Hepa1-6 Cells Research Articles

Hepatic serum- and glucocorticoid-regulated protein kinase 1 (SGK1) regulates insulin sensitivity in mice via extracellular-signal-regulated kinase 1/2 (ERK1/2).

Insulin resistance is a major hallmark of metabolic syndromes, including Type2 diabetes. Although numerous functions of SGK1 (serum- and glucocorticoid-regulated kinase 1) have been identified, a direct effect of SGK1 on insulin sensitivity has not been previously reported. In the present study, we generated liver-specific SGK1-knockout mice and found that these mice developed glucose intolerance and insulin resistance. We also found that insulin signalling is enhanced or impaired in Hep1-6 cells infected with adenoviruses expressing SGK1 (Ad-SGK1) or shRNA directed against the coding region of SGK1 (Ad-shSGK1) respectively. In addition, we determined that SGK1 inhibits ERK1/2 (extracellular-signal-regulated kinase 1/2) activity in liver and Ad-shERK1/2-mediated inhibition of ERK1/2 reverses the attenuated insulin sensitivity in Ad-shSGK1 mice. Finally, we found that SGK1 functions are compromised under insulin-resistant conditions and overexpression of SGK1 by Ad-SGK1 significantly ameliorates insulin resistance in both glucosamine-treated HepG2 cells and livers of db/db mice, a genetic model of insulin resistance.

Application of double-tagging in monitoring of tumorigenesis and tracking of mesenchymal stem cells in mice

To establish murine liver cancer cell line (Hepa1-6) and human mesenchymal stem cells (hMSCs) with dual-modality imaging lentiviral vector transfection and monitor the development of liver tumors and dynamically tracking of hMSCs by bioluminescent imaging in vivo. The cell lines of Hepa 1-6 and hMSCs were constructed abd tranfected with RL-eGFP and FL2-mKate2 lentiviruses respectively. Then the eGFP(+)-Hepa1-6 and mKate2(+)-hMSCs cell lines were selected by fluorescence activated cell sorting (FACS). The bioluminescent imaging technology was employed to evaluate their bioluminescent abilities in vitro and in vivo. And the capacity of mKate2(+)-hMSCs migrating to HCC cell line (eGFP(+)-Hepa1-6) was evaluated by Transwell migration assay. DNA sequencing analysis confirmed that gene sequences of lentiviral vectors were correct without mutation. We successfully constructed the lentivirus vector, produced lentivirus and infected Hepa1-6 and hMSCs respectively. Subsequently eGFP(+)-Hepa1-6 cells and mKate2(+)-hMSCs were obtained by FACS. The expressions of fluorescent protein eGFP and mKate were confirmed by fluorescence microscope. By bioluminescent imaging system, bioluminescence intensity strongly correlated with cell number. The Transwell assay showed that hMSCs migrated toward heptocellular carcinoma (HCC) cells. A lentivirus vector has been successfully constructed for infecting Hepa1-6 and hMSCs and analyzed by fluorescence and bioluminescent imaging. And that makes it technically feasible to further monitor the interaction of hMSCs and liver tumor cells. These results provide rationales for further studying the mechanism of MSCs in the development and progression of HCC.

Dihydromyricetin induces mouse hepatoma Hepal-6 cell apoptosis via the transforming growth factor-β pathway.

Dihydromyricetin (DHM) is a flavonoid compound which possesses potent antitumor activity. In the present study, it was demonstrated that DHM significantly inhibited proliferation and induced apoptosis in mouse hepatocellular carcinoma Hepal-6 cells. Transforming growth factor β (TGF-β) is recognized as a major profibrogenic cytokine and is therefore a common target for drugs in the treatment of liver disease. The present study aimed to investigate whether TGF-β was involved in DHM-triggered cell-viability inhibition and apoptosis induction. An MTT assay was used to evaluate the viability of Hepal-6 cells following DHM treatment. TGF-β signalling is mediated by Smads and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is a crucial regulator of reactive oxygen species ROS production. TGF-β, Smad3, phosphorylated (p)-Smad2/3 and NOX4 protein expression levels were evaluated by western blot analysis. TGF-β and NOX4 gene expression levels were determined by quantitative polymerase chain reaction. The results indicated that DHM downregulated TGF-β, Smad3, p-Smad2/3 and NOX4 in a concentration-dependent manner. A cell counting assay indicated that DHM also inhibited Hepal-6 cell growth in a concentration-dependent manner. TGF-β expression was significantly decreased following DHM treatment. In conclusion, the results of the present study defined and supported a novel function for DHM, indicating that it induced cell apoptosis by downregulating ROS production via the TGF-β/Smad3 signaling pathway in mouse hepatocellular carcinoma Hepal-6 cells.

