Rat Hepatoma Cells Research Articles

03:18 PM Abstract No. 220 Retinoic acid enhances doxorubicin-induced cytotoxicity in rat hepatocellular carcinoma cell lines

03:18 PM Abstract No. 220 Retinoic acid enhances doxorubicin-induced cytotoxicity in rat hepatocellular carcinoma cell lines

Glutamate–oxaloacetate transaminase activity promotes palmitate lipotoxicity in rat hepatocytes by enhancing anaplerosis and citric acid cycle flux

Hepatocyte lipotoxicity is characterized by aberrant mitochondrial metabolism, which predisposes cells to oxidative stress and apoptosis. Previously, we reported that translocation of calcium from the endoplasmic reticulum to mitochondria of palmitate-treated hepatocytes activates anaplerotic flux from glutamine to α-ketoglutarate (αKG), which subsequently enters the citric acid cycle (CAC) for oxidation. We hypothesized that increased glutamine anaplerosis fuels elevations in CAC flux and oxidative stress following palmitate treatment. To test this hypothesis, primary rat hepatocytes or immortalized H4IIEC3 rat hepatoma cells were treated with lipotoxic levels of palmitate while modulating anaplerotic pathways leading to αKG. We found that culture media supplemented with glutamine, glutamate, or dimethyl-αKG increased palmitate lipotoxicity compared with media that lacked these anaplerotic substrates. Knockdown of glutamate-oxaloacetate transaminase activity significantly reduced the lipotoxic effects of palmitate, whereas knockdown of glutamate dehydrogenase (Glud1) had no effect on palmitate lipotoxicity. 13C flux analysis of H4IIEC3 cells co-treated with palmitate and the pan-transaminase inhibitor aminooxyacetic acid confirmed that reductions in lipotoxic markers were associated with decreases in anaplerosis, CAC flux, and oxygen consumption. Taken together, these results demonstrate that lipotoxic palmitate treatments enhance anaplerosis in cultured rat hepatocytes, causing a shift to aberrant transaminase metabolism that fuels CAC dysregulation and oxidative stress.

Anti-tumor effects of mAb against L-type amino acid transporter 1 (LAT1) bound to human and monkey LAT1 with dual avidity modes.

l‐Type amino acid transporter 1 (LAT1) disulfide linked to CD98 heavy chain (hc) is highly expressed in most cancer cells, but weakly expressed in normal cells. In the present study, we developed novel anti‐LAT1 mAbs and showed internalization activity, inhibitory effects of amino acid uptake and cell growth and antibody‐dependent cellular cytotoxicity, as well as in vivo antitumor effects in athymic mice. Furthermore, we examined the reactivity of mAbs with LAT1 of Macaca fascicularis to evaluate possible side‐effects of antihuman LAT1 mAbs in clinical trials. Antihuman LAT1 mAbs reacted with ACHN human and MK.P3 macaca kidney‐derived cells, and this reactivity was significantly decreased by siRNAs against LAT1. Macaca LAT1 cDNA was cloned from MK.P3, and only two amino acid differences between human and macaca LAT1 were seen. RH7777 rat hepatoma and HEK293 human embryonic kidney cells expressing macaca LAT1 were established as stable transfectants, and antihuman LAT1 mAbs were equivalently reactive against transfectants expressing human or macaca LAT1. Dual (high and low) avidity modes were detected in transfectants expressing macaca LAT1, MK.P3, ACHN and HCT116 human colon cancer cells, and KA values were increased by anti‐CD98hc mAb, suggesting anti‐LAT1 mAbs detect an epitope on LAT1‐CD98hc complexes on the cell surface. Based on these results, LAT1 may be a promising anticancer target and Macaca fascicularis can be used in preclinical studies with antihuman LAT1 mAbs.

