Hepatocyte Cell Line Research Articles

Essential phospholipids decrease apoptosis and increase membrane transport in human hepatocyte cell lines

BackgroundEssential phospholipids (EPL) have hepatoprotective effects across many liver diseases/conditions. The impact of EPL on hepatocyte function in vitro was investigated.MethodsEffects of noncytotoxic concentrations of EPL (0.1 and 0.25 mg/ml), and its constituents, polyenylphosphatidylcholine (PPC) and phosphatidylinositol (PI) (both at 0.1 and 1 mg/ml), on membrane fluidity, apoptosis and extracellular transport versus controls were investigated in human hepatocyte cell lines (HepG2, HepaRG, steatotic HepaRG). ResultsSignificantly increased membrane fluidity occurred with all 3 phospholipids (PLs) in HepG2 cultures, and with PI (1 mg/ml) in steatotic HepaRG cells. Significantly decreased tamoxifen-induced apoptosis was observed in HepG2 cells with EPL, PPC and PI. Breast cancer resistance protein (BCRP) activity was significantly increased by EPL and PI in HepG2 cells. Multidrug resistance-associated protein 2 (MRP-2) activity was unaffected by any PL in HepG2 cells, and significantly increased by EPL, PI and PPC (1 mg/ml) in HepaRG cells, and by PI (1 mg/ml) in steatotic HepaRG cells. Bile salt export protein (BSEP) activity in HepG2 cells and steatotic HepaRG cells was significantly increased by EPL (0.25 mg/ml), and PPC (both concentrations), but not by PI. The PLs had no effects on HepaRG cell BSEP activity. P-glycoprotein (P-GP) activity was significantly increased by all compounds in HepG2 cells. PI (1 mg/ml) significantly increased P-GP activity in HepaRG and steatotic HepaRG cells.ConclusionsEPL, PPC, and PI increased hepatocyte membrane fluidity, decreased apoptosis and increased hepatocellular export, all of which may improve liver function. These in-vitro investigations provide valuable insights into the mechanism of action of EPL.

PSVIII-20 Patatin-Like Phospholipase Domain-Containing Protein 3 Abundance is Affected by Knockdown of Sterol Regulatory Element-Binding Protein 1c but not of Carbohydrate Response Element Binding Protein

Abstract Patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a key lipase involved in fatty liver syndrome onset and recovery in humans and bovine. Regulation of PNPLA3 is mediated by carbohydrate response element binding protein (ChREBP) in rodents and sterol regulatory element-binding protein 1c (SREBP1c) in humans. The objectives of our study were to optimize an siRNA knockdown of ChREBP and SREBP1c and to determine if either transcription factor knockdown would affect PNPLA3 abundance in an immortalized bovine hepatocyte cell line, BFH12. Hepatocytes were cultured in monolayer and at least 80% confluent after 24h when cells were randomly assigned in triplicate to treatment with ChREBP-targeted (siC1, siC2, siC3) or SREBP1c-targeted (siS1, siS2, siS3) siRNA at either 20, 60, or 80µM, or a nonspecific-siRNA control (siNON). Media was replenished after 4h and cells incubated for 20h before harvest in TRIzol for subsequent protein isolation. Protein abundances were determined by Western blot and normalized to total lane protein. Data were analyzed (PROC MIXED, SAS 9.4) with fixed effect of siRNA and concentration. Differences were declared at P≤0.05; tendencies at 0.05

Diallyl Trisulfide attenuates alcohol-induced hepatocyte pyroptosis via elevation of hydrogen sulfide.

Garlic is a popular culinary herb for the prevention and treatment of alcoholic liver disease (ALD). Diallyl Trisulfide (DATS) is the major organosulfur compound of garlic. Latest studies indicated that the hepatocyte pyroptosis serves a primary role in the pathogenesis of ALD. The present study aims to assess the inhibitory effect of DATS on alcohol-induced hepatocyte pyroptosis, and to elucidate the potential mechanism by using the hepatocyte cell line HL-7702. Our study found that DATS inhibited alcohol-induced pyroptosis by decreasing gasdermin D (GSDMD) activation. Results illuminated that DATS inhibited alcohol-induced (NOD)-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation by reducing intracellular reactive oxygen species (ROS) accumulation. Furthermore, DATS upregulated hydrogen sulfide (H2S) to resist ROS overproduction. The present study demonstrated that DATS mitigated alcohol-induced hepatocyte pyroptosis by increasing the intracellular level of H2S.