SIN3A, Generally Regarded as a Transcriptional Repressor, Is Required for Induction of Gene Transcription by the Aryl Hydrocarbon Receptor

CYP1A1 bioactivates several procarcinogens and detoxifies several xenobiotic compounds. Transcription of CYP1A1 is highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the aryl hydrocarbon receptor. We recently described an RNAi high throughput screening performed in the Hepa-1 mouse hepatoma cell line, which revealed that SIN3A is necessary for the induction of CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) enzymatic activity by TCDD. In the current studies, we sought to provide insight into the role of SIN3A in this process, particularly because studies on SIN3A have usually focused on its repressive activity on transcription. We report that ectopic expression of human SIN3A in Hepa-1 cells enhanced EROD induction by TCDD and efficiently rescued TCDD induction of EROD activity in cells treated with an siRNA to mouse SIN3A, thus validating a role for SIN3A in CYP1A1 induction. We demonstrate that SIN3A is required for TCDD induction of the CYP1A1 protein in Hepa-1 cells but not for expression of the aryl hydrocarbon receptor protein. In addition, siRNAs for SIN3A decreased TCDD-mediated induction of CYP1A1 mRNA and EROD activity in human hepatoma cell line Hep3B. We establish that TCDD treatment of Hepa-1 cells rapidly increases the degree of SIN3A binding to both the proximal promoter and enhancer of the Cyp1a1 gene and demonstrate that increased binding to the promoter also occurs in human Hep3B, HepG2, and MCF-7 cells. These studies establish that SIN3A physically interacts with the CYP1A1 gene and extends the transcriptional role of SIN3A to a gene that is very rapidly and dramatically induced.

Hepatoma SK Hep-1 cells exhibit characteristics of oncogenic mesenchymal stem cells with highly metastatic capacity.

BackgroundSK Hep-1 cells (SK cells) derived from a patient with liver adenocarcinoma have been considered a human hepatoma cell line with mesenchymal origin characteristics, however, SK cells do not express liver genes and exhibit liver function, thus, we hypothesized whether mesenchymal cells might contribute to human liver primary cancers. Here, we characterized SK cells and its tumourigenicity.Methods and Principal FindingsWe found that classical mesenchymal stem cell (MSC) markers were presented on SK cells, but endothelial marker CD31, hematopoietic markers CD34 and CD45 were negative. SK cells are capable of differentiate into adipocytes and osteoblasts as adipose-derived MSC (Ad-MSC) and bone marrow-derived MSC (BM-MSC) do. Importantly, a single SK cell exhibited a substantial tumourigenicity and metastatic capacity in immunodefficient mice. Metastasis not only occurred in circulating organs such as lung, liver, and kidneys, but also in muscle, outer abdomen, and skin. SK cells presented greater in vitro invasive capacity than those of Ad-MSC and BM-MSC. The xenograft cells from subcutaneous and metastatic tumors exhibited a similar tumourigenicity and metastatic capacity, and showed the same relatively homogenous population with MSC characteristics when compared to parental SK cells. SK cells could unlimitedly expand in vitro without losing MSC characteristics, its tumuorigenicity and metastatic capacity, indicating that SK cells are oncogenic MSC with enhanced self-renewal capacity. We believe that this is the first report that human MSC appear to be transformed into cancer stem cells (CSC), and that their derivatives also function as CSCs.ConclusionOur findings demonstrate that SK cells represent a transformation mechanism of normal MSC into an enhanced self-renewal CSC with metastasis capacity, SK cells and their xenografts represent a same relative homogeneity of CSC with substantial metastatic capacity. Thus, it represents a novel mechanism of tumor initiation, development and metastasis by CSCs of non-epithelial and endothelia origin.

Effect of interleukin-15 and dendritic cells on the phenotype and cytotoxicity of cytokine-induced killer cells