SAR and molecular mechanism studies of monoamine oxidase inhibition by selected chalcone analogs

The present study describes the synthesis of a series of 22 chalcone analogs. These compounds were evaluated as potential human MAO-A and MAO-B inhibitors. The compounds showed varied selectivity against the two isoforms. The IC50 values were found to be in the micromolar to submicromolar range. The Ki values of compound 16 were determined to be 0.047 and 0.020 μM for the inhibition of MAO-A and MAO-B, respectively. Dialysis of enzyme-inhibitor mixtures indicated a reversible competitive mode of inhibition. Most of the synthesized chalcone analogs showed a better selectivity toward MAO-B. However, introducing of 2,4,6-trimethoxy substituents on ring B shifted the selectivity toward MAO-A. In addition, we investigated the molecular mechanism of MAO-B inhibition by selected chalcone analogs. Our results revealed that these selected chalcone analogs increased dopamine levels in the rat hepatoma (H4IIE) cells and decreased the relative mRNA expression of the MAO-B enzyme.

Orthotopic hepatocellular carcinoma: molecular imaging-monitored intratumoral hyperthermia-enhanced direct oncolytic virotherapy

Objective: To validate the feasibility of molecular imaging-monitored intratumoral radiofrequency hyperthermia (RFH) enhanced direct oncolytic virotherapy for hepatocellular carcinoma (HCC).Methods: This study included in vitro experiments using luciferase-labeled rat HCC cells and in vivo validation experiments on rat models with orthotopic HCCs. Both cells and HCCs in four groups (n = 6/group) were treated by: (1) combination therapy of oncolytic virotherapy (T-VEC) plus RFH at 42 °C for 30 min; (2) oncolytic virotherapy alone; (3) RFH alone; and (4) saline. For in vitro confirmation, confocal microscopy and bioluminescence optical imaging were used to evaluate the cell viabilities. For in vivo validation, oncolytic viruses were directly infused into rat HCCs through a multi-functional perfusion-thermal RF electrode, followed by RFH. Ultrasound and optical imaging were used to follow up size and bioluminescence signal changes of tumors overtime, which were correlated with subsequent laboratory examinations.Results: For in vitro experiments, confocal microscopy showed the lowest number of viable cells, as well as a significant decrease of bioluminescence signal intensity of cells with combination therapy group, compared to other three groups (p < .001). For in vivo experiments, ultrasound and optical imaging showed the smallest tumor volume, and significantly decreased bioluminescence signal intensity in combination therapy group compared to other three groups (p < .05), which were well correlated with pathologic analysis.Conclusion: It is feasible of using molecular imaging to guide RFH-enhanced intratumoral oncolytic virotherapy of HCC, which may open new avenues to prevent residual or recurrent disease of thermally ablated intermediate-to-large HCCs.

Comparative analyses of cellular physiological responses of non-target species to cypermethrin and its formulated product: Contribution to mode of action research

Physiological responses of bacterial, fish, rat and human hepatoma cells to the technical cypermethrin (AS), cypermethrin-based plant protection product (PPP), and the major co-formulant (solvent) were compared. The endpoints included: bioluminescence, total protein content, activity of mitochondrial dehydrogenase and cytochrome P450 (CYP) enzymes CYP1A and CYP1B, and expression of several genes encoding different CYP enzyme isoforms. Toxicity of PPP was compared with the toxicity predicted using concentration addition model. Cypermethrin disturbs the activity of mitochondrial dehydrogenase. Induction of CYP1A1-, CYP1A2- and CYP1B1-associated activity was more pronounced in PPP than in cypermethrin treatment. The predominant biotransformation pathway of cypermethrin is related to Cyp3a1 induction. Deviations between observed and predicted toxicity of PPP indicate synergistic effects of cypermethrin and a solvent. In vitro cellular assays may serve as rapid pre-screening tool and provide for a good indication of mixture effects and prompt further in vivo testing of PPPs when really needed.

