- Email: [email protected]
505 RAPAMYCIN PREVENTS PROLIFERATION OF DAMAGES HEPATOCYTES DURING CHRONIC LIVER INJURY AND THEREBY LIVER TUMOR DEVELOPMENT
S188
Poster Session − Friday, April 24
03a: LIVER TUMORS − a) EXPERIMENTAL
502 POLYCHLORINATED BIPHENYLS IN BILE OF PATIENTS WITH BILIARY TRACT CANCER A. Adenugba1 , S.A. Khan2 , S.D. Taylor-Robinson2 , I.J. Cox3 , M.B. Toledano4 , A.V. Thillainayagam2 , D.S. Bansi2 , R.W. Gibson1 , H.C. Thomas2 , A.J. Beck1 . 1 Faculty of Natural Sciences, Imperial College London, Wye, Ashford, Kent, 2 Hepatology & Gastroenterology, 3 Imaging Sciences, 4 Department of Epidemiology & Public Health, Imperial College London, London, UK E-mail: [email protected] Introduction: Polychlorinated biphenyls (PCBs) are anthropogenic, organic compounds. Although banned in the 1970s, PCBs are poorly biodegradable and hence ubiquitous in the environment. They accumulate in adipose tissue and are implicated various malignancies, including breast and pancreatic cancer. The hepatobiliary system is the main excretory route for such xenobiotic toxins. Incidence rates of intrahepatic biliary tract cancer are increasing worldwide. Measurement and comparison of PCB levels in bile from human patients with benign and malignant bile duct disease has not previously been done. Aims and Methods: The aims were to compare PCB concentrations in bile from patients with malignant (n = 8) and non-malignant (n = 7) biliary disease. 15 human bile samples, collected endoscopically, were analysed using gas chromatography mass spectrometry for seven target PCB congeners (28, 52, 101, 118, 153, 138, and 180), known to occur in the environment and food. Results: Amongst males, total PCB concentrations in bile ranged from 6.ng/mL (aged 73 years) to 49 ng/mL (aged 90 years); and in females between 8 ng/mL (aged 33 years) to 43 ng/ml (aged 67 years) bile. Although there was no overall difference in mean PCB levels between non-cancer and cancer patients, levels of congener 28 were significantly higher in patients with biliary tract cancer (p < 0.05). Conclusions: Despite the banning of PCBs over 30 years ago, these xenobiotics are present in the bile of patients with biliary disease. PCB levels tend to increase with age, suggesting chronic bioaccumulation. Further research is necessary to investigate the relevance of increased levels of congener 28 in bile in biliary tract cancer. 503 MOLECULAR MECHANISMS INVOLVED IN HEPATOCARCINOMA CELLS RESISTANCE TO HYPOXIA: ROLE OF HIF-1 AND OF CARBONIC ANHYDRASE IX E. Alchera1 , C. Dal Ponte1 , C. Imarisio1 , L. Tacchini2 , E. Albano1 , R. Carini1 . 1 Department of Medical Sciences, University ‘A. Avogadro’ of Piemonte Orientale, Novara, 2 Institute of General Pathology, University of Milan, Milan, Italy E-mail: [email protected] Background and Aims: Hypoxia inducible factor 1 (HIF-1) is a pleiotropic mediator of tissue adaptation to ischemia. Intra-tumoral hypoxia and genetic alterations can lead to HIF-1 over-expression which is associated to increased cancer cell survival, angiogenesis and infiltration. Understanding the role of HIF-1 in tumor cell resistance to hypoxia might be crucial to develop new therapies to enforce tumour regression. Methods: Mouse hepatocarcinoma C1C7 cells and their C4 HIF-1defective variant harboring a non-functional HIF-1beta subunit were exposed to hypoxia and the expression of HIF-1 and of its target gene carbonic anhydrase IX (CAIX) was evaluated together with necrotic cell death and intracellular Na+ and pH levels. Results: In C1C7 cells hypoxia induced the nuclear translocation of HIF-1 and the expression of carbonic anhydrase IX (CAIX), a transmembrane enzyme that catalyzes bicarbonate production. No expression of CAIX was
detectable in C4 cells. Cell killing by hypoxia was significantly lower in C1C7 than in C4 cells and such an effect was associated to a reduction of intracellular acidosis and Na+ accumulation. The inhibition of CAIX with acetazolamide or the block of bicarbonate influx with disodium-4acetamido-4-isothiocyanato-stilben-2,2-disulfonate abolished the increased resistance of C1C7 cells to hypoxia and caused alterations of intracellular pH and Na+ to a similar extent to those observable in C4 cells. Conclusions: HIF-1 induced expression of CAIX is associated to the reduction of the intracellular acidosis and the consequent increase of intracellular Na+ that precipitates cell necrosis during hypoxia. Our results indicate that CAIX acting as a pH buffering system contributes to the HIF1 induced tolerance to hypoxia by cancer cells and point to CAIX inhibitors as new promising anti-tumor agents.