Objective To investigate the effect of interleukin 15 (IL-15) and dendritic cells (DC) on the phenotype and cytotoxicity of cytokine-induced killer (CIK) cells.Methods Spleen mononuclear cells were harvested from C57BL/6 mice and cultured in anti-CD3 antibody-coated plate with interferon-γ (IFN-γ) and IL-2 containing medium to induce CIK cells.Bone marrow derived-DC was prepared.IL-15,DC or both were added to the CIK culture medium.CIK,CIK,IL-15-CIK,DC-CIK,DC/IL-15-CIK cells were produced,respectively.The percentage of CD3 + CD8 +,CD3 + NK1.1 + cells in these CIK cells were analyzed with FCM.Cytotoxicity of different CIK cells on mouse liver carcinoma hepal-6 cells were determined through detection of lactate dehydrogenase (LDH) release.Results The ratio of CD3 + CD8 + and CD3 + NK1.1 + cells in the spleen mononuclear cells of C57BL/6 mice before stimulation was (12.3 ± 2.1) ％ and (1.1 ± 0.5) ％,respectively.After culturing for 14 days in vitro,the ratio of CD3 + CD8 + cells in CIK,IL-15-CIK,DC-CIK and DC/IL-15-CIK cells was (18.8 ± 2.4)％,(22.1 ± 2.0)％,(28.2 ±2.6)％ and (34.7 ±2.3)％,respectively.The ratio of CD3 +NK1.1 + cells was (17.1±2.6)％,(18.9 ±3.4)％,(22.5±1.5)％ and (25.9±1.8)％.There was significant difference between IL-15 treatment group and control.The cytotoxicity of DC-CIK and DC/IL-15-CIK cells on liver carcinoma Hepal-6 cells were significantly higher than CIK cells as effector-target ration range from 2.5∶1.0 to 20.0∶1.0 (P ＜ 0.05).Cytotoxicity of DC-CIK and DC/IL-15-CIK was (54.9 ± 3.1) ％ and (58.75 ± 4.34) ％ as effector-target ratio was 20.0∶ 1.0.Conclusion Both IL-15 and DC increase the ratio of CD3 + CD8 + and CD3 + NK1.1 + cells in CIK cells,and synergetic effects also present.CIK cells stimulated with DC showed increased anti-tumor effect,and IL-15 can further enhance the antitumor effect of DC-CIK cells. Key words: Cytokine-induced killer; Interleukin-15 ; Dendritic cells ; Cytotoxicity

Obesity increases histone H3 lysine 9 and 18 acetylation at Tnfa and Ccl2 genes in mouse liver.

Obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD), which is characterized by the upregulated expression of two key inflammatory mediators: tumor necrosis factor (Tnfa) and monocyte chemotactic protein 1 (Mcp1; also known as Ccl2). However, the chromatin make-up at these genes in the liver in obese individuals has not been explored. In this study, to identify obesity-mediated epigenetic changes at Tnfa and Ccl2, we used a murine model of obesity induced by a high-fat diet (HFD) and hyperphagic (ob/ob) mice. Chromatin immunoprecipitation (ChIP) assay was used to determine the abundance of permissive histone marks, namely histone H3 lysine 9 and 18 acetylation (H3K9/K18Ac), H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 36 trimethylation (H3K36me3), in conjunction with polymerase 2 RNA (Pol2) and nuclear factor (Nf)-κB recruitment in the liver. Additionally, to correlate the liver tissue-derived ChIP measurements with a robust in vitro transcriptional response at the Tnfa and Ccl2 genes, we used lipopolysaccharide (LPS) treatment to induce an inflammatory response in Hepa1-6 cells, a cell line derived from murine hepatocytes. ChIP revealed increased H3K9/K18Ac at Tnfa and Ccl2 in the obese mice, although the differences were only statistically significant for Tnfa (p<0.05). Unexpectedly, the levels of H3K4me3 and H3K36me3 marks, as well as Pol2 and Nf-κB recruitment, did not correspond with the increased expression of these two genes in the obese mice. By contrast, the acute treatment of Hepa1-6 cells with LPS significantly increased the H3K9/K18Ac marks, as well as Pol2 and Nf-κB recruitment at both genes, while the levels of H3K4me3 and H3K36me3 marks remained unaltered. These results demonstrate that increased Tnfa and Ccl2 expression in fatty liver at the chromatin level corresponds to changes in the level of histone H3 acetylation.

The regulation of hepcidin expression by serum treatment: Requirements of the BMP response element and STAT- and AP-1-binding sites