Vanadate inhibits transcription of the rat insulin receptor gene via a proximal sequence of the 5′flanking region

Vanadate, a protein tyrosine phosphatase inhibitor which elicits insulin-like effects, has previously been shown to inhibit expression of the insulin receptor gene at the transcriptional level in rat hepatoma cells. In an attempt to identify the DNA sequence and transcription factors potentially involved in this effect, a fragment of the proximal 5′flanking region of the IR gene (−1143/−252 upstream the ATG codon) has been cloned and functionally characterized. RNase protection allowed the identification of several transcription start sites in the conserved region of the gene, among which two major sites at −455 and −396. Upon fusion to the luciferase gene and transient transfection into hepatoma cells, the −1143/−252 fragment showed promoter activity. This was unaffected by deletion of the −1143/−761 sequence, but markedly decreased (90%) by additional deletion of the −760/−465 sequence. Treatment of hepatoma cells with vanadate led to a dose-dependent decrease in promoter activity of the 1143/−252, −760/−252 and −464/−252 constructs (change relative to untreated cells, 40, 55 and 23% at 125 μM, and 70, 85 and 62% at 250 μM, respectively). These data suggest that although the entire DNA sequence upstream the transcription start sites is probably involved in vanadate-induced inhibition, the short sequence downstream of position −464 and is sufficient for inhibition. Potential targets of vanadate are the transcription factors FoxO1 and HMGA1, two downstream targets of the insulin signaling pathway which have been shown to mediate the inhibitory effect of insulin on IR gene expression.

A potential role as a suppressor protein of Regucalcin in the development of human carcinogenesis

Regucalcin was discovered in 1978 as a calcium-binding protein. After that, regucalcin was demonstrated to play a multifunctional role as a suppressor protein in signal transduction in various types of cell and tissues. The regucalcin gene (rgn) is localized on the X chromosome. Regucalcin was found to suppress nuclear deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis in liver cells. Overexpression of endogenous regucalcin possessed suppressive effects on proliferation in the modeled rat hepatoma cells by inhibiting G1 and G2/M cell cycle arrests. Suppressed regucalcin gene expression was found to be associated with progression of hepatocarcinogenesis by proteosome analysis. Moreover, regucalcin mRNA expression was found to suppress in various human normal and tumor tissues including hepatocellular carcinoma, kidney transitional cell carcinoma, brain malignant meningioma, and lung non-small cell carcinoma of human subjects. Suppressed regucalcin gene expression may be a key in development of carcinogenesis. Development of the regucalcin gene deliver system will be expected as a novel gene therapy in clinical aspects for cancer treatment.

Inhibition of tumor formation and metastasis by a monoclonal antibody against lymphatic vessel endothelial hyaluronan receptor 1.

Although cancer metastasis is associated with poor prognosis, the mechanisms of this event, especially via lymphatic vessels, remain unclear. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1) is expressed on lymphatic vessel endothelium and is considered to be a specific marker of lymphatic vessels, but it is unknown how LYVE‐1 is involved in the growth and metastasis of cancer cells. We produced rat monoclonal antibodies (mAb) recognizing the extracellular domain of mouse LYVE‐1, and investigated the roles of LYVE‐1 in tumor formation and metastasis. The mAb 38M and 64R were selected from hybridoma clones created by cell fusion between spleen cells of rats immunized with RH7777 rat hepatoma cells expressing green fluorescent protein (GFP)‐fused mouse LYVE‐1 proteins and mouse myeloma cells. Two mAb reacted with RH7777 and HEK293F human embryonic kidney cells expressing GFP‐fused mouse LYVE‐1 proteins in a GFP expression‐dependent manner, and each recognized a distinct epitope. On immunohistology, the 38M mAb specifically stained lymphatic vessels in several mouse tissues. In the wound healing assay, the 64R mAb inhibited cell migration of HEK293F cells expressing LYVE‐1 and mouse lymphatic endothelial cells (LEC), as well as tube formation by LEC. Furthermore, this mAb inhibited primary tumor formation and metastasis to lymph nodes in metastatic MDA‐MB‐231 xenograft models. This shows that LYVE‐1 is involved in primary tumor formation and metastasis, and it may be a promising molecular target for cancer therapy.