Occult infection with hepatitis B virus PreS variants synergistically promotes hepatocellular carcinoma development in a high-fat diet context by generating abnormal ceramides

BackgroundSome occult hepatitis B virus (HBV) infections are resulted from PreS mutations that reduce secretion of envelope protein (HBsAg). We investigated the ceramide amounts and species in hepatocytes infected with PreS variants that were isolated from HBsAg-seronegative patients with hepatocellular carcinoma (HCC) and the ceramide effects on autochthonous HCC development in murine models.MethodsHBV PreS/S regions from 35 HBsAg-seronegative HCC patients were sequenced. Hepatocyte cell lines and male C57BL/6J mouse livers were transfected with two PreS variant representatives. The ceramides with variated lengths of fatty acyl chains were quantified. Tumour development was examined in the HBV-transfected mice fed different diet types.ResultsIn HBsAg-seronegative HCC patients, nonneoplastic liver tissues harboured HBsAg and replication-competent HBV. The most frequently detected PreS/S variants carried mutations of altered amino acid properties in HBsAg compared with an isolate from one HBsAg-seronegative HCC patient. Hepatocyte infection with PreS variants caused HBsAg retention within the endoplasmic reticulum and generated more amounts of ceramides with C16:0 ceramide elevated the highest. Saturated fatty acids aggravated the PreS variant-infected hepatocytes to generate abnormal amounts and species of ceramides, which with HBV proteins synergistically activated NLRP3 inflammasome in liver inflammatory macrophages. Liver tumours were only detected in HBV-transfected mice fed high-fat diet, with higher tumour loads in the PreS variant-transfected, associated with abnormal ceramide generation.ConclusionsHBV PreS mutations which altered amino acid properties of envelope proteins inhibited HBsAg secretion. Hepatocyte infection with PreS variants generated abnormal ceramides which with HBV proteins coactivated NLRP3 inflammasome in liver macrophages to promote autochthonous HCC development.

LncRNA 1700020I14Rik promotes AKR1B10 expression and activates Erk pathway to induce hepatocyte damage in alcoholic hepatitis

Alcoholic hepatitis (AH), a kind of alcoholic liver disease, shows poor prognosis. Long noncoding RNAs (lncRNAs) exert critical role in liver diseases. Here, we intended to investigate the possible molecular mechanism that 1700020I14Rik-based regulation of microRNA (miR)-137/Aldo-keto reductase family 1 member B10 (AKR1B10) affecting the inflammatory response and hepatocyte damage in AH. AH-related genes and the down-stream regulatory pathway were screnned by bioinformatics. Mouse normal hepatocyte cell line AML12 was selected to construct an ethanol-induced hepatocyte injury model for in vitro mechanistic validation, while we also established an AH mouse model using the ethanol with gradually increased concentration of 2–4% (v/v) for in vivo study. Specific role of 1700020I14Rik/miR-137/AKR1B10 in AML12 cell viability, proliferation and apoptotic capacity as well as inflammation and liver damage in mice were analyzed following ectopic and depletion approaches. We found elevated AKR1B10 and 1700020I14Rik but reduced miR-137 in AH. 1700020I14Rik was able to elevated miR-137-mediated AKR1B10. In vitro cell experiments and in vivo animal experiments validated that 1700020I14Rik reduced ethanol-induced hepatocyte damage and inflammation in AH mice through regulation of miR-137–mediated AKR1B10/Erk axis. The current study underlied that 1700020I14Rik could activate AKR1B10/Erk signaling through inhibition of miR-137, thereby promoting the hepatocyte damage in AH mice.