Expression of hepcidin, a central regulator of systemic iron metabolism, is transcriptionally regulated by the bone morphogenetic protein (BMP) pathway. However, the factors other than the BMP pathway also participate in the regulation of hepcidin expression. In the present study, we show that serum treatment increased hepcidin expression and transcription without inducing the phosphorylation of Smad1/5/8 in primary hepatocytes, HepG2 cells or Hepa1-6 cells. Co-treatment with LDN-193189, an inhibitor of the BMP type I receptor, abrogated this hepcidin induction. Reporter assays using mutated reporters revealed the involvement of the BMP response element-1 (BMP-RE1) and signal transducers and activator of transcription (STAT)- and activator protein (AP)-1-binding sites in serum-induced hepcidin transcription in HepG2 cells. Serum treatment induced the expression of the AP-1 components c-fos and junB in primary hepatocytes and HepG2 cells. Forced expression of c-fos or junB enhanced the response of hepcidin transcription to serum treatment. By contrast, the expression of dominant negative (dn)-c-fos and dn-junB decreased hepcidin transcription. The present study reveals that serum contains factors stimulating hepcidin transcription. Basal BMP activity is essential for the serum-induced hepcidin transcription, although serum treatment does not stimulate the BMP pathway. The induction of c-fos and junB by serum treatment stimulates hepcidin transcription, through possibly cooperation with BMP-mediated signaling. Considering that AP-1 is induced by various stimuli, the present results suggest that hepcidin expression is regulated by more diverse factors than had been previously considered.

Protective role of macrophage migration inhibitory factor in nonalcoholic steatohepatitis.

MIF is an inflammatory cytokine but is hepatoprotective in models of hepatotoxin-induced liver fibrosis. Hepatic fibrosis can also develop from metabolic liver disease, such as nonalcoholic fatty liver disease (NASH). We investigated the role of MIF in high-fat or methionine- and choline-deficient diet mouse models of NASH. Mif(-/-) mice showed elevated liver triglyceride levels (WT, 53±14 mg/g liver; Mif(-/-), 103±7 mg/g liver; P<0.05) and a 2-3-fold increased expression of lipogenic genes. Increased fatty degeneration in the livers of Mif(-/-) mice was associated with increased hepatic inflammatory cells (1.6-fold increase in F4/80(+) macrophages) and proinflammatory cytokines (e.g., 2.3-fold increase in Tnf-α and 2-fold increase in Il-6 expression). However, inflammatory cells and cytokines were decreased by 50-90% in white adipose tissue (WAT) of Mif(-/-) mice. Subset analysis showed that macrophage phenotypes in livers of Mif(-/-) mice were skewed toward M2 (e.g., 1.7-fold and 2.5-fold increase in Arg1 and Il-13, respectively, and 2.5-fold decrease in iNos), whereas macrophages were generally reduced in WAT of these mice (70% reduction in mRNA expression of F4/80(+) macrophages). The protective MIF effect was scrutinized in isolated hepatocytes. MIF reversed inflammation-induced triglyceride accumulation in Hepa1-6 cells and primary hepatocytes and also attenuated oleic acid-elicited triglyceride increase in 3T3-L1 adipocytes. Protection from fatty hepatocyte degeneration was paralleled by a 2- to 3-fold reduction by MIF of hepatocyte proinflammatory cytokine production. Blockade of MIF receptor cluster of differentiation 74 (CD74) but not of CXCR2 or CXCR4 fully reverted the protective effect of MIF, comparable to AMPK inhibition. In summary, we demonstrate that MIF mediates hepatoprotection through the CD74/AMPK pathway in hepatocytes in metabolic models of liver injury.

Effects of 2,3-dimercaptosuccinic acid-coated Fe3O4 nanoparticles on genes in two mouse cell lines.

To evaluate the effects of iron oxide nanoparticles on genes from different cell lines in this study, mouse macrophage RAW264.7 and hepatocyte Hepa1-6 cell lines were treated with DMSA-coated Fe3O4 nanoparticles. Two doses were used, 50 microg/mL and 100 microg/mL, respectively, for 24 hr and gene expression profile was detected by DNA microarrays. The gene expression patterns for the two cell lines greatly differed from each other when compared, revealing distinct cell-specific effects of the nanoparticles on the genes. It was found that the nanoparticles significantly influenced expression of genes in the RAW264.7 cells, showing complete difference from those of Hepa1-6 cells. More genes were downregulated in the RAW264.7 cells by two doses of the nanoparticles but up-regulated in the Hepa1-6 cells by two doses of the same nanoparticles. Moreover, the increase of nanoparticle dose greatly decreased up-regulation of genes but increased down-regulation of genes in the RAW264.7 cells. The influence of nanoparticles did not result in similar effect in the Hepa1-6 cells. Apart from the difference in gene expression patterns in the two cell lines, there were eight and seven genes which were consistently down-regulated and up-regulated by low-dose and high-dose of nanoparticles in the two cell lines, respectively, revealing the common effects of the nanoparticles on the genes in the two cell lines. These common effects on genes were caused by homeostatic processes, influenced by down-regulation of genes and immune responses, and also cell death, influenced by up-regulation of genes. The data from this study has shed new insights into the potential in vivo nanotoxicolgy of the DMSA-coated Fe3O4 nanoparticles.

