335 BCL-2 FAMILY MEMBER MCL-1 INTERACTS WITH THE P53 FAMILY OF TRANSCRIPTION FACTORS

335 BCL-2 FAMILY MEMBER MCL-1 INTERACTS WITH THE P53 FAMILY OF TRANSCRIPTION FACTORS

POSTERS treatment with Ocoxin+Viusid. Using specific inhibitors for these enzymes, p38 MAPK was identified as a key mediator of the apoptotic effect of ...

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POSTERS treatment with Ocoxin+Viusid. Using specific inhibitors for these enzymes, p38 MAPK was identified as a key mediator of the apoptotic effect of Ocoxin+Viusid on HSC apoptosis. Treatment of HepG2 cells with a combination of Ocoxin+viusid and different concentrations of the cytotoxic agent Sorafenib, showed an increased negative effect on cell viability compared to the effect caused by Sorafenib alone. Conclusion: Ocoxin+viusid diminished cell viability of human hepatoma cells and hepatic stellate cells by inducing apoptosis. The apoptotic effect of Ocoxin+viusid on HSC was mediated by the activation of p38 MAPK. In hepatocytes, Ocoxin+viusid showed a differential effect on transformed versus primary cells and potentiate the effect of Sorafenib in cell viability of hepatoma cells. Taken together, these results point to the possibility of Ocoxin+viusid exerting a beneficial effect on liver damage. 333 PRIMARY HUMAN HEPATOCYTES ARE DAMAGED BY HYDROPHOBIC BILE ACIDS IN A CASPASE-INDEPENDENT MECHANISM C.P. Kleiss, R. Wimmer, T. Vennegeerts, G.U. Denk, C. Rust. Department of Medicine II, Klinikum Grosshadern, LudwigMaximilians-University, Munich, Germany E-mail: [email protected] Background and Aims: Hydrophobic bile acids induce hepatocyte damage. However, it remains unclear, if this is mainly due to apoptosis or necrosis, or a novel form of cell death termed “necroptosis”, a death-receptor dependent, but caspaseindependent mechanism. In addition, most of the existing studies are based on hepatoma cell lines or rodent hepatocytes. Therefore the aim of this study was to determine the effect of glycochenodeoxycholic acid (GCDCA) on cell death in primary human hepatocytes. Methods: Primary human hepatocytes were obtained from “Human tissue and cell research foundation (HTCR)” and from Invitrogen. After 24 hours of culture, cells were incubated with GCDCA, tauroursodeoxycholic acid (TUDCA) (100, 200, 500 mmol/l) or a combination for 24 hours. TNFa (28 mg/l) and anti-Apo-1 (500 ng/ml) served as positive controls. Apoptosis was quantified by a caspase-3/-7-assay. Cell damage was measured by LDH leakage in medium supernatant and metabolic activity with a WST assay. To verify the unknown relevance of necroptosis in the liver, primary rat hepatocytes were incubated with TNFa (28 mg/l) and the pancaspase inhibitor zVED (0.5 mmol/l) with or without the RIP-1 inhibitor necrostatin-1 (100 mmol/l). Results: GCDCA did not induce a significant caspase 3/7-activity in human hepatocytes. In contrast, TNFa induced a 3-fold and Apo-1 a 6-fold increase in caspase activity. However, GCDCA at 100, 200 and 500 mmol/l led to a 30-fold, 50-fold and 80-fold release of LDH, respectively. Furthermore, GCDCA 500 mmol/l reduced the metabolic activity significantly from 100% to 24%. Simultaneous incubation with TUDCA showed a protective effect on both LDH leakage and metabolic activity. In primary rat hepatocytes, TNFa induced a 200-fold increase of LDH, which was only reduced to control levels when necrostatin-1 was used in addition to zVED. Conclusions: Primary human hepatocytes showed a caspaseindependent form of cell death induced by GCDCA. The high LDH leakage and decreased metabolic activity could be due to necrosis or another caspase-independent mechanism. Since necroptosis is detectable in primary rat hepatocytes, human hepatocytes might also be damaged by this form of cell death. Thus, necroptosis should be further investigated as a possible mechanism of liver injury in cholestasis.