Transcriptional level evaluation of osteopontin/miRNA-181a axis in hepatocellular carcinoma cell line-secreted extracellular vesicles

Recent evidence suggested the role of secreted extracellular vesicles (EVs) in the intracellular signalling within the liver becoming a promising candidate as biomarker in hepatocellular carcinoma (HCC). Osteopontin (OPN) seems to play a relevant role both for early diagnosis of HCC than on the mechanisms that drive oncogenesis but, to date, information on the expression levels of OPN in EVs secreted by HCC tumor cell line are missing. The study aimed to verify, by transcriptional and proteomic study, the presence of OPN in EVs secreted by tumorigenic (HepG2) and non-tumorigenic hepatocyte cell line (WRL68), and to analyse the expression variations of OPN, its isoforms and miRNA-181a in both these EVs. “In silico analysis” was also performed via the Gene expression Profiling Interactive analysis (GEPIA) and Hepatocellular Carcinoma Database (HCCDB). An up-regulation of OPN in EVs secreted by HepG2 with respect to WRL68 was found in line with the results obtained by the “in silico analysis”. The study demonstrates, for the first time, the OPN isoforms and its modulator miRNA-181a expression in EVs secreted by both cell lines, highlighting high levels of OPN isoforms in EVs secreted by HepG2 and identifying OPN as a promising biomarker for HCC diagnosis.

Cellular senescence in hepatocytes contributes to metabolic disturbances in NASH.

Cellular senescence is a state of irreversible cell cycle arrest and has been shown to play a key role in many diseases, including metabolic diseases. To investigate the potential contribution of hepatocyte cellular senescence to the metabolic derangements associated with non-alcoholic steatohepatitis (NASH), we treated human hepatocyte cell lines HepG2 and IHH with the senescence-inducing drugs nutlin-3a, doxorubicin and etoposide. The senescence-associated markers p16, p21, p53 and beta galactosidase were induced upon drug treatment, and this was associated with increased lipid storage, increased expression of lipid transporters and the development of hepatic steatosis. Drug-induced senescence also led to increased glycogen content, and increased VLDL secretion from hepatocytes. Senescence was also associated with an increase in glucose and fatty acid oxidation capacity, while de novo lipogenesis was decreased. Surprisingly, cellular senescence caused an overall increase in insulin signaling in hepatocytes, with increased insulin-stimulated phosphorylation of IR, Akt, and MAPK. Together, these data indicate that hepatic senescence plays a causal role in the development of NASH pathogenesis, by modulating glucose and lipid metabolism, favoring steatosis. Our findings contribute to a better understanding of the mechanisms linking cellular senescence and fatty liver disease and support the development of new therapies targeting senescent cells for the treatment of NASH.

MicroRNA-378 inhibits hepatocyte apoptosis during acute liver failure by targeting caspase-9 in mice

BackgroundAcute liver failure (ALF) is a severe and potentially lethal clinical syndrome. It has been demonstrated that micro ribonucleic acids (miRNAs) are crucial mediators of nearly all pathological processes, including liver disease. ObjectiveThe present study investigates the role of miR-378 in ALF. An ALF mouse model was induced using intraperitoneal injections of d-galactosamine/lipopolysaccharide (d-GalN/LPS). A hepatocyte cell line and miR-378 analogue were used in vitro to investigate the possible roles of miR-378 in ALF. MethodsThe expressions of miR-378 and predicted target genes were measured via reverse transcription-quantitative polymerase chain reaction and western blotting, and cell apoptosis was assayed using flow cytometry. ResultsCompared with mice in the control group, the mice challenged with d-GalN/LPS showed higher levels of alanine aminotransferase, aspartate aminotransferase, tumour necrosis factor-alpha and interleukin-6, more severe liver damage and increased numbers of apoptotic hepatocytes. Hepatic miR-378 was distinctly downregulated, while messenger RNA and protein levels of cysteinyl aspartate specific proteinase 9 (caspase-9) were upregulated in the ALF model. Furthermore, miR-378 was downregulated in d-GalN/TNF-induced hepatocyte cells, and miR-378 was found to inhibit hepatocyte apoptosis by targeting caspase-9. ConclusionTogether, the present results indicate that miR-378 is a previously unrecognised post-ALF hepatocyte apoptosis regulator and may be a potential therapeutic target in the context of ALF.