E2F1 is a novel fibrogenic gene that regulates cholestatic liver fibrosis through the Egr-1/SHP/EID1 network

E2F transcription factor 1 (E2F1) is an important regulator of metabolic diseases; however, its role in liver function remains elusive. This study unraveled a regulatory cascade involving E2F1, early growth response-1 (Egr-1), nuclear receptor small heterodimer partner (SHP, NR0B2), and EIA-like inhibitor of differentiation 1 (EID1) in cholestatic liver fibrosis. Liver E2F1 messenger RNA (mRNA) and protein expression was strongly up-regulated in human nonalcoholic steatohepatitis (NASH) and alcohol cirrhosis; the latter was inversely correlated with diminished SHP expression. E2F1 was also highly induced by 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) feeding and bile-duct ligation (BDL) in mice. E2F1-/- mice exhibited reduced biliary fibrosis by DDC as determined by Masson Trichrome and Picro Sirius red staining, and decreased serum bile acid (BA), BA pool size, and fecal BA excretion. In addition, cholestatic liver fibrosis induced by BDL, as determined by immunohistochemistry analysis of a1 collagen expression, was increased in SHP-/- mice but attenuated in hepatocyte SHP-overexpressed transgenic (STG) mice. Egr-1 exhibited marked induction in livers of SHP-/- mice compared to the wild-type mice in both sham and BDL groups, and reduction in STG livers. Egr-1 promoter was activated by E2F1, and the activation was abrogated by expression of SHP and its co-repressor EID1 in hepatoma cells Huh7, Hepa1, and stellate cells LX2. Chromatin immunoprecipitation assays further confirmed the association of E2F1, SHP, and EID1 proteins with the Egr-1 promoter, and their direct protein interactions were determined by glutathione S-transferase pull-down assays. Interestingly, E2F1 activated Egr-1 expression in a biphasic fashion as described in both human and mouse hepatocytes. E2F1 is a fibrogenic gene and could serve as a potential new diagnostic marker for nonalcoholic and alcoholic liver fibrosis/cirrhosis.

Inhibitory action of benzo[α]pyrene on hepatic lipoprotein receptors in vitro and on liver lipid homeostasis in mice.

BackgroundDyslipidemia associated with obesity often manifests as increased plasma LDL and triglyceride-rich lipoprotein levels suggesting changes in hepatic lipoprotein receptor status. Persistent organic pollutants have been recently postulated to contribute to the obesity etiology by increasing adipogenesis, but little information is available on their potential effect on hepatic lipoprotein metabolism.ObjectiveThe objective of this study was to investigate the effect of the common environmental pollutant, benzo[α]pyrene (B[α]P) on two lipoprotein receptors, the LDL-receptor and the lipolysis-stimulated lipoprotein receptor (LSR) as well as the ATP-binding cassette transporter A1 (ABCA1) using cell and animal models.ResultsLSR, LDL-receptor as well as ABCA1 protein levels were significantly decreased by 26–48% in Hepa1-6 cells incubated (<2 h) in the presence of B[α]P (≤1 µM). Real-time PCR analysis and lactacystin studies revealed that this effect was due primarily to increased proteasome, and not lysosomal-mediated degradation rather than decreased transcription. Furthermore, ligand blots revealed that lipoproteins exposed to 1 or 5 µM B[α]P displayed markedly decreased (42–86%) binding to LSR or LDL-receptor. B[α]P-treated (0.5 mg/kg/48 h, i.p. 15 days) C57BL/6J mice displayed higher weight gain, associated with significant increases in plasma cholesterol, triglycerides, and liver cholesterol content, and decreased hepatic LDL-receptor and ABCA1 levels. Furthermore, correlational analysis revealed that B[α]P abolished the positive association observed in control mice between the LSR and LDL-receptor. Interestingly, levels of other proteins involved in liver cholesterol metabolism, ATP-binding cassette transporter G1 and scavenger receptor-BI, were decreased, while those of acyl-CoA:cholesterol acyltransferase 1 and 2 were increased in B[α]P-treated mice.ConclusionsB[α]P demonstrates inhibitory action on LSR and LDL-R, as well as ABCA1, which we propose leads to modified lipid status in B[α]P-treated mice, thus providing new insight into mechanisms underlying the involvement of pollutants in the disruption of lipid homeostasis, potentially contributing to dyslipidemia associated with obesity.