504 GLUT1 AND GLUT3 EXPRESSION ARE INCREASED IN HEPATOCELLULAR CARCINOMA AND PROMOTE TUMORIGENICITY T. Amann1 , U. Maegdefrau U1 , A. Hartmann2 , O. Stoeltzing1 , T. Weiss1 , J. Sch¨olmerich1 , A. Bosserhoff1 , C. Hellerbrand1 . 1 University of Regensburg, Regensburg, 2 University of Erlangen, Erlangen, Germany E-mail: [email protected] Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoforms 1 and 3 (GLUT1 and GLUT3) are key rate-limiting factors in the transport of glucose into cancer cells but the expression and the biological significance of GLUT1 and GLUT3 expression in HCC hepatocellular carcinoma (HCC) are unknown. The aim of this study was to analyze the expression and function of GLUT1 and GLUT3 in HCC. Methods and Results: In HCC tissues and cell lines GLUT1 and GLUT3 expression were significantly higher than in primary human hepatocytes (PHH) and non-tumorous tissue. Immunohistochemical analysis of a tissue microarray containing HCC and corresponding non-cancerous liver tissue of 85 patients revealed that GLUT1 as well as GLUT3 positive HCC tissue showed a significantly higher Ki67 labeling index, more advanced tumor stages and less differentiated tumor grading. In accordance, suppression of GLUT1 or GLUT3 expression in HCC cells by siRNA significantly impaired attachment dependent and independent growth, and the migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression inhibited both glucose uptake and lactate secretion, indicating impaired anaeobic glycolysis. Pharmacological induction of hypoxia further increased HIF1alpha and GLUT expression in HCC cells and further increased the tumorigenicity of the HCC cells, while the hypoxia induced induction was abrogated by inhibition of HIF1alpha activation. Summary and Conclusion: GLUT1 and GLUT3 expression in HCC cells functionally affect tumorigenicity of HCC cells via induction of anaerobic glycolysis, and these mechanism is further increased during hypoxia via induction of HIF-1alpha. These findings indicate GLUT1 and GLUT3 as a prognostic markers for HCC progression and as innovative therapeutic targets for this highly aggressive tumor.