Gemigliptin exerts protective effects against doxorubicin-induced hepatotoxicity by inhibiting apoptosis via the regulation of fibroblast growth factor 21 expression

AimDoxorubicin is a highly effective anticancer agent that causes hepatotoxicity and cardiotoxicity in patients. Fibroblast growth factor 21, a well-known regulator of glucose and lipid metabolism, exerts cardioprotective effects. Gemigliptin and dipeptidyl peptidase-4 inhibitors are widely used in the treatment of patients with type 2 diabetes. The protective effects of gemigliptin on hepatotoxicity via the increase in fibroblast growth factor 21 expression has not yet been elucidated. This study was designed to investigate the protective effects of gemigliptin against doxorubicin-induced hepatotoxicity via the upregulation of fibroblast growth factor 21 expression in the cultured murine hepatocyte cell line, AML12. MethodsMurine hepatocyte AML12 cells were treated with doxorubicin, fibroblast growth factor 21 and gemigliptin in 0.5% fetal bovine serum medium for 24 h at the indicated doses. Cells were transfected with the fibroblast growth factor 21 small interfering RNA for 24 h, followed by protein isolation. ResultsFibroblast growth factor 21 expression levels were increased during doxorubicin-induced hepatotoxicity in the murine hepatocyte AML12 cells. Fibroblast growth factor 21 treatment prevented doxorubicin-induced hepatotoxicity by attenuating apoptosis. Gemigliptin prevented doxorubicin-induced hepatotoxicity by upregulating fibroblast growth factor 21 expression. However, the protective effects of gemigliptin were blocked by fibroblast growth factor 21 inhibition in doxorubicin-treated AML12 cells. ConclusionThese results indicate that gemigliptin exhibits protective effects against doxorubicin-induced hepatotoxicity by upregulating the fibroblast growth factor 21 expression levels in the cultured murine hepatocyte AML12 cells.

Synthesis and antitumor activity of a series of novel N-aryl-5-(2,2,2-trifluoroethoxy)-1,5-dihydro-2H-pyrrol-2-ones derivatives

With the help of the establishment of novel reaction methodology, a series of N-Aryl-5-(2,2,2-trifluoroethoxy)-1,5-dihydro-2H-pyrrol-2-one conjugates were designed and synthesized in 2–4 steps, and subsequent anticancer activity of these compounds was evaluated. Preliminary results showed that these compounds have moderate to potent activities against human acute leukemia cells K562, human lung cancer A549, human breast cancer MDA-MB-231, and human cervical cancer HeLa cancer cell lines. Among them, compounds 2d and 2k were the most potent against K562 cell line with IC50 values of 0.07 and 0.52 µM, respectively, and the toxicity of 2d to the normal of hepatocytes (LO2) cell line was low (the survival rate 81 %). Flow cytometry analysis showed that 2d arrested K562 cells in the G2/M phase potently, even much better than Combretastatin A4 (CA4). In addition, the results demonstrated the involvement of the caspase-dependent or independent pathways of apoptosis, evidenced by the upregulation of FADD, pro-caspase 3, cleaved-caspase 3, HTRA2/Omi, SMAC/Diablo and the ratio of Bax/Bcl-2.The biological effects founding of 2d in this work point to prospective uses against acute leukemia.

Glycidamide and cis-2-butene-1,4-dial (BDA) as potential carcinogens and promoters of liver cancer - An in vitro study.