Enantiospecific Effects of Ketoconazole on Aryl Hydrocarbon Receptor

Azole antifungal ketoconazole (KET) was demonstrated to activate aryl hydrocarbon receptor (AhR). Since clinically used KET is a racemic mixture of two cis-enantiomers (2R,4S)-(+)-KET and (2S,4R)-(−)-KET, we examined the effects of KET enantiomers on AhR signaling pathway. (+)-KET dose-dependently activated AhR in human gene reporter cell line AZ-AHR, and displayed 5–20× higher agonist activity (efficacy), as compared to (−)-KET; both enantiomers were AhR antagonists with equal potency (IC50). Consistently, (+)-KET strongly induced CYP1A1 mRNA and protein in human HepG2 cells, while (−)-KET exerted less than 10% of (+)-KET activity. In primary human hepatocytes, both enantiomers preferentially induced CYP1A2 over CYP1A1 mRNA and protein, and the potency of (+)-KET was slightly higher as compared to (−)-KET. Ligand binding assay with guinea pig liver cytosols revealed that both (+)-KET and (−)-KET are weak ligands of AhR that displaced [3H]-TCDD with comparable potency. Similarly, both enantiomers weakly transformed AhR to DNA-binding form with similar potency, as showed by EMSA, in guinea pig liver cytosolic extracts and nuclear extracts from mouse Hepa-1 cells. We also examined effects of KET on glucocorticoid receptor (GR), a regulator of AhR activity. Both KET enantiomers antagonized GR with similar potency, as revealed by gene reporter assay in AZ-GR cell line and down-regulation of tyrosine aminotransferase mRNA in human hepatocytes. Finally, we demonstrate enantiospecific antifungal activities of KET enantiomers in six Candida spp. strains. In conclusion, the significance of current study is providing the first evidence of enatiospecific effects of cis-enantiomers of ketoconazole on AhR-CYP1A pathway.

Blockade of CD47-Signaling Regulatory Protein alpha Signaling Enhances the Macrophage Phagocytic Activity Against Cancer Cells.

Background: Liver transplantation (LT) is the best treatment for unresectable, early-stage hepatocellular carcinoma (HCC). However, HCC recurrence is common, due to either occult metastasis development or circulating tumor cell engraftment. We previously demonstrated that the phagocytic activity of human liver macrophages is inhibited by the binding of signaling regulatory protein alpha (SIRPα) on macrophages to CD47 on target cells, a major regulator of phagocytic responses (2006 PNAS). Using in vitro and in vivo mouse syngeneic models, we examined whether the inhibition of CD47-SIRPα signaling would increase macrophage phagocytic activity against cancer cells. Methods: For the in vitro phagocytosis assay, Hepa1-6 and CMT93 target cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and incubated with mouse peritoneal cavity (PerC) macrophages and either anti-mouse SIRPα-blocking monoclonal antibodies (mAbs) (SIRPα mAbs) or isotype-matched control antibodies (isotype Abs). The phagocytosis of CFSE-labeled target cells was measured by flow cytometry. For the in vivo phagocytosis assay, CFSE-labeled target cells were injected into the peritoneal cavity, together with SIRPα mAbs, CD47 mAbs, or isotype Abs. CD47 knock-down (KD) was achieved via lentiviral particle-mediated shRNA delivery to Hepa1-6 cells, which were used in both in vitro and in vivo assays. Results: In the in vitro phagocytosis assay, anti-SIRPα mAb addition markedly enhanced the phagocytic activity of PerC macrophages against Hepa1-6 and CMT93, compared with isotype Abs treatment (p < 0.01). In the in vivo phagocytosis assay, a significant increase in phagocytic activity (p < 0.01) was observed with SIRPα mAb treatment, but not with CD47 mAb treatment. CD47 KD Hepa1-6 cells were significantly more sensitive to macrophage phagocytosis than parental Hepa1-6 cells in both in vitro and in vivo assays (p = 0.01), demonstrating the pivotal role of the CD47-SIRPα interaction in restricting tumor cell killing. In vivo CD47 mAb administration led to a remarkable decrease in CD80, CD86, and MHC class II expression on macrophages, implying their functional impairment. This finding might explain why treatment with CD47 mAbs alone did not enhance macrophage phagocytic activity against cancer cells. Conclusions: CD47-SIRPα interaction could be a therapeutic target for preventing HCC recurrence in LT recipients.