The aryl hydrocarbon receptor (AhR) activity and DNA-damaging effects of chlorinated polycyclic aromatic hydrocarbons (Cl-PAHs)

An increasing number of studies have indicated that environmental contamination with chlorinated polycyclic aromatic hydrocarbons (Cl-PAHs) has been underestimated. However, insufficient available toxicological information on Cl-PAHs makes evaluating their risks to health challenging. Two in vitro bioassays were used in the present study to characterize the aryl hydrocarbon receptor (AhR) activity and DNA-damaging effects of 22 low-molecular-weight PAHs and their Cl-PAHs by using the EROD assay in rat hepatoma (H4IIE) cells and the SOS/umu test (S. typhimurium TA1535/pSK1002). Compared with their parent PAHs, most of the Cl-PAHs enhanced AhR-mediated activity in the EROD assay. 1,3,6,8-Tetrachloro-pyrene (1,3,6,8-Tetracl-Py) induced the greatest potency of EROD activity (83.1%-TCDD-max) and its single point ReP was 6.64 × 10−6. Compared with their parent PAHs, several Cl-PAHs showed significant DNA-damaging effects in the SOS/umu test with the addition of S9, and the toxic equivalency of benzo[a]pyrene (TEQBaP) was calculated for them. 9-Chloroanthracene (9-Ant) and 5,6-Dichloroacenaphthene (5,6-Dicl-Ace) had relatively high TEQBaP (0.62 and 0.54, respectively). However, only 1,3,6,8-Tetracl-Py elicited strong DNA-damaging effects in the absence of S9. The degree of chlorination, the position of chlorine substitutions, and the structure of parent PAHs influenced the potency of low-molecular-weight PAHs with regard to their AhR activity and DNA-damaging effects. More concern should be raised for these environmentally relevant pollutants.

An effective protocol for pharmacological defatting of primary human hepatocytes which is non-toxic to cholangiocytes or intrahepatic endothelial cells.

IntroductionPharmacological defatting of rat hepatocytes and hepatoma cell lines suggests that the same method could be used to ameliorate macrovesicular steatosis in moderate to severely fatty livers. However there is no data assessing the effects of those drugs on primary human liver cells. We aimed to determine the effectiveness of a pharmacological cocktail in reducing the in vitro lipid content of primary human hepatocytes (PHH). In addition we sought to determine the cytotoxicity of the cocktail towards non-parenchymal liver cells.MethodsSteatosis was induced in PHH by supplementation with a combination of saturated and unsaturated free fatty acids. This was followed by addition of a defatting drug cocktail for up to 48 hours. The same experimental method was used with human intra-hepatic endothelial cells (HIEC) and human cholangiocytes. MTT assay was used to assess cell viability, triglyceride quantification and oil red O staining were used to determine intracellular lipids content whilst ketone bodies were measured in the supernatants following experimentation.ResultsIncubation of fat loaded PHH with the drugs over 48 hours reduced the intracellular lipid area by 54%, from 12.85% to 5.99% (p = 0.002) (percentage of total oil red O area), and intracellular triglyceride by 35%, from 28.24 to 18.30 nmol/million of cells (p<0.001). Total supernatant ketone bodies increased 1.4-fold over 48 hours in the defatted PHH compared with vehicle controls (p = 0.002). Moreover incubation with the drugs for 48 hours increased the viability of PHH by 11%, cholangiocytes by 25% whilst having no cytotoxic effects on HIEC.ConclusionThese data demonstrate that pharmacological intervention can significantly decrease intracellular lipid content of PHH, increase fatty acids β-oxidation whilst being non-toxic to PHH, HIEC or cholangiocytes.