Acrylamide and furan are environmental and food contaminants that are metabolized by cytochrome P450 2E1 (CYP2E1), giving rise to glycidamide and cis-2-butene-1,4-dial (BDA) metabolites, respectively. Both glycidamide and BDA are electrophilic species that react with nucleophilic groups, being able to introduce mutations in DNA and perform epigenetic remodeling. However, whereas these carcinogens are primarily metabolized in the liver, the carcinogenic potential of acrylamide and furan in this organ is still controversial, based on findings from experimental animal studies. With the ultimate goal of providing further insights into this issue, we explored in vitro, using a hepatocyte cell line and a hepatocellular carcinoma cell line, the putative effect of these metabolites as carcinogens and cancer promoters. Molecular alterations were investigated in cells that survive glycidamide and BDA toxicity. We observed that those cells express CD133 stemness marker, present a high proliferative capacity and display an adjusted expression profile of genes encoding enzymes involved in oxidative stress control, such as GCL-C, GSTP1, GSTA3 and CAT. These molecular changes seem to be underlined, at least in part, by epigenetic remodeling involving histone deacetylases (HDACs). Although more studies are needed, here we present more insights towards the carcinogenic capacity of glycidamide and BDA and also point out their effect in favoring hepatocellular carcinoma progression.

Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.

SUMMARYExcessive hepatic glucose production contributes to the development of hyperglycemia and is a key feature of type 2 diabetes. Here, we report that activation of hepatocyte Rap1a suppresses gluconeogenic gene expression and glucose production, whereas Rap1a silencing stimulates them. Rap1a activation is suppressed in obese mouse liver, and restoring its activity improves glucose intolerance. As Rap1a′s membrane localization and activation depends on its geranylgeranylation, which is inhibited by statins, we show that statin-treated hepatocytes and the human liver have lower active-Rap1a levels. Similar to Rap1a inhibition, statins stimulate hepatic gluconeogenesis and increase fasting blood glucose in obese mice. Geranylgeraniol treatment, which acts as the precursor for geranylgeranyl isoprenoids, restores Rap1a activity and improves statin-mediated glucose intolerance. Mechanistically, Rap1a activation induces actin polymerization, which suppresses gluconeogenesis by Akt-mediated FoxO1 inhibition. Thus, Rap1a regulates hepatic glucose homeostasis, and blocking its activity, via lowering geranylgeranyl isoprenoids, contributes to statin-induced glucose intolerance.

Acetaminophen-induced reduction of NIMA-related kinase 7 expression exacerbates acute liver injury

Background & AimsAcetaminophen (APAP)-induced acute liver injury (ALI) is a global health issue characterised by an incomplete understanding of its pathogenesis and unsatisfactory therapies. NEK7 plays critical roles in both cell cycle regulation and inflammation. In the present study, we investigated the role and mechanism of NEK7 in APAP-induced ALI.MethodsIn mice with NEK7 overexpression (hydrodynamic tail vein injection of NEK7 plasmids), hepatocyte-specific NEK7 knockout (cKO), and inducible NEK7 knockout (iKO), an overdose of APAP was administered to induce ALI. Liver injury was determined by an analysis of serum liver enzymes, pathological changes, inflammatory cytokines, and metabonomic profiles. In vitro, hepatocyte damage was evaluated by an analysis of cell viability, the reactive oxygen species levels, and mitochondrial function in different cell lines. Hepatocyte proliferation and the cell cycle status were determined by Ki-67 staining, EdU staining, and the cyclin levels.ResultsNEK7 was markedly downregulated in APAP-induced injured liver and damaged hepatocytes. NEK7 overexpression in the liver significantly alleviated APAP-induced liver injury, as shown by the restored liver function, reduced pathological injury, and decreased inflammation and oxidative stress, which was confirmed in a hepatocyte cell line. Moreover, both NEK7 cKO and iKO mice exhibited exacerbation of APAP-induced ALI. Finally, we determined that cyclin B1-mediated cell cycle progression could mediate the protective effect of NEK7 against APAP-induced ALI.ConclusionsReduced NEK7 contributes to APAP-induced ALI, possibly by dysregulating cyclins and disturbing cell cycle progression.Lay summaryAcetaminophen-induced acute liver injury is one of the major global health issues, owing to its high incidence, potential severity, and limited therapeutic options. Our current understanding of its pathogenesis is incomplete. Herein, we have shown that reduced NEK7 (a protein with a key role in the cell cycle) exacerbates acetaminophen-induced acute liver injury. Hence, NEK7 could be a possible therapeutic target for the prevention or treatment of this condition.

Deepening the Whole Transcriptomics of Bovine Liver Cells Exposed to AFB1: A Spotlight on Toll-like Receptor 2.

Aflatoxin B1 (AFB1) is a food contaminant metabolized mostly in the liver and leading to hepatic damage. Livestock species are differently susceptible to AFB1, but the underlying mechanisms of toxicity have not yet been fully investigated, especially in ruminants. Thus, the aim of the present study was to better characterize the molecular mechanism by which AFB1 exerts hepatotoxicity in cattle. The bovine fetal hepatocyte cell line (BFH12) was exposed for 48 h to three different AFB1 concentrations (0.9 µM, 1.8 µM and 3.6 µM). Whole-transcriptomic changes were measured by RNA-seq analysis, showing significant differences in the expression of genes mainly involved in inflammatory response, oxidative stress, drug metabolism, apoptosis and cancer. As a confirmatory step, post-translational investigations on genes of interest were implemented. Cell death associated with necrosis rather than apoptosis events was noted. As far as the toxicity mechanism is concerned, a molecular pathway linking inflammatory response and oxidative stress was postulated. Toll-Like Receptor 2 (TLR2) activation, consequent to AFB1 exposure, triggers an intracellular signaling cascade involving a kinase (p38β MAPK), which in turn allows the nuclear translocation of the activator protein-1 (AP-1) and NF-κB, finally leading to the release of pro-inflammatory cytokines. Furthermore, a p38β MAPK negative role in cytoprotective genes regulation was postulated. Overall, our investigations improved the actual knowledge on the molecular effects of this worldwide relevant natural toxin in cattle.

Low syntaxin 17 expression in donor liver is associated with poor graft prognosis in recipients of living donor liver transplantation.

Liver transplantation (LT) is the only curative therapy for decompensated liver cirrhosis. For recipients of living donor LT (LDLT), restoration of liver function after transplantation is highly dependent on liver regenerative capacity, which requires large amounts of intracellular energy. Mitochondrial metabolism provides a stable supply of adenosine 5'-triphosphate (ATP) for liver regeneration. Mitophagy is a selective process in which damaged, non-functional mitochondria are degraded and replaced with new functional mitochondria. We investigated the relationship between expression of Syntaxin17 (STX17), a key protein in mitophagy regulation, in donor livers and graft survival. We examined STX17 expression in grafts from 143 LDLT donors who underwent right lobe resection and investigated the relationship between STX17 expression and graft function. We investigated the correlations among STX17 expression, mitochondrial membrane potential and cell proliferation, using a STX17-knockdown hepatocyte cell line. Recipients transplanted with low STX17-expression grafts had significantly lower graft survival rates than recipients transplanted with high STX17-expression grafts (88.9% vs. 100%, p<0.01). Multivariate analysis showed that low STX17 expression (HR: 10.7, CI: 1.29-88.0, p<0.05) and the absence of splenectomy (HR: 6.27, CI: 1.59-24.8, p<0.01) were independent predictive factors for small-for-size graft syndrome, which is the severe complication in LDLT. In the vitro experiments, the percentage of depolarized damaged mitochondria was increased in the STX17-knockdown hepatocyte cell line, suggesting decreased mitophagy and ATP synthesis. Cell proliferation was significantly decreased in the STX17-knockdown hepatocyte cell line. STX17 contributes to mitophagy and maintenance of mitochondrial function in hepatocytes and may be a predictor of graft dysfunction in LDLT patients.

Circular dorsal ruffles disturb the growth factor-induced PI3K-AKT pathway in hepatocellular carcinoma Hep3B cells