505 RAPAMYCIN PREVENTS PROLIFERATION OF DAMAGES HEPATOCYTES DURING CHRONIC LIVER INJURY AND THEREBY LIVER TUMOR DEVELOPMENT L.E. Buitrago-Molina1 , J. Laml`e1 , S. Marhenke1 , S. Sch¨ungel1 , U. Kossatz1 , K. Breuhahn2 , M.P. Manns1 , N. Malek1 , A. Vogel1 . 1 Hepatology, Gastroenterology and Endocrinology, Medical School Hannover, Hannover, 2 Pathology, University of Heidelberg, Heidelberg, Germany E-mail: [email protected] Background and Aims: mTOR is a serine/threonine kinase that integrates mitogenic and nutrient signaling to regulate proliferation and survival. Aberrant mTOR signaling has been recently identified in almost 50% of HCC cases suggesting that mTOR signaling plays a pivotal role in the
03a: LIVER TUMORS − a) EXPERIMENTAL pathogenesis of HCC. To delineate the role of mTOR in hepatocarcinogenesis, the ability of RAD001 to delay tumor development was tested in a mouse model of hereditary tyrosinemia type-1 (HT1), which is associated with the highest risk for HCCs of any human disease. Methods: To evaluate the role of mTOR in liver tumor development, WT and Fah deficient mice were treated with the rapamacin analogue RAD001. Results: Treatment with RAD001 markedly delayed liver tumor development in Fah deficient mice. RAD001 did not significantly reduce liver injury inflicted by FAA and RAD001 treated mice displayed similar markers of oxidative stress as placebo treated controls. Remarkably however, RAD001 efficiently inhibited proliferation of hepatocytes with DNA damage and sustained their apoptosis sensitivity during chronic liver injury. Furthermore, our data uncover an important interaction between the mTOR and the p53 pathway in hepatocytes. First, RAD001 treated Fah−/− mice displayed significantly lower p53 and/or p21 levels than untreated controls suggesting that activation of mTOR contributes to p53 accumulation in hepatocytes during chronic liver injury. Second, the effect of RAD001 on hepatocytes proliferation was markedly attenuated in Fah/p53−/− mice indicating that activation of the p53 checkpoint is required to suppress proliferation of hepatocytes with DNA damage. Mechanistically, we show that RAD001 affects the expression of multiple cell cycle related proteins and the assembly and activation of cyclin-cdk complexes facilitating the transition of cells from G0 to G1 phase of the cell cycle. Finally, we provide evidence that loss of p21 expression in RAD001 treated mice sustained the apoptosis sensitivity of hepatocytes during chronic liver injury. Conclusions: Our data show that RAD001 specifically prevents proliferation of damaged hepatocytes and sustains their apoptosis sensitivity during chronic liver injury. Long-term treatment with RAD001 significantly delayed liver tumor development.
506 THE RAS INHIBITOR FARNESYLTHIOSALICYLIC ACID INHIBITS THE GROWTH OF HEPATOCARCINOMA CELL LINES IN VITRO AND IN VIVO N. Charette1 , V. Lannoy1 , C. De Saeger1 , I. Leclercq1 , Y. Horsmans1,2 , P. St¨arkel1,2 . 1 Laboratory of Gastroenterology, Universit´e Catholique de Louvain, 2 Department of Gastroenterology, Saint Luc University Hospital, Brussel, Belgium E-mail: [email protected] Background and Aims: Ras activation has been shown to be a frequent event in hepatocarcinoma (HCC). Its downstream targets, the raf/MEK/ERK and PI3K/AKT/mTOR pathways, are currently considered as promising therapeutic targets. Farnesylthiosalicylic acid (FTS) inhibits ras by dislodging its activated form from its membrane docking sites. The aim of this study was to evaluate the effect of FTS in two HCC cell lines, Huh7 and HepG2, and to examine its impact on cell proliferation, apoptosis, and signaling through ERK and AKT-mTOR. Methods: Cells were cultured for up to 5 days in culture medium containing dimethylsulfoxyde (solvent) or FTS at concentrations ranging from 50 to 150 mM. Cell number, viability and proliferation were assessed by standard cell count and colorimetric WST-1 and BrdU incorporation assays, respectively. Apoptosis induction was evaluated by a caspase 3/7 activity assay after 24 hours of treatment. ERK, phospho-Erk, AKT, phospho-AKT, p70 and phospho-p70 (mTOR activation) were determined by immunoblotting. Finally, HepG2 cells were subcutaneously implanted in nude mice. FTS (10 mg/kg) was administered intraperitoneally daily when tumors became palpable. Response to treatment was assessed by tumor weight at sacrifice compared with solvent-treated controls. Results: FTS induced a significant time- and dose-dependent decrease in the number of viable cells in both cell lines. A dose-dependent decrease in BrdU incorporation occurred before the effect on cell number became apparent. In addition, an increase in caspase 3/7 activity was observed after 24 hours of treatment. In FTS-treated cells, ERK phosphorylation decreased in both cell lines and a decrease in AKT phosphorylation was observed in Huh7 cells. p70 phosphorylation did not change in both FTStreated cell lines. Finally, FTS treatment in xenografted mice for 10 days reduced tumor growth by 55% compared with untreated controls.