Lymphocyte-Specific Protein-1 Controls Sorafenib Sensitivity and Hepatocellular Proliferation through Extracellular Signal-Regulated Kinase 1/2 Activation

The gene leukocyte-specific protein-1 (LSP1), encodes an F-actin binding protein that directly interacts with the mitogen-activated protein kinase pathway. LSP1 has copy number variations in 52% of human hepatocellular carcinoma (HCC). LSP1 suppresses proliferation and migration in hepatocytes. LSP1 binds to the rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein/extracellular signal-regulated kinase (ERK)/ERK signaling cassette, the target for sorafenib, a crucial chemotherapeutic agent for HCC. This study addresses the role of LSP1 in liver regeneration and sensitivity to sorafenib in normal and neoplastic hepatocytes. Two mouse models, an Lsp1 global knockout (LSP1KO) and a hepatocyte-specific Lsp1 transgenic (LSP1TG) mouse, were used. After two-thirds hepatectomy (PHx), LSP1KO mice displayed increased proliferation and ERK activation, whereas LSP1TG mice displayed suppressed proliferation and decreased ERK activation. LSP1KO hepatocytes cultured without growth factors exhibited increased proliferation, whereas LSP1TG hepatocytes showed decreased proliferation. Rat and human hepatoma cells expressing Lsp1 shRNA displayed increased sensitivity to sorafenib, as evidenced by decreased cell numbers and phosphorylated ERK expression compared with control. LSP1 KO mice treated with sorafenib before PHx displayed decreased hepatocyte proliferation. Our data show that loss of LSP1 function, observed in HCC, leads to increased sensitivity to sorafenib treatment and enhanced hepatocellular proliferation after PHx in vivo and in cultured cells.

In vitro toxicity and in silico docking analysis of two novel selective AH-receptor modulators

The mediator of dioxin toxicity, aryl hydrocarbon receptor (AHR), has also important physiological functions. Selective AHR modulators (SAHRMs) share some effects of dioxins, except for their marked toxicity. We recently characterised toxicologically two novel SAHRMs, prodrugs IMA-08401 and IMA-07101 in rats, demonstrating that they are far less deleterious than the most toxic AHR-agonist, TCDD. Here, we analysed the in vitro toxicity and in silico AHR binding of the respective active, deacetylated metabolites, IMA-06201 (N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-quinoline-3-carboxamide) and IMA-06504 (N-(4-trifluoromethylphenyl)-1,2-dihydro-4-hydroxy-5-methoxy-1-methyl-2-oxo-quinoline-3-carboxamide). In H4IIE rat hepatoma cells, IMA-06201 and IMA-06504 induced CYP1A1 with comparable potencies and efficacies to those of TCDD. They had little effect on cell viability as assessed by LDH leakage and MTT reduction assays, and were not mutagenic in the Ames test, but IMA-06504 elicited a maximally 2.7-fold increase in micronuclei. Molecular docking simulations showed that similar to TCDD, they occupy the central region of AHR ligand binding cavity. Hence, while showing low to negligible in vitro toxicity, these novel SAHRMs bind to the AHR qualitatively in a similar fashion to TCDD, and appear comparably powerful AHR agonists. Combined with our earlier results demonstrating that they seem considerably less toxic in vivo than TCDD, these compounds are thus highly interesting new SAHRMs.

In vitro and in vivo efficacies of carbazole aminoalcohols in the treatment of alveolar echinococcosis

Benzimidazoles, including albendazole and mebendazole, are the major drugs for clinical chemotherapy of echinococcosis. They mainly exert parasitostatic effects depending on high dosages for long-term. Previous studies have identified carbazole aminoalcohols as novel anti-CE (cystic echinococcosis) agents. However, it is still to be confirmed whether it is effective on alveolar echinococcosis (AE) or not. In the present study, efficacies of novel carbazole aminoalcohols, propylamine, R-propylamine and S-propylamine were evaluated under in vitro and in vivo conditions. Carbazole aminoalcohols were tested against Echinococcus multilocularis (E. multilocularis) protoscoleces (PSC) in vitro. The effects of propylamine and R-propylamine exhibited a time-dependent manner at different concentrations, while the effect of S-propylamine was very poor. At a concentration of 20 μM, the mortality of PSC achieved to 100% on the 11th day after exposure to R-propylamine. The treatment of carbazole aminoalcohols to infected mice resulted in statistically significant reductions in the cyst weights compared with those obtained from negative control mice (p < 0.05), and no significant differences were found between albendazole and carbazole aminoalcohols (p > 0.05). The cytotoxicity examination in rat hepatoma (RH) cells indicated that propylamine and R/S-propylamine were lower that of albendazole at a low concentration (5 μM). In addition, histopathological observation of organs (liver, spleen and kidney) for experimental mice showed mild inflammatory changes in the liver and spleen. This study reveals the potential of carbazole aminoalcohols as a class of novel anti-AE agents.

Abstract 178: Bioactive Compounds in Fenugreek Ameliorate ER Stress and VLDL Overproduction via the Mediation of Insig Signaling

A number of health-enhancing bioactive compounds have been identified in the seed of fenugreek, an annual legume. These compounds have been shown to exert multiple health beneficial effects on anti-obesity and type 2 diabetes. In this study, we characterized the molecular mechanisms of fenugreek seed in regulating lipid and lipoprotein metabolism and metabolic inflammatory stress. Two groups of hyperlipidemic mice induced by depletion of cAMP responsive element binding protein H were fed either a chow containing 2% fenugreek seed or vehicle control for 7 weeks. Q-RT-PCR and immunoblotting analysis demonstrated that fenugreek seed containing diet inhibited hepatic SREBP-1c activation and the subsequent de novo lipogenesis by enhancing expression of insulin-inducible gene-1 (Insig-1) and gene-2 (Insig-2). mRNA expression of PPARα and its target genes that were involved in fatty acid β-oxidation were also upregulated in the fenugreek seed treated-mice which was accompanied by significantly reduced hepatic lipid accumulation and VLDL secretion and improved endoplasmic reticulum (ER) stress. These actions ameliorated hepatic steatosis and systemic hyperlipidemia. Fenugreek seed further improved insulin sensitivity by upregulating expression of glucose transporters Glut-2 and Glut-4 and enhancing the activity of hepatic inulin signaling molecules, insulin receptor and AKT. In vitro, treating a rat hepatoma cell line, McA-RH7777, with trigonelline, a phytochemical compound in fenugreek seed, increased expression of Insig-2 which prevented the activation of SREBP-1c and the subsequent de novo lipid synthesis, resulting in less secretion of VLDL. Trigonelline treatment also attenuated ER stress induced by a free fatty acid, palmitic acid, indicated by the downregulation of GRP94/78 and reduced phosphorylation of JNK and eIF2α. This study unveiled a novel mechanism of the bioactive compound trigonelline in ameliorating metabolic inflammatory stress, hepatic steatosis, VLDL overproduction and insulin resistance via the mediation of Insig signaling. This evidence lands support for developing fenugreek seed as a nutraceutical supplement for the prevention and treatment of nutrient-surplus associated metabolic disorders.

Abstract 601: MicroRNAs and Hedgehog Signaling in the Pathogenesis of Progressive Liver Injury from NAFLD to Fibrosis

Non-alcoholic fatty liver disease (NAFLD) is very common (~70%) in subjects with diabetes and is associated, independently of several confounding factors, with an increased risk of cardiovascular disease (CVD). Diabetes is an independent risk factor for progression of liver fibrosis. Recent studies in human subjects with NAFLD and animal models have demonstrated that microRNAs, a group of noncoding small RNAs, are intimately involved in the development and progression of liver injury, and act to alter expression of genes involved in lipid metabolism and apoptosis. MiR-34a and miR-29 family were among the most frequently dysregulated miRNAs in NAFLD. Thus, the objective of this study was to determine the role of miR-34a and miR-29 in the onset and progression of hepatic steatosis to liver fibrosis in a diabetic mouse model. We showed that streptozotocin (STZ)-induced diabetes predisposed mice to a high-fat-diet (HFD) induced severe liver injury in a much short time course compared to non-diabetic mice. The livers exhibited the development of a spectrum pathological change, with hepatic steatosis at week 6, NASH at week 8 and liver fibrosis at week 12 upon high fat feeding. More importantly, the progressive live injury was closely associated with significantly increased or reduced expression of miR-34a and miR-29 family, respectively, resulting in the activation of hedgehog signaling and the markers of fibrogenesis, Col1A1 and alpha-SMA. Treatment with nanoparticles carrying miR-29b1 mimics improved insulin sensitivity and prevented HFD induced liver fibrosis. In vitro, treatment of McA-RH7777 cells, a rat hepatoma cell line, with a hedgehog signaling inhibitor MDB5 prevented overexpression of miR-34a induced by a free fatty acid, palmitic acid but significantly enhanced expression of miR-29 family in both hepatocytes and hepatic stellate cells. This action hindered the activation of fibrogenesis by lipotoxicity and inflammatory cytokine TNFα. Our study, for the first time, delineates the interaction between miR-34a and miR-29 family and hedgehog signaling in the development of NAFLD and liver fibrosis. This novel finding may provide rationale for developing miRNAs as pharmaceutical targets for the prevention and treatment of liver injury.

Role of COUP‐TFII in Glucose Homeostasis in Dahl Salt Sensitive Rats

The Interaction between genetic and environmental factors plays an important role in disease progression. One of the major environmental factor includes diets, which governs significant attention based on the broad spectrum of effect in cardiovascular and associated diseases. Consumptions of diets that are rich in calories and salts can significantly increases the risk for developing life threatening cardiovascular events including hypertension and metabolic syndrome. Through GWAS studies, a locus with significant association for hypertension was found in human chromosome 15 containing the gene Chicken Ovalbumin Upstream Promotor Transcription Factor II (COUP‐TFII). To validate this gene, a mutant rat was developed with a 15‐base pair deletion in Dahl Salt Sensitive (SS) rat background. Under high salt diet regimen, the Coup‐TfIImutant rat had an improved cardiac and renal function when compared to SS rats. Being a known transcriptional regulator, Coup‐TfII also plays a role in other physiological events including glucose homeostasis and energy metabolism. In H4IIE rat hepatoma cells, Coup‐TfII expression modulated in response to glucose levels; under high glucose condition Coup‐TfII expression was significantly down‐regulated (p&lt; 0.05); whereas in low glucose condition Coup‐TfII expression was significantly up‐regulated (p&lt; 0.05). In addition, Coup‐TfII protein expression is found to negatively correlate with insulin regardless on the amount of glucose present in H4IIE cells (p&lt; 0.05). Similar to our in‐vitro findings, Coup‐TfII gene expression was significantly up‐regulated in SS rat liver during fasting as compared to re‐fed condition (p&lt; 0.05). Coup‐TfIImutant rat have also showed a significant decrease in fasting blood glucose levels (p&lt; 0.05) compared to SS rat along with a significant decrease in their body weight (p&lt; 0.05). Furthermore, the Coup‐TfIImutant rat demonstrated a superior glucose response and insulin response as measured by glucose tolerance test (GTT) and Insulin tolerance test (ITT) (p&lt; 0.05). Further, under fasting condition the mRNA expression analysis in livers of SS and CoupTfIImutant rats revealed that the key enzymes involved in both beta oxidation and gluconeogenesis was significantly down regulated in Coup‐TfIImutant rats compared to SS rats (p&lt; 0.05). Overall, our preliminary findings suggest that Coup‐TfII is important for glucose homeostasis in SS rats. Further understanding of Coup‐TfII function will help to dissect the relationship between metabolic syndrome and blood pressure regulation in SS rats.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glypican 3 (GPC3)‐CD81 axis regulates Ezrin mediated Hippo pathway via cross talking with HGF/c‐Met axis in hepatocytes and hepatocellular carcinoma (HCC)