BackgroundCircular dorsal ruffles (CDRs) are rounded membrane ruffles induced on the dorsal surfaces of cells stimulated by growth factors (GF). They can serve as signal platforms to activate AKT protein kinase. After GF stimulation, phosphatidylinositol 3-kinase (PI3K) generates phosphatidylinositol (3,4,5)-triphosphate (PIP3) in the plasma membrane. PIP3 accumulates inside CDRs, recruits AKT into the structures, and phosphorylates them (pAKT). Given the importance of the PI3K-AKT pathway in GF signaling, CDRs are likely involved in cell growth. Interestingly, some cancer cell lines express CDRs. We hypothesized that CDRs contribute to carcinogenesis by modulating the AKT pathway. In the present study, we identified CDR-expressing cancer cell lines and investigated their cellular functions.MethodsCDR formation was examined in six cancer cell lines in response to epidermal growth factor (EGF) and insulin. The morphology of the CDRs was characterized, and the related signaling molecules were observed using confocal and scanning electron microscopy. The role of CDRs in the AKT pathway was studied using biochemical analysis. The actin inhibitor cytochalasin D (Cyto D) and the PI3K inhibitor TGX221 were used to block CDRs.ResultsGF treatment induced CDRs in the hepatocellular carcinoma (HCC) Hep3B cell line, but not in others, including HCC cell lines HepG2 and Huh7, and the LO2 hepatocyte cell line. Confocal microscopy and western blot analysis showed that the PI3K-PIP3-AKT pathway was activated at the CDRs and that receptor proteins were recruited to the structures. Cyto D and TGX221 completely blocked CDRs and partially attenuated GF-induced pAKT. These results indicate that CDRs regulate the receptor-mediated PI3K-AKT pathway in Hep3B cells and the existence of CDR-independent pAKT mechanisms.ConclusionsOur results showed that CDRs modulate the AKT pathway in Hep3B cells. Since CDRs were not observed in other HCC and hepatocyte cell lines, we propose that CDRs in Hep3B would determine the carcinoma characteristic of the cell by aberrantly triggering the AKT pathway. Signaling molecules involved in CDR formation are promising therapeutic targets for some types of HCC.9eb9aTWDk6mAQeLNgQGeJaVideo abstract

Geraniol ve vitamin C’nin dietilnitrozamin kaynaklı deneysel hepatoselüler karsinogenez üzerindeki etkisi

Purpose: This study aimed to investigate the protective effect of geraniol and vitamin C on the experimental hepatocellular carcinogenesis (HCC) model by inducing FL83B hepatocyte cell lines with diethylnitrosamine (DENA).&#x0D; Materials and Methods: The cells prepared in the medium were incubated with DENA (5 μM), geraniol (5 μM), and vitamin C (50 μM) for 48 hours in an incubator at 37 °C and 5% CO2. Groups were designed as follows: Group 1 (Control), group 2 (DENA Control), group 3 (DENA+Geraniol), group 4 (DENA+Vitamin C), and group 5 (DENA+Geraniol+Vitamin C) on standard cell culture plates. Six plates from each experimental group were studied. After the homogenization was centrifuged, analyses of pathway mediators NF-ĸB, AIF, caspase-3, BCL-2, bax, gadd153, GRP78, and COX were performed by the Elisa method. &#x0D; Results: The expression of Bax, caspase-3, COX-2, NFkB, GADD153, AIF, and GRP78 increased in cancer cells when compared to group 1 and decreased in other groups where antiproliferative agents were applied. Bcl-2 expression is decreased when compared to group 1, and expression is increased in other groups where antiproliferative agents are applied. &#x0D; Conclusion: There was a significant hepatoprotective effect in the groups administered geraniol+vitamin C on pathway mediators in a DENA-induced HCC model.

3D-Epigenomic Regulation of Gene Transcription in Hepatocellular Carcinoma.

The fundamental cause of transcription dysregulation in hepatocellular carcinoma (HCC) remains elusive. To investigate the underlying mechanisms, comprehensive 3D-epigenomic analyses are performed in cellular models of THLE2 (a normal hepatocytes cell line) and HepG2 (a hepatocellular carcinoma cell line) using integrative approaches for chromatin topology, genomic and epigenomic variation, and transcriptional output. Comparing the 3D-epigenomes in THLE2 and HepG2 reveal that most HCC-associated genes are organized in complex chromatin interactions mediated by RNA polymerase II (RNAPII). Incorporation of genome-wide association studies (GWAS) data enables the identification of non-coding genetic variants that are enriched in distal enhancers connecting to the promoters of HCC-associated genes via long-range chromatin interactions, highlighting their functional roles. Interestingly, CTCF binding and looping proximal to HCC-associated genes appear to form chromatin architectures that overarch RNAPII-mediated chromatin interactions. It is further demonstrated that epigenetic variants by DNA hypomethylation at a subset of CTCF motifs proximal to HCC-associated genes can modify chromatin topological configuration, which in turn alter RNAPII-mediated chromatin interactions and lead to dysregulation of transcription. Together, the 3D-epigenomic analyses provide novel insights of multifaceted interplays involving genetics, epigenetics, and chromatin topology in HCC cells.

Analysis of time-series gene expression data to explore mechanisms of isoniazid-induced liver toxicity.

Isoniazid (INH) is a first-line anti-tuberculosis drug which can cause idiosyncratic liver injury, while the underlying mechanisms need to be further elucidated. In this study, we explored the time series gene expression profiling of a hepatocyte cell line under isoniazid treatment. Through cluster analysis and enrichment analysis of differentially expressed genes, we revealed a total of 6 gene clusters and a series of pathways related to hepatotoxicity, and 13 key candidate genes were identified according to the protein-protein interaction (PPI) network analysis and maSigPro analysis. These findings lay a foundation for understanding the mechanisms of isoniazid -induced liver toxicity and provide new target genes for the monitoring and treatment of INH-induced hepatotoxicity in the future.

Abstract 5398: Apelinergic signalling in hepatocellular carcinoma (HCC): A new therapeutic treatment option

Abstract HCC is the third most frequent cancer-related death worldwide and incidence continues to rise. Most patients presenting with advanced stage disease where their treatment goal is palliation, emphasizing the requirement for new treatment options. HCC is characterized by arterialization of its blood supply deemed pathognomonic for its development. We investigated the neoangiogenic peptide apelin as a driver of carcinogenesis in HCC followed by the efficacy of novel apelin antagonists as a therapeutic strategy in-vitro. Firstly, meta-analysis conducted through the TCGA database showed that apelin is significantly upregulated in liver cancer compared to healthy tissue and this correlated with a worse mean overall survival. Additionally, enzyme-linked immunosorbent assay (ELISA) of patient serum samples corroborated that apelin concentration significantly increases from healthy liver to cirrhosis and then further through tumorigenesis. In-vitro studies across multiple hepatocyte cell lines in media conditioned with elevated serum apelin levels were then performed to investigate the effects of increased apelin. All cell-lines demonstrated an increase in migration and proliferative properties with naturally higher basal apelin expressing cell lines showing the most significant changes. Using knockdown models, we elucidated hypoxia a well-known driver of angiogenesis, up-regulates apelin expression through a HIF-1α dependent mechanism. Furthermore, increased apelin expression resulted in upregulation of its G-protein coupled receptor, APJ which initiates its downstream signaling pathways, together referred to as apelinergic signaling, demonstrating an autocrine loop. Co-culture of hepatocytes with hepatic stellate cells (HSCs), activated in cirrhosis, further elucidated a paracrine signaling loop whereby secreted chemokines from HSCs, which including apelin, enhanced hepatocyte proliferation and migration. Assessing apelin as a therapeutic target, several novel antagonists were designed in-silico and investigated across multiple hepatocyte cell lines. MM315 showed the most promise with significant cytostatic properties at nanomolar levels coupled with a high binding affinity to APJ at picomolar levels demonstrated through blocking studies. As HCC is a highly vascularized cancer, we explored the likely effects of these antagonists on endothelial cells, cultured in the presence of IC50 concentrations of each antagonist. MM315 treatment resulted in a significant reduction of abnormal blood vessel formation. Overall, our results suggest inhibiting apelinergic signaling is a promising therapeutic treatment for HCC, with MM315 being a lead compound. In-vivo work which will be presented. Citation Format: Jamie Beaumont, Eric Aboagye, Beata Wojciak-Stothard, Gilberto Serrano-De-Almeida, Robert Glen, Rohini Sharma. Apelinergic signalling in hepatocellular carcinoma (HCC): A new therapeutic treatment option [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5398.