S189
Conclusion: FTS inhibits proliferation in a dose-dependent manner and rapidly induces apoptosis in HepG2 and Huh7 cells. This effect is associated with disruption of ERK and possibly AKT signalling, but not mTOR activation. Moreover, FTS treatment in vivo inhibits tumor growth in a HepG2 xenograft model. Acknowledgments: This work was supported by grants from the Fondation Saint-Luc and the FSR, Belgium. 507 CCR1, SYNDECANS 1 AND 4, ARE INVOLVED IN RANTES/CCL5-MEDIATED MIGRATION OR INVASION OF HUMAN HEPATOMA CELLS F. Charni1 , V. Friand1 , N. Suffee1 , O. Haddad1 , O. Oudar1 , L. Martin2 , R. Vassy3 , L. Gattegno1,4 , A. Sutton1,4 , N. Charnaux1,4 . 1 EA3410 Bioth´erapies B´en´efices et Risques, Universit´e Paris 13, Bobigny, 2 CEA Saclay, Gif-sur-Yvette, 3 CNRS UMR 7033, Universit´e Paris 13, Bobigny, 4 Laboratoire de Biochimie, Hopital Jean Verdier AP-HP, Bondy, France E-mail: [email protected] Background and Aims: In addition of their physiologic effects in inflammation and angiogenesis, chemokines such as the CCchemokine: regulated on activation, normal T-cell expressed, and secreted (RANTES)/CCL5 are involved in cancer pathology. We recently demonstrated that the human Huh7 hepatoma cells express RANTES/CCL5 G protein-coupled receptor CCR1 but not CCR3 nor CCR5. RANTES/CCL5 binding to these cells depends on CCR1 and glycosaminoglycans (GAGs). Moreover, RANTES/CCL5 strongly stimulates the migration and the invasion of Huh7 cells at a 3 nmol/L physiological concentration. The aim of this study is to elucidate some of the molecular mechanisms including the involvement of signalling pathways or the role of two membrane proteoglycans belonging to the syndecans (SDC) family (SDC-1 and SDC4), in RANTES/CCL5-induced migration and invasion of human hepatoma cells. Methods: Semi-quantitative RT-PCR and flow cytometry experiments were performed to analyse the expression of CCR1, SDC-1 and SDC4 by Huh7 hepatoma cells. We studied Huh7 migration or invasion by using Bio-coat cell migration chambers. A spreading experiment was performed after cell incubation with anti-CCR1, anti-SDC-1, or anti-SDC4 antibodies or specific RNA interference. Results: RANTES/CCL5-induced migration, invasion and spreading of Huh7 cells are both strongly inhibited by anti-CCR1, anti-SDC-1 and anti-SDC-4 antibodies. Reducing CCR-1, SDC-1 and SDC-4 expression by RNA interference also decreased RANTES/CCL5-mediated migration, invasion and spreading of Huh7 cells. Therefore, CCR1 and the two syndecans (SDC-1 and SDC-4) are involved in these main events of HCC progression. RANTES/CCL5 also induces the phosphorylation of focal adhesion kinase, JNK/SAPK and MAPK. These transduction pathways, PKC and Pi3K are involved in RANTES/CCL5-mediated migration, invasion and spreading as treatments of the cells with specifics inhibitors strongly reduced these effects. Conclusions: our data indicate that the RANTES/CCL5 may play an important role in the pathogenesis of human hepatocellular carcinoma (HCC). Targeting the RANTES-glycosaminoglycan interaction could be a new therapeutic approach for HCC. 508 EPIGENETIC METHYLATIONS AND EXPRESSIONS OF CASPASE 8 AND SURVIVIN IN HEPATOCELLULAR CARCINOMA S. Cho1 , S.B. Cho1 , W.S. Lee1 , Y.E. Joo1 , H.S. Kim1 , J.H. Lee2 , J.S. Rew1 , S.K. Choi1 . 1 Internal Medicine, 2 Pathology, Chonnam National University Medical School, Gwang ju, South Korea E-mail: [email protected] Background and Aims: Failure of apoptosis is an important cause in tumor development and progression in hepatocellular carcinoma (HCC). Caspase 8 has been known to the induction of apoptosis and survivin, an
Poster Session − Friday, April 24
03a: LIVER TUMORS − a) EXPERIMENTAL
502 POLYCHLORINATED BIPHENYLS IN BILE OF PATIENTS WITH BILIARY TRACT CANCER A. Adenugba1 , S.A. Khan2 , S.D. Taylor-Robinson2 , I.J. Cox3 , M.B. Toledano4 , A.V. Thillainayagam2 , D.S. Bansi2 , R.W. Gibson1 , H.C. Thomas2 , A.J. Beck1 . 1 Faculty of Natural Sciences, Imperial College London, Wye, Ashford, Kent, 2 Hepatology & Gastroenterology, 3 Imaging Sciences, 4 Department of Epidemiology & Public Health, Imperial College London, London, UK E-mail: [email protected] Introduction: Polychlorinated biphenyls (PCBs) are anthropogenic, organic compounds. Although banned in the 1970s, PCBs are poorly biodegradable and hence ubiquitous in the environment. They accumulate in adipose tissue and are implicated various malignancies, including breast and pancreatic cancer. The hepatobiliary system is the main excretory route for such xenobiotic toxins. Incidence rates of intrahepatic biliary tract cancer are increasing worldwide. Measurement and comparison of PCB levels in bile from human patients with benign and malignant bile duct disease has not previously been done. Aims and Methods: The aims were to compare PCB concentrations in bile from patients with malignant (n = 8) and non-malignant (n = 7) biliary disease. 15 human bile samples, collected endoscopically, were analysed using gas chromatography mass spectrometry for seven target PCB congeners (28, 52, 101, 118, 153, 138, and 180), known to occur in the environment and food. Results: Amongst males, total PCB concentrations in bile ranged from 6.ng/mL (aged 73 years) to 49 ng/mL (aged 90 years); and in females between 8 ng/mL (aged 33 years) to 43 ng/ml (aged 67 years) bile. Although there was no overall difference in mean PCB levels between non-cancer and cancer patients, levels of congener 28 were significantly higher in patients with biliary tract cancer (p < 0.05). Conclusions: Despite the banning of PCBs over 30 years ago, these xenobiotics are present in the bile of patients with biliary disease. PCB levels tend to increase with age, suggesting chronic bioaccumulation. Further research is necessary to investigate the relevance of increased levels of congener 28 in bile in biliary tract cancer. 503 MOLECULAR MECHANISMS INVOLVED IN HEPATOCARCINOMA CELLS RESISTANCE TO HYPOXIA: ROLE OF HIF-1 AND OF CARBONIC ANHYDRASE IX E. Alchera1 , C. Dal Ponte1 , C. Imarisio1 , L. Tacchini2 , E. Albano1 , R. Carini1 . 1 Department of Medical Sciences, University ‘A. Avogadro’ of Piemonte Orientale, Novara, 2 Institute of General Pathology, University of Milan, Milan, Italy E-mail: [email protected] Background and Aims: Hypoxia inducible factor 1 (HIF-1) is a pleiotropic mediator of tissue adaptation to ischemia. Intra-tumoral hypoxia and genetic alterations can lead to HIF-1 over-expression which is associated to increased cancer cell survival, angiogenesis and infiltration. Understanding the role of HIF-1 in tumor cell resistance to hypoxia might be crucial to develop new therapies to enforce tumour regression. Methods: Mouse hepatocarcinoma C1C7 cells and their C4 HIF-1defective variant harboring a non-functional HIF-1beta subunit were exposed to hypoxia and the expression of HIF-1 and of its target gene carbonic anhydrase IX (CAIX) was evaluated together with necrotic cell death and intracellular Na+ and pH levels. Results: In C1C7 cells hypoxia induced the nuclear translocation of HIF-1 and the expression of carbonic anhydrase IX (CAIX), a transmembrane enzyme that catalyzes bicarbonate production. No expression of CAIX was
detectable in C4 cells. Cell killing by hypoxia was significantly lower in C1C7 than in C4 cells and such an effect was associated to a reduction of intracellular acidosis and Na+ accumulation. The inhibition of CAIX with acetazolamide or the block of bicarbonate influx with disodium-4acetamido-4-isothiocyanato-stilben-2,2-disulfonate abolished the increased resistance of C1C7 cells to hypoxia and caused alterations of intracellular pH and Na+ to a similar extent to those observable in C4 cells. Conclusions: HIF-1 induced expression of CAIX is associated to the reduction of the intracellular acidosis and the consequent increase of intracellular Na+ that precipitates cell necrosis during hypoxia. Our results indicate that CAIX acting as a pH buffering system contributes to the HIF1 induced tolerance to hypoxia by cancer cells and point to CAIX inhibitors as new promising anti-tumor agents.