The experiments that integrate different pathways in normal and abnormal circumstances will help us to develop optimum targeted therapies because of multiple pathways involved in the solid tumor carcinogenesis. We have shown that activation of CD81 is associated with phosphorylation of Ezrin, which is overexpressed in many types of solid tumors and has been identified as metastasis related protein for many years. We have also shown that GPC3 binds to the membrane tetraspanin CD81, which is one of the portals of entry of hepatitis C virus (HCV). Previous studies have shown that GPC3 is associated with inhibition of hepatocyte growth and liver regeneration by inhibiting CD81‐mediated p‐Ezrin/Hippo/Yap pathway. It also has been shown that HGF/Met axis which promotes growth in HCC can phosphorylate Ezrin as well by different kinases. Since we have demonstrated the predominant GPC+++/CD81− pattern in HCC, the purpose of this study is to explore the regulation of expression and phosphorylation of Ezrin in HCC. We have found that CD81 expression could be detected in membrane and organelle fraction in human hepatoma cell lines HepG2, Huh7 and Hep3B, but immunoprecipitation experiment suggests that CD81 from latter two human hepatoma cell lines has lost association with GPC3. Human hepatoma cell lines have dramatically up regulated Ezrin expression and Yap activity. We found that treatment of primary rat hepatocytes with CD81 agonist antibody or HGF led to higher levels of phosphorylated Ezrin and lower Hippo activity. Mice overexpressing GPC3 had decreased p‐Ezrin but increased Hippo activity. In mice with post‐natal Met knock out there was less Yap expression at baseline level. Rat hepatoma cell lines JM1 and JM2 had dramatically down regulated CD81 expression and Hippo activity, and up regulated p‐Ezrin. Treatment with 20 umol of Ezrin inhibitor led to significant cell death selectively in JM2 cells in vitro.ConclusionsBoth HGF/Met and CD81 mediate p‐Ezrin activity and enhance nuclear Yap levels. These pathways in normal hepatocytes are inhibited by GPC3 and low HGF/Met activity. In human hepatocytes, GPC3 inhibits the CD81‐mediated p‐Ezrin effect on Hippo pathway and cause a decrease in nuclear Yap. The different distribution pattern of HGF/Met and GPC3/CD81 between normal hepatocytes and HCC cells provides the chance to develop precise anti‐HCC therapies. And Ezrin is a promising target of anti‐HCC therapies including primary lesion and metastasis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biotransformation and oxidative stress responses in rat hepatic cell-line (H4IIE) exposed to racemic ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP)

Pharmaceuticals such as racemate ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP) are highly detectable non-steroidal anti-inflammatory drugs (NSAIDs) in the aquatic environment and therefore are designated as one of the most emerging groups of pollutants that can affect environmental and human health. The potential impact of these pharmaceuticals was assessed for the first time in vitro using a rat hepatocellular carcinoma cell line (H4IIE). Cells were exposed to low and high concentrations of these drugs. Cytotoxicity was determined by MTT reduction assay; CYP1A1 transcriptional and enzymatic levels together with canonical oxidative stress responsive markers (GPx, GR, GST and CAT) were also investigated. Cells exposed to RS-KP and S(+)-KP did not show cytotoxicity effect at the concentrations tested. However, this study highlighted differences between RS-KP and S(+)-KP in most of the evaluated markers, showing compound-, concentration- and time-specific effect patterns which suggest a potential stereo-selective toxicity of these drugs.

Abstract No. 445 Hypoxic stress induces the overexpression of programmed death ligand 1 and chemokine ligand 17 on rat hepatoma cell lines

Abstract No. 445 Hypoxic stress induces the overexpression of programmed death ligand 1 and chemokine ligand 17 on rat hepatoma cell lines