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334 LAMPREY HEPATOCYTES AS AN EVOLUTIONARY MODEL FOR STUDY OF PROGRAMMED CELL DEATH S. Konovalova, M. Savina. I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry of Russian Academy of Sciences, Saint Petersburg, Russia E-mail: [email protected] Background and Aim: Dysregulation of the programmed cell death process is accompanied by many liver diseases (hepatitis, liver failure, cirrhosis, cancer). The lamprey liver in the last winter of life cycle (lampreys are monocyclic and after spawning they die) is a unique evolutionary model to study the mechanisms of programmed cell death. The study was aimed to evaluate possible relationship between different pathways of programmed cell death on the lamprey hepatocytes model. Methods: The suspension of lamprey hepatocytes was obtained by collagenase method. To assay cell death by necrosis and apoptosis automated fluorescence method with sequential acridine orange and propidium iodidae staining using confocal microscopy was applied. Cytochrome c release from mitochondria into cytosol was determined by Western blotting using the cytochrome c rabbit polyclonal antibody. For detection of the intracellular cathepsin B and caspases (3, 7, 9) activity Magic Red and FLICA Apoptosis detection kits were used. Results: In spring period of pre-spawning migration we observed a sharp increase of apoptotic hepatocytes (about 80% cells have bright orange lysosomes and nuclei staining by acridine orange). At the same time cytochrome c was released from mitochondria and clearly detected in cytosolic fraction of liver cells. The activity of key proteolytic enzymes of mitochondria apoptotic pathway (caspase 9 and caspases 3, 7) was increased. Besides we determined the activity of main autophagy protease (cathepsin B) in this period. Conclusions: Thus apoptosis develops in parallel with autophagy in lamprey hepatocytes. This fact indicates interaction between different pathways of programmed cell death in liver. 335 BCL-2 FAMILY MEMBER MCL-1 INTERACTS WITH THE P53 FAMILY OF TRANSCRIPTION FACTORS M. Meinhard1 , L. Koenig1 , A. Pelc1 , A. Lovas1 , N. Joschko1 , 1 1 H.H. Schulze-Bergkamen2 , W. Stremmel1 , M. Muller ¨ . Internal Medicine IV, University Hospital Heidelberg, 2 National Center for Tumor Diseases, University Hospital Heidelberg, German Cancer Research Center, Heidelberg, Germany E-mail: [email protected] Members of the p53 family proteins are master regulators of apoptosis. The three transcriptionally active (TA) isoforms, TAp53, TAp63, and TAp73 induce apoptosis while the dominant negative (DN) isoforms DNp63 and DNp73 antagonize the TA isoforms and block apoptosis. We have previously shown that a disturbed balance between the TA and DN isoforms in favour of the DN isoforms leads to oncogenic events: development, progression and therapy resistance of hepatocellular carcinoma (HCC). Furthermore, we have shown that expression of myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the Bcl2 family, leads to chemoresistance in HCC. Here, we aimed to study the interplay of the p53 familydependent apoptosis pathways with the Mcl-1 signalling pathways on the transcriptional and post-translational level. To identify putative p53 and p63 binding sites we applied HUSAR und MatchTM . Luciferase-Reporter Assay, qPCR and Western-blot were applied to confirm regulation of the Mcl-1 gene by the different isoforms. Protein-protein interactions of the TA isoforms with Mcl-1 were studied by LUMIER-Assay (LUminescence-based Mammalian IntERactome mapping). We identified a putative p53 binding site in the promoter and two additional putative p53 response elements in intron 1 and

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POSTERS intron 2 of the Mcl-1 gene. Luciferase reporter assays revealed that wild-type p53 and the TA isoforms of p63 and p73 repress the Mcl-1 gene, while the DN isoforms of the p53 family transactivate the Mcl-1 gene via a p53 responsive element in the promoter. The antagonistic regulation of the Mcl-1 gene by the TA and DN isoforms of the p53 family was further validated by qPCR and Western-blot. Furthermore, we showed by LUMIER-Assay that the TA isoforms of the p53 family directly interact with Mcl-1 on the protein level, and thereby inactivate this anti-apoptotic protein. The interaction of the p53 family and Mcl-1 so far has been incompletely characterized. Here we show that the Mcl-1 gene is a target gene of p53 and p63. Furthermore, the p53 family proteins and Mcl-1 can interact via protein-protein interaction and thus influence the mitochondria membrane potential in mitochondriamediated apoptosis signalling. 336 THE SYSTEMIC IRON HORMONE HEPCIDIN IS INDUCED IN MACROPHAGES BY REACTIVE OXYGEN SPECIES AND DURING PHAGOCYTOSIS OF OXIDIZED ERYTHROCYTES T. Peccerella, H.-K. Seitz, S. Mueller, G. Millonig. Salem Medical Center, University of Heidelberg, Heidelberg, Germany E-mail: [email protected] Background: The peptide and liver-secreted hormone hepcidin is the master regulator of the body iron metabolism. It is secreted in response to elevated body iron stores or inflammatory conditions (IL-6). By blocking the iron exporter ferroportin, hepcidin not only prevents intestinal iron uptake but also iron release from macrophages that efficiently recycle iron by phagocytosing and degrading aged red blood cells (RBCs). Thus far, however, it is poorly understood why macrophages, besides hepatocytes, actively secrete hepcidin. Material and Methods: THP-1 monocytes were differentiated into macrophages by PMA 5 ng/ml over 48 h. RBCs were isolated by a percoll gradient and oxidized by incubation with CuCl2 and ascorbic acid at 37°C for 2 h. THP-1macrophages were then incubated with oxidized RBCs for 24 h. Non-phagocytosed RBCs were lyzed by hypotonic PBS. Oxidative burst via NADPH-oxidase was blocked by diphenylene iodonium chloride (DPI). Macrophages were lyzed in Trizol for RNA isolation. Hepcidin was quantified by real time PCR. Results: PMA-differentiation induced hepcidin in a dose-dependent way in THP-1 cells. PMA-mediated induction of hepcidin was completely abolished by treatment with DPI indicating a direct involvement of reactive oxygen species. Phagocytosis of oxidized RBCs led to a further increase in hepcidin production while hepcidin was downregulated by co-incubation with naïve RBCs. Hepcidin upregulation was specific for phagocytosis of oxidized RBCs and could neither be reproduced by phagocytosis of oxidized other cell types nor by incubation with lyzed RBCs. Conclusion: Hepcidin induction in macrophages is dependent on their production of reactive oxygen species. They only upregulate hepcidin when phagocytosing extensively oxidized RBCs. Decreased iron export from macrophages after ingestion of oxidized RBCs with low iron availability for erythropoeisis may be a major mechanism of so called anemia of chronic inflammation where large numbers of RBCs are oxidized.

337 SECRETED CYCLOPHILIN A VIA INTERACTING WITH CD147 INDUCES CELL PROLIFERATION THROUGH ERK1/2-MEDIATED G1/S TRANSITION IN CHOLANGIOCARCINOMA S. Obchoei1,2 , K. Sawanyawisuth1 , C. Chalermwat1,2 , C. Wongkham1 , C. Chen2 , S. Wongkham1 . 1 Department of Biochemistry, The Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 2 Molecular Surgeon Research Center, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA E-mail: [email protected] Background: Our previous study showed that cyclophilin A (CypA) and its membrane receptor-CD147 are up-regulated in cholangiocarcinoma (CCA) patients’ tissues. Suppression of intracellular CypA significantly reduces cell proliferation in vitro and tumor growth in nude mice and also attenuates ERK1/2 activity in CCA cell lines. In the present study, we investigated the effects and potential mechanism of secreted CypA (sCypA) and CD147 on cell proliferation of CCA cell lines. Methods: The CCA cells (KKU100 and M139) were treated with sCypA-containing conditioned media (CM) or purified recombinant human CypA (rhCypA) at different time points. Cell proliferation and/or ERK1/2 activity were determined by MTS assay and western blot respectively. Results: CypA was detected in CM from both CCA cell lines and the sCypA level corresponded to the intracellular CypA level. Both sCypA-containing CM and rhCypA significantly increased cell proliferation (P < 0.05 versus control). Reduction of CD147 expression by shRNA-based knockdown or neutralizing with monoclonal antibody significantly abolished rhCypA-induced cell proliferation. Upon rhCypA treatment, phosphorylated-ERK1/2 was rapidly increased whereas neutralizing CD147 inhibited ERK1/2 phosphorylation in the presence of rhCypA. Since RAF-MEK-ERK1/2 cascade plays a pivotal role in G1/S cell cycle progression, therefore, the expression levels of cell cycle mediators including cyclin D1, p21Cip1/WAF1 , RB, and phosphorylated-RB were monitored. The results showed up-regulation of cyclin D1 and phosphorylated-RB proteins and down-regulation of p21Cip1/WAF1 in rhCypA-treated cells as compared with the untreated controls. Conclusions: We demonstrated that CypA is secreted from CCA cells and enhances cell proliferation in an autocrine/paracrine manner at least via direct binding with CD147, a signaling receptor on cell membrane. Upon CypA/CD147 binding, ERK1/2 pathway is rapidly activated and resulted in cyclin D1 production, a typically ratelimiting step of cyclin D-cdk4/6 complexes formation of quiescent cells re-enter the cycle. Supported: By the RGJ-PhD Program, TRF (PHD/0210/2548) to SO and SW and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Health Cluster (SHePGMS), Khon Kaen University. 338 THE SYNERGISTIC EFFECT OF CO-ADMINISTRATION OF HMG-COA REDUCTASE INHIBITOR AND SELECTIVE COX-2 INHIBITOR AGAINST HEPATOCELLULAR CARCINOMA CELLS IS MEDIATED BY INHIBITION OF NF-KB/AKT PATHWAY S.J. Lee, H.J. Yim, J.W. Hwang, H.J. Lee, E.L. Yoon, S.J. Suh, J.J. Hyun, S.W. Jung, J.S. Koo, R.S. Choung, J.H. Kim, Y.S. Seo, J.E. Yeon, S.H. Um, K.S. Byun, S.W. Lee, J.H. Choi. Department of Internal Medicine, Division of Gastroenterology and Hepatology, Korea University College of Medicine, Seoul, Republic of Korea E-mail: [email protected] Background and Aims: Both NS-398, a selective cyclo-oxygenase-2 (COX-2) inhibitor and simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor exert anti-cancer effect

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