504 GLUT1 AND GLUT3 EXPRESSION ARE INCREASED IN HEPATOCELLULAR CARCINOMA AND PROMOTE TUMORIGENICITY T. Amann1 , U. Maegdefrau U1 , A. Hartmann2 , O. Stoeltzing1 , T. Weiss1 , J. Sch¨olmerich1 , A. Bosserhoff1 , C. Hellerbrand1 . 1 University of Regensburg, Regensburg, 2 University of Erlangen, Erlangen, Germany E-mail: [email protected] Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoforms 1 and 3 (GLUT1 and GLUT3) are key rate-limiting factors in the transport of glucose into cancer cells but the expression and the biological significance of GLUT1 and GLUT3 expression in HCC hepatocellular carcinoma (HCC) are unknown. The aim of this study was to analyze the expression and function of GLUT1 and GLUT3 in HCC. Methods and Results: In HCC tissues and cell lines GLUT1 and GLUT3 expression were significantly higher than in primary human hepatocytes (PHH) and non-tumorous tissue. Immunohistochemical analysis of a tissue microarray containing HCC and corresponding non-cancerous liver tissue of 85 patients revealed that GLUT1 as well as GLUT3 positive HCC tissue showed a significantly higher Ki67 labeling index, more advanced tumor stages and less differentiated tumor grading. In accordance, suppression of GLUT1 or GLUT3 expression in HCC cells by siRNA significantly impaired attachment dependent and independent growth, and the migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression inhibited both glucose uptake and lactate secretion, indicating impaired anaeobic glycolysis. Pharmacological induction of hypoxia further increased HIF1alpha and GLUT expression in HCC cells and further increased the tumorigenicity of the HCC cells, while the hypoxia induced induction was abrogated by inhibition of HIF1alpha activation. Summary and Conclusion: GLUT1 and GLUT3 expression in HCC cells functionally affect tumorigenicity of HCC cells via induction of anaerobic glycolysis, and these mechanism is further increased during hypoxia via induction of HIF-1alpha. These findings indicate GLUT1 and GLUT3 as a prognostic markers for HCC progression and as innovative therapeutic targets for this highly aggressive tumor.
505 RAPAMYCIN PREVENTS PROLIFERATION OF DAMAGES HEPATOCYTES DURING CHRONIC LIVER INJURY AND THEREBY LIVER TUMOR DEVELOPMENT L.E. Buitrago-Molina1 , J. Laml`e1 , S. Marhenke1 , S. Sch¨ungel1 , U. Kossatz1 , K. Breuhahn2 , M.P. Manns1 , N. Malek1 , A. Vogel1 . 1 Hepatology, Gastroenterology and Endocrinology, Medical School Hannover, Hannover, 2 Pathology, University of Heidelberg, Heidelberg, Germany E-mail: [email protected] Background and Aims: mTOR is a serine/threonine kinase that integrates mitogenic and nutrient signaling to regulate proliferation and survival. Aberrant mTOR signaling has been recently identified in almost 50% of HCC cases suggesting that mTOR signaling plays a pivotal role in the
03a: LIVER TUMORS − a) EXPERIMENTAL pathogenesis of HCC. To delineate the role of mTOR in hepatocarcinogenesis, the ability of RAD001 to delay tumor development was tested in a mouse model of hereditary tyrosinemia type-1 (HT1), which is associated with the highest risk for HCCs of any human disease. Methods: To evaluate the role of mTOR in liver tumor development, WT and Fah deficient mice were treated with the rapamacin analogue RAD001. Results: Treatment with RAD001 markedly delayed liver tumor development in Fah deficient mice. RAD001 did not significantly reduce liver injury inflicted by FAA and RAD001 treated mice displayed similar markers of oxidative stress as placebo treated controls. Remarkably however, RAD001 efficiently inhibited proliferation of hepatocytes with DNA damage and sustained their apoptosis sensitivity during chronic liver injury. Furthermore, our data uncover an important interaction between the mTOR and the p53 pathway in hepatocytes. First, RAD001 treated Fah−/− mice displayed significantly lower p53 and/or p21 levels than untreated controls suggesting that activation of mTOR contributes to p53 accumulation in hepatocytes during chronic liver injury. Second, the effect of RAD001 on hepatocytes proliferation was markedly attenuated in Fah/p53−/− mice indicating that activation of the p53 checkpoint is required to suppress proliferation of hepatocytes with DNA damage. Mechanistically, we show that RAD001 affects the expression of multiple cell cycle related proteins and the assembly and activation of cyclin-cdk complexes facilitating the transition of cells from G0 to G1 phase of the cell cycle. Finally, we provide evidence that loss of p21 expression in RAD001 treated mice sustained the apoptosis sensitivity of hepatocytes during chronic liver injury. Conclusions: Our data show that RAD001 specifically prevents proliferation of damaged hepatocytes and sustains their apoptosis sensitivity during chronic liver injury. Long-term treatment with RAD001 significantly delayed liver tumor development.
506 THE RAS INHIBITOR FARNESYLTHIOSALICYLIC ACID INHIBITS THE GROWTH OF HEPATOCARCINOMA CELL LINES IN VITRO AND IN VIVO N. Charette1 , V. Lannoy1 , C. De Saeger1 , I. Leclercq1 , Y. Horsmans1,2 , P. St¨arkel1,2 . 1 Laboratory of Gastroenterology, Universit´e Catholique de Louvain, 2 Department of Gastroenterology, Saint Luc University Hospital, Brussel, Belgium E-mail: [email protected] Background and Aims: Ras activation has been shown to be a frequent event in hepatocarcinoma (HCC). Its downstream targets, the raf/MEK/ERK and PI3K/AKT/mTOR pathways, are currently considered as promising therapeutic targets. Farnesylthiosalicylic acid (FTS) inhibits ras by dislodging its activated form from its membrane docking sites. The aim of this study was to evaluate the effect of FTS in two HCC cell lines, Huh7 and HepG2, and to examine its impact on cell proliferation, apoptosis, and signaling through ERK and AKT-mTOR. Methods: Cells were cultured for up to 5 days in culture medium containing dimethylsulfoxyde (solvent) or FTS at concentrations ranging from 50 to 150 mM. Cell number, viability and proliferation were assessed by standard cell count and colorimetric WST-1 and BrdU incorporation assays, respectively. Apoptosis induction was evaluated by a caspase 3/7 activity assay after 24 hours of treatment. ERK, phospho-Erk, AKT, phospho-AKT, p70 and phospho-p70 (mTOR activation) were determined by immunoblotting. Finally, HepG2 cells were subcutaneously implanted in nude mice. FTS (10 mg/kg) was administered intraperitoneally daily when tumors became palpable. Response to treatment was assessed by tumor weight at sacrifice compared with solvent-treated controls. Results: FTS induced a significant time- and dose-dependent decrease in the number of viable cells in both cell lines. A dose-dependent decrease in BrdU incorporation occurred before the effect on cell number became apparent. In addition, an increase in caspase 3/7 activity was observed after 24 hours of treatment. In FTS-treated cells, ERK phosphorylation decreased in both cell lines and a decrease in AKT phosphorylation was observed in Huh7 cells. p70 phosphorylation did not change in both FTStreated cell lines. Finally, FTS treatment in xenografted mice for 10 days reduced tumor growth by 55% compared with untreated controls.
S189
Conclusion: FTS inhibits proliferation in a dose-dependent manner and rapidly induces apoptosis in HepG2 and Huh7 cells. This effect is associated with disruption of ERK and possibly AKT signalling, but not mTOR activation. Moreover, FTS treatment in vivo inhibits tumor growth in a HepG2 xenograft model. Acknowledgments: This work was supported by grants from the Fondation Saint-Luc and the FSR, Belgium. 507 CCR1, SYNDECANS 1 AND 4, ARE INVOLVED IN RANTES/CCL5-MEDIATED MIGRATION OR INVASION OF HUMAN HEPATOMA CELLS F. Charni1 , V. Friand1 , N. Suffee1 , O. Haddad1 , O. Oudar1 , L. Martin2 , R. Vassy3 , L. Gattegno1,4 , A. Sutton1,4 , N. Charnaux1,4 . 1 EA3410 Bioth´erapies B´en´efices et Risques, Universit´e Paris 13, Bobigny, 2 CEA Saclay, Gif-sur-Yvette, 3 CNRS UMR 7033, Universit´e Paris 13, Bobigny, 4 Laboratoire de Biochimie, Hopital Jean Verdier AP-HP, Bondy, France E-mail: [email protected] Background and Aims: In addition of their physiologic effects in inflammation and angiogenesis, chemokines such as the CCchemokine: regulated on activation, normal T-cell expressed, and secreted (RANTES)/CCL5 are involved in cancer pathology. We recently demonstrated that the human Huh7 hepatoma cells express RANTES/CCL5 G protein-coupled receptor CCR1 but not CCR3 nor CCR5. RANTES/CCL5 binding to these cells depends on CCR1 and glycosaminoglycans (GAGs). Moreover, RANTES/CCL5 strongly stimulates the migration and the invasion of Huh7 cells at a 3 nmol/L physiological concentration. The aim of this study is to elucidate some of the molecular mechanisms including the involvement of signalling pathways or the role of two membrane proteoglycans belonging to the syndecans (SDC) family (SDC-1 and SDC4), in RANTES/CCL5-induced migration and invasion of human hepatoma cells. Methods: Semi-quantitative RT-PCR and flow cytometry experiments were performed to analyse the expression of CCR1, SDC-1 and SDC4 by Huh7 hepatoma cells. We studied Huh7 migration or invasion by using Bio-coat cell migration chambers. A spreading experiment was performed after cell incubation with anti-CCR1, anti-SDC-1, or anti-SDC4 antibodies or specific RNA interference. Results: RANTES/CCL5-induced migration, invasion and spreading of Huh7 cells are both strongly inhibited by anti-CCR1, anti-SDC-1 and anti-SDC-4 antibodies. Reducing CCR-1, SDC-1 and SDC-4 expression by RNA interference also decreased RANTES/CCL5-mediated migration, invasion and spreading of Huh7 cells. Therefore, CCR1 and the two syndecans (SDC-1 and SDC-4) are involved in these main events of HCC progression. RANTES/CCL5 also induces the phosphorylation of focal adhesion kinase, JNK/SAPK and MAPK. These transduction pathways, PKC and Pi3K are involved in RANTES/CCL5-mediated migration, invasion and spreading as treatments of the cells with specifics inhibitors strongly reduced these effects. Conclusions: our data indicate that the RANTES/CCL5 may play an important role in the pathogenesis of human hepatocellular carcinoma (HCC). Targeting the RANTES-glycosaminoglycan interaction could be a new therapeutic approach for HCC. 508 EPIGENETIC METHYLATIONS AND EXPRESSIONS OF CASPASE 8 AND SURVIVIN IN HEPATOCELLULAR CARCINOMA S. Cho1 , S.B. Cho1 , W.S. Lee1 , Y.E. Joo1 , H.S. Kim1 , J.H. Lee2 , J.S. Rew1 , S.K. Choi1 . 1 Internal Medicine, 2 Pathology, Chonnam National University Medical School, Gwang ju, South Korea E-mail: [email protected] Background and Aims: Failure of apoptosis is an important cause in tumor development and progression in hepatocellular carcinoma (HCC). Caspase 8 has been known to the induction of apoptosis and survivin, an