACA metabolites were recommended as urinary markers for 4F-MDMB-BINACA intake.

Ethanol-mediated upregulation of APOA1 gene expression in HepG2 cells is independent of de novo lipid biosynthesis

BackgroundModerate alcohol intake in human increases HDL-cholesterol, and has protective effects against cardiovascular disease (CVD). Although de novo lipid synthesis inhibitors are highly effective in lowering total and LDL-cholesterol they have only modest effects on raising HDL-C. A better understanding of the mechanism of ethanol-mediated HDL-C regulation could suggest new therapeutic approaches for CVD.MethodsHuman hepatoblastoma (HepG2) and colorectal epithelial adenocarcinoma (Caco-2) cells were incubated in the presence of varying concentrations of ethanol in the culture medium, with or without addition of de novo lipid synthesis (DNLS) inhibitors (atorvastatin and/or TOFA). ApoA1 protein was measured by Western blot, and RNA of lipid pathway genes APOA1, APOC3, APOA4, APOB100, HMGCR, LDLR, and SREBF2 by quantitative RT-PCR. Lipoproteins (VLDL, LDL, and HDL) and lipids were also monitored.ResultsEthanol stimulated ApoA1 protein (both cytoplasmic and secreted) and APOA1 RNA levels in HepG2 cells in a dose sensitive way, with ~ 50% upregulation at 100 mM ethanol in the medium. The effect was not observed in intestinal-derived Caco-2 cells. DNLS inhibitors did not block the upregulation of ApoA1 RNA by ethanol; TOFA alone produced a modest increase in ApoA1 RNA. Ethanol had no effect on ABCA1 protein levels. Addition of ethanol to the cell medium led to modest increases in de novo synthesis of total cholesterol, cholesteryl esters and triglycerides, and as expected these increases were blocked when the lipid synthesis inhibitors were added. Ethanol stimulated a small increase in HDL and VLDL but not LDL synthesis. Ethanol in the cell medium also induced modest but measurable increases in the RNA of APOC3, APOA4, APOB, LDLR, and HMGCR genes. Unlike APOA1, induction of RNA from APOC3 and APOA4 was also observed in Caco-2 cells as well as HepG2 cells.ConclusionThis study has verified the previously reported upregulation of APOA1 by exposure of HepG2, but not Caco-2 cells, to ethanol in the culture medium. It is shown for the first time that the effect is dependent on RNA polymerase II-mediated transcription, but not on de novo biosynthesis of cholesterol or fatty acids, and therefore is not a generalized metabolic response to ethanol exposure. Some other lipid pathway genes are also modulated by ethanol exposure of cells. The results reported here suggest that the proximal signaling molecule leading to increased APOA1 gene expression in response to ethanol exposure may be free acetate or acetyl-CoA.Take homeUpregulation of ApoA1 gene expression in hepatoma cells in culture, upon exposure to moderate ethanol concentrations in the medium, occurs at the level of RNA and is not dependent on new cholesterol or fatty acid synthesis. The primary signaling molecule may be free acetate or acetyl-CoA. These results are important for understanding the mechanism by which moderate alcohol consumption leads to upregulation of serum HDL-cholesterol in humans, and suggests new approaches to targeting HDL as a risk factor for cardiovascular disease.

MK-571, a Cysteinyl Leukotriene Receptor 1 Antagonist, Inhibits Hepatitis C Virus Replication.

The quinoline MK-571 is the most commonly used inhibitor of multidrug resistance protein-1 (MRP-1) but was originally developed as a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist. While studying the modulatory effect of MRP-1 on anti-hepatitis C virus (HCV) direct-acting antiviral (DAA) efficiency, we observed an unexpected anti-HCV effect of compound MK-571 alone. This anti-HCV activity was characterized in Huh7.5 cells stably harboring a subgenomic genotype 1b replicon. A dose-dependent decrease of HCV RNA levels was observed upon MK-571 administration, with a 50% effective concentration (EC50 ± standard deviation) of 9 ± 0.3 μM and a maximum HCV RNA level reduction of approximatively 1 log10 MK-571 also reduced the replication of the HCV full-length J6/JFH1 model in a dose-dependent manner. However, probenecid and apigenin homodimer (APN), two specific inhibitors of MRP-1, had no effect on HCV replication. In contrast, the CysLTR1 antagonist SR2640 increased HCV-subgenomic replicon (SGR) RNA levels in a dose-dependent manner, with a maximum increase of 10-fold. In addition, a combination of natural CysLTR1 agonist (LTD4) or antagonists (zafirlukast, cinalukast, and SR2640) with MK-571 completely reversed its antiviral effect, suggesting its anti-HCV activity is related to CysLTR1 rather to MRP-1 inhibition. In conclusion, we showed that MK-571 inhibits HCV replication in hepatoma cell cultures by acting as a CysLTR1 receptor antagonist, thus unraveling a new host-virus interaction in the HCV life cycle.