Tumor-associated macrophages promote cancer stem cell-like properties via transforming growth factor-beta1-induced epithelial–mesenchymal transition in hepatocellular carcinoma

Tumor-associated macrophages (TAMs), a crucial component of immune cells infiltrated in tumor microenvironment, have been found to be associated with progression and metastasis of hepatocellular carcinoma (HCC). In this study, we aimed to clarify the mechanism underlying the crosstalk between TAMs and cancer stem cells (CSCs) in HCC. Mouse macrophage cell line RAW264.7 cells were used to investigate the effects of TAMs on mouse hepatoma cell line Hepa1-6 cells in vivo and vitro. A total of 90 clinical samples had pathology-proven HCC were used to evaluate the distribution of TAMs and CSCs and analyze their value in predicting the prognosis. In the study, we have found that the number of TAMs has a positive correlation with the density of CSCs in the marginal of human HCC. Our results show that, cocultured with TAM-conditioned medium (CM) promoted CSC-like properties in Hepa1-6 cells, which underwent EMT and gained higher invasive capability. TAMs secreted more transforming growth factor- beta1 (TGF-beta1) than other phenotypes of macrophage. Furthermore, depletion of TGF-beta1 blocked acquisition of CSC-like properties by inhibition of TGF-beta1-induced EMT. High expression of CD68 in the EpCAM positive expression HCC tissues was strongly associated with both poor cancer-free survival and overall survival in patients. Our results indicate that the TAMs promote CSC-like properties via TGF-beta1-induced EMT and they may contribute to investigate the prognosis of HCC.

Immunotherapy for Hepatoma Using a Dual-Function Vector with Both Immunostimulatory and Pim-3–Silencing Effects

Tumorigenesis is an immortalization process in which the growth of normal cells is uncontrolled and programmed cell death is suppressed. Molecular biologic and immunologic studies have revealed that the aberrant expression of some proto-oncogenes boosts proliferation and inhibits apoptosis, which is vital for tumor development. The hypofunction of the host immune system also drives the development and metastasis of malignant tumors. Pim-3, a member of the Pim family, is aberrantly expressed in several cancers. Data suggest that Pim-3 inhibits apoptosis by phosphorylating the proapoptotic BH3-only protein Bad. Here, we constructed a dual-function small hairpin RNA (shRNA) vector containing an shRNA targeting Pim-3 and a TLR7-stimulating ssRNA. Stimulation with this bi-functional vector in vitro promoted significant apoptosis of Hepa1-6 cells by regulating the expression of apoptosis-related proteins and induced secretion of type I IFNs. Most importantly, this bi-functional vector more effectively inhibited subcutaneous Hepa1-6 cell growth than did single shRNA and ssRNA treatment in vivo. Natural killer (NK), CD4(+) T, and CD8(+) T cells and macrophages were required for effective tumor suppression, and CD4(+) T cells were shown to play a helper role in the activation of NK cells, possibly by regulating the secretion of Th1 or Th2 cytokines. This ssRNA-shRNA bi-functional vector may represent a promising approach for tumor therapy.

Hepcidin expression in liver cells: evaluation of mRNA levels and transcriptional regulation.

Hepcidin produced in the liver negatively regulates intestinal iron absorption, and the bone morphogenetic protein (BMP) pathway is well-known to stimulate hepcidin expression. However, the regulation of hepcidin expression has not been fully elucidated. In this study, we evaluate different systems that can be used to determine how hepcidin expression is regulated. The basal expression of hepcidin in liver cell lines, such as HepG2 cells and Hepa1-6 cells, was lower than that in the liver and primary hepatocytes; the expression levels of hepcidin in the cell lines were near the limit of detection for RT-PCR and RT-qPCR analyses. Treatment with trichostatin A, RNAlater, or MG-132 enhanced the expression of hepcidin in HepG2 cells, suggesting that histone deacetylation, instability of mRNA, or proteosomal degradation of the protein(s) that positively regulate hepcidin expression may be responsible for the decreased expression of hepcidin in HepG2 cells. In luciferase-based reporter assays, BMP induced the transcription of a reporter, hepcidin(-2018)-luc, that contains nt -2018 through nt -35 of the hepcidin promoter in HepG2 cells and Hepa1-6 cells. However, BRE-luc, a representative reporter used to evaluate BMP signaling, was unresponsive to BMP in HepG2 cells. These results suggest that hepcidin transcription can be best evaluated in liver cell lines and that the hepcidin promoter senses BMP signaling with high sensitivity. The present study demonstrates that studies regarding the regulation of hepcidin expression at the mRNA level should be evaluated in primary hepatocytes, and liver cell lines are well-suited for studies examining the transcriptional regulation of hepcidin.

Mitochondrial-mediated inflammasome activation controls production of Hepatoma-derived IL-1β (TUM7P.942)

Abstract Obesity is emerging as a critical driver of chronic inflammation and inflammation-associated cancers, such as hepatocellular carcinoma. The obesity-associated proinflammatory cytokine IL-1β is upregulated in multiple solid tumors and correlates with poor overall prognosis. Recently, fatty acid-induced inflammasome activation in macrophages has been shown to be dependent on mitochondrial reactive oxygen species (ROS) production. Here we asked whether in addition to traditional sources of IL-1β, the tumor itself might participate in maintenance of the inflammatory state. Stimulation of murine hepatoma line Hep1-6 cells with LPS substantially increased the mRNA level of IL-1β. Addition of LPS and palmitate together significantly elevated Hep1-6 IL-1β mRNA and mature IL-1β abundance. Inflammasome constituents were detectable in Hep1-6 cells with Nlrp3 mRNA content increased in LPS/palmitate treated cells. Cellular ROS levels were significantly increased in the LPS/palmitate treated group. AICAR, a potent activator of AMPK and mitophagy, decreased cellular ROS and mature IL-1β production by LPS/palmitate treated Hep1-6 cells, but did not affect IL-1β mRNA levels. Collectively, our data indicates that fatty acid-induced mitochondrial ROS results in inflammasome activation and cooperates with LPS-driven IL-1β mRNA resulting in tumor cell production of mature IL-1β. Through this mechanism, plasma fatty acids may promote tumor-associated inflammation.

Mo1772 Lipopolysaccharides Can Accelerate the Development of Nonalcoholic Fatty Liver Disease, Caused by the Up-Regulation of Lipogenic Gene Expressions

Background/Aim: Obesity is rapidly becoming a pandemic and is associated with increased prevalence of iron deficiency anaemia (IDA). Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related IDA. Since leptin shares a number of common biological features with IL-6, a major factor in the development of anaemia of chronic inflammation (ACI), we speculated that leptin and its metabolic counterpart adiponectin might play a role in regulating iron metabolism in the overweight population. Methods: The human hepatoma cells HuH7 and Hep G2 were exposed to IL-6 [10-50ng/ml], leptin [0.1-10μg/ml] and adiponectin [5μg/ml] for 16 hours. mRNA expression was determined using real-time quantitative RT-PCR, protein levels were detected by western blot analysis. Both cell lines were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. Results: Similar to IL-6 (***p<0.001), leptin causes a significant dose-dependent increase of hepcidin promoter activity (***p<0.001) in both cell lines, leading to elevated mRNA-levels. The activation of transcription factor STAT3 seems to play a crucial role. This was confirmed with specific STAT3-Inhibitor S3I201 [50μM] (***p<0.001). Furthermore, we were able to show for the first time that adiponectin completely abolished leptin effects on hepcidin promoter activation (***p<0.001 in HepG2, *p<0.05 in Huh7). Conclusion: Collectively, these data provide a novel insight into the molecular link between obesity and IDA and further demonstrate that adiponectin has the molecular potential to inhibit leptininduced hepcidin expression.

Mo1774 Sitagliptin Can Inhibit the Acceleration of the Development in NAFLD Caused by Lipopolysaccharides Through Inhibiting Expression of MYD88 and NfκB

Background/Aim: Obesity is rapidly becoming a pandemic and is associated with increased prevalence of iron deficiency anaemia (IDA). Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related IDA. Since leptin shares a number of common biological features with IL-6, a major factor in the development of anaemia of chronic inflammation (ACI), we speculated that leptin and its metabolic counterpart adiponectin might play a role in regulating iron metabolism in the overweight population. Methods: The human hepatoma cells HuH7 and Hep G2 were exposed to IL-6 [10-50ng/ml], leptin [0.1-10μg/ml] and adiponectin [5μg/ml] for 16 hours. mRNA expression was determined using real-time quantitative RT-PCR, protein levels were detected by western blot analysis. Both cell lines were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. Results: Similar to IL-6 (***p<0.001), leptin causes a significant dose-dependent increase of hepcidin promoter activity (***p<0.001) in both cell lines, leading to elevated mRNA-levels. The activation of transcription factor STAT3 seems to play a crucial role. This was confirmed with specific STAT3-Inhibitor S3I201 [50μM] (***p<0.001). Furthermore, we were able to show for the first time that adiponectin completely abolished leptin effects on hepcidin promoter activation (***p<0.001 in HepG2, *p<0.05 in Huh7). Conclusion: Collectively, these data provide a novel insight into the molecular link between obesity and IDA and further demonstrate that adiponectin has the molecular potential to inhibit leptininduced hepcidin expression.