- Email: [email protected]
[287] INDUCTION OF THE INTRINSIC APOPTOSIS PATHWAY BY TNP-470 IN AN IN VIVO MODEL OF HEPATOCARCINOMA
POSTERS
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alpha. In all groups of patients investigated, a constant 4-8 fold increase of interferon gamma production was observed by coincubation of adenosine with interferon alpha as compared to stimulation with interferon alpha alone. This increased TFN gamma production required the presence of CD14+ cells and was independent from CD56+ cells. Conclusion: Thus, OUT data suggest that adenosine and interferon alpha may synergistically increase effector functions during acute infections but also prevent overwhelming immune responses by inhibiting proliferation and induction of apoptosis of activated lymphoid cells. The potential therapeutic role of adenosine receptor agonists in the treatment with interferon alpha-based therapies in viral hepatitis requires further investigation.
12851 UP-REGULATION OF A20, AN ANTI-APOPTOTIC PROTEIN, IS PART OF THE PROTECTIVE RESPONSE TO HYPOXIC STRESS IN THE LIVER A. Keogh, M. Gass, D. Candinas, D. Stroka. Clinic of Visceral and Transplantation Surgev, Unioersiiy of Bern, Bern, Switzerland E-mai I : adrian. [email protected] i be .ch Background and Aim: A20 was originally described as a TNF-inducible protein whose expression protects against apoptosis and limits inflammation via inhibition of NF-kB. We previously demonstrated that A20 mRNA is induced in livers of mice during surgically induced hypoxia. We identified within the A20 promoter putative binding sites for the hypoxia-inducible transcription factor (H1F)-1. The aim of this study was to determine whether hypoxia directly effects the expression of A20 and if the mechanism was dependent on HIF-1. Methods: Isolated primary human hepatocytes were cultured in William’s E medium for 24-48 hours followed by either incubated in a hypoxic (1.5% 0 2 ) or normoxic environment for 6 hours. To investigate the effects of inflammation, hepatocytes were incubated with TNF ( 1 Ong/mL) with and without hypoxia. To determine HTF dependent activation, cells were incubated with the HTF- 1 activators, dimethyloxaloylglycine (DMOG) ( I 25mM), CoC12 ( 1 00mM) or desferoxamine (DFX) ( I 00mM) for 4 hours under normal culture conditions. RNA and protein were isolated for each condition for real time PCR and western blot analysis respectively. Results: Hypoxia increased A20 mRNA 33-foldf10 (n=21) compared to normoxic hepatocytes (p < 0.001). The median fold increase by hypoxia was significantly higher than cells cultured in the presence of TNF 9-fold&3 (p < 0.001) (n=9) alone. However, the combination of hypoxia and TNF resulted in a 1 7 8 f 2 0 median fold increase. Western blot analysis confirms the respective increase of A20 protein. Stabilization of HTF-I a protein after treated with DMOG, CoC12 or DFX is confirmed by western, however there was no resulting increase of A20 mRNA 1.2-fold+0.3 (n = 6) or protein. Conclusion: Here we present the first evidence that A20 is induced by hypoxia albeit in a HTF-independent manner. Originally described as a TNF-inducible protein, hypoxic stress is a more potent stimulus of A20 expression in hepatocytes and hypoxia augments the TNF-induced response. These finding broaden the anti-apoptotic scope of A20 to include hepatoprotection under low-oxidative stress.
12861 BIFUNCTIONAL ROLE OF MITOCHONDRIAL REACTIVE OXYGEN SPECIES IN DETERMINING THE SURVlVALlSUSCEPTlBlLITY OF HEPATOMA CELLS TO HY POXlA J.M. L1uis’,2, A. Morales’,2, C. Blasco’, A. Colell’,2, M. Mari’,2, C. Garcia-Ruiz’,2, J.C. Fernandez-Checa’,2. ’Liver Unit, Instituto Mablties Digestiues, Hospital Clinic, IDIBAPS, Barcelona; ’Department of‘ Cell Death And Prolif2vrution, IIBB-CSK, Barcