331 ACUTE LIVER FAILURE IN MICE FOLLOWING DELETION OF CFLIP - A KEY ROLE OF APOPTOSIS SIGNALING PATHWAYS IN TISSUE HOMEOSTASIS

POSTERS results demonstrated that decreased Pin1 expression could inhibit matrix metalloproteinase-2 (MMP-2) synthesis and secretion. Conclusions: Pin1 could up-regulate the MMP-2 to enhance the aggressive invasion in colon cancer. This can establish new idea to colon cancer gene therapeutic. 330 ONCOGENIC HBX PROTEIN INDUCES PROLIFERATION OF HEPATOMA CELLS BY ENHANCING MICRORNA-21 EXPRESSION P. Damania1 , B. Sen1 , A. Kumari1 , A. Singh1 , S.B. Dar1 , E. Gupta1 , S.K. Sarin2 , S.K. Venugopal1,3 . 1 Department of Research, 2 Hepatology, Institute of Liver and Biliary Sciences (ILBS), 3 Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India E-mail: [email protected] Background: Hepatitis B viral infection is one of the major health problems in developing countries. One of the HBV proteins, HBx, modulates the host gene expression via several mechanisms. Recent studies have shown that HBV modulates the expression of host microRNAs. MicroRNA-21 has been shown to induce proliferation in hepatic cells. We hypothesized that HBx enhances cell proliferation, at least in part, by increasing the expression of microRNA-21 in hepatic cells. Methods: Hep G2 and Huh7 cells were used in all the experiments. HBx gene was over-expressed in these cells using lipofectamine LTX transfection reagent. After 72 hours, the cells were collected and used for all the assays. The transfection efficiency was analyzed by expressing eGFP-containing expression plasmid in parallel experiments. The expression of HBx was analyzed by Western blots and b-actin was used as the loading control. The total RNA enriched with miRNA was isolated and the expression of microRNA-21 was analyzed using real time RT-PCR reaction. Apoptosis induction was studied by TUNEL assay and cell proliferation was estimated by WST1 assay. The cells were transfected with pre-miR-21 oligos using NeoFX transfection reagent and cellular proliferation was analyzed. Results: The expression of HBx in Hep G2 and Huh 7 cells was confirmed by Western blot for HBx, and found to be highly expressed. The transfection efficiency was checked in the cells expressing eGFP and it was found that more than 70% of the cells are expressing the eGFP protein. Over-expression of HBx protein in Hep G2 and Huh7 cells resulted in a significant increase in proliferation (five-fold increase; n = 3; p < 0.01) and microRNA-21 expression (44-fold increase, normalized with 5S rRNA; n = 4; p < 0.001) but there was no effect on apoptosis. Over-expression of microRNA-21 resulted in significant increase in proliferation (2.8-fold increase over control cells; n = 2; p < 0.05) of these cells. Conclusion: The data show that HBx induces proliferation by enhancing the expression of microRNA-21 in hepatoma cells. We conclude that HBV infection enhances cell proliferation, at least in part, via HBx-induced miRNA-21 expression during hepatocellular carcinoma progression. 331 ACUTE LIVER FAILURE IN MICE FOLLOWING DELETION OF CFLIP – A KEY ROLE OF APOPTOSIS SIGNALING PATHWAYS IN TISSUE HOMEOSTASIS 1 J.M. Schattenberg1 , D. Garcia Bardon1 , I. Wagner1 , M.A. Worns ¨ , T. Zimmermann1 , A. Schad2 , Y.W. He3 , P.R. Galle1 , M. Schuchmann1 . 1 I. Medizinische Klinik und Poliklinik, 2 Department of Pathology, Universit¨ atsmedizin Mainz, Mainz, Germany, 3 Department of Immunology, Duke University Medical Center, Durham, NC, USA E-mail: [email protected] Introduction: The caspase 8 homologue cFLIP blocks caspase activation and deletion in mice results in embryonic lethality. The dysregulation of apoptosis signaling pathways has been observed in a variety of liver diseases including acute liver failure and

hepatocarcinogenesis. We have previously shown, that deletion of cFLIP in hepatocytes results in increased sensitivity towards apoptosis from CD95 and TNF. Methods: To investigate the role of cFLIP in-vivo, we crossed mice expressing the cre-recombinase under control of the estrogen receptor and mice with lox-P sites in the cFLIP gene (ER-cre:FLIPf /f ). Deletion of cFLIP was achieved by injection of tamoxifen in 8-week old mice. Results: Following tamoxifen injection ER-Cre: FLIPf /f mice exhibited a sharp rise in serum ALT (ER-Cre: FLIPf /f vs control: 20716 vs 13 U/l; p < 0.001) that did not occur in wild type mice. Up to 72 h no toxic effects of tamoxifen could be observed in wild type mice. In parallel, ER-Cre: FLIPf /f developed significant hypoglycemia (ERCre: FLIPf /f vs control: 49 vs 244 mg/dl; p < 0.00001). The dynamic of liver failure following Tam-injection started at 36 h and ERCre: FLIPf /f mice exhibited increased mortality at 48 h. Histological analysis showed apoptotic bodies in the liver, spleen and colon, while the heart, lung, kidneys or brain tissue were unaffected. TUNEL staining and immunohistochemistry for activated caspase 3 and 8 confirmed caspase activation in these organs, especially in the white pulp of the spleen. Liver injury was sensitive to inhibition with the pancaspase inhibitor zVAD at 48 h (ALT: ERCre: FLIPf /f ±zVAD: 20716 vs 2408 U/l; p < 0.006, n = 8). In parallel to liver injury increased phosphorylation of the cJun N-terminal kinase (JNK) occurred in ER-Cre: FLIP mice. In conclusion, deletion of cFLIP in mice leads to acute liver failure and hypoglycemia involving activation of caspases and the MAPK JNK. Injury occurred specifically in the liver, spleen and colon, while other organs were unaffected. These findings stress the importance of cFLIP in tissue homeostasis and point towards an involvement of the liver, colon and lymphatic organs in this type of multiorgan failure. 332 OCOXIN+VIUSID INDUCES APOPTOSIS OF HEPATOMA AND HEPATIC STELLATE CELLS AND POTENTIATES THE EFFECT OF SORAFENIB ON HEPATOMA CELLS E. Arriazu, M. Ruiz de Galarreta, M.P. Perez de Obanos, M. Iraburu. Biochemistry and Molecular Biology, University of Navarra, Pamplona, Spain E-mail: [email protected] Background: Ocoxin+viusid are natural antioxidants and amino acids that have been shown to exert antioxidant and immunomodulatory responses in liver. Aim: The present work was aimed to determine the effect of Ocoxin+viusid on different hepatic cells: primary mouse hepatocytes, activated rat hepatic stellate cells (HSC) and human hepatoma cells. Materials and Methods: Cells were incubated with increasing amounts of Ocoxin+viusid. Cell viability was analyzed by neutral red exclussion assay and apoptosis by ELISA detection of cytosolic histone-associated fragments. ERK, p38MAPK and JNK activation was analysed by Western blot of the active phosphorylated forms. In some experiments cells were pre-treated with specific inhibitors for the different kinases. Caspase-3 activation was measured using a commertial kit. Results: Ocoxin+viusid caused a dose-response negative effect on viability of HepG2 as well as in HSC cells at almost all the doses tested. However, only high doses of Ocoxin+viusid diminished the viability of primary mouse hepatocytes, suggesting tumoral and activated hepatic stellate cells to be more sensitive to this compound. The study of different biochemical and morfological parameters on Ocoxin+viusid-treated HSC showed an apoptotic effect characterized by accumulation of histone-associated cytosolic DNA fragments and caspase-3 activation. Incubation of HSC with Ocoxin+Viusid caused a transient and dramatic enhancement on ERK, JNK and p38 MAPK phosphorylation levels 15 minutes after

Journal of Hepatology 2012 vol. 56 | S71–S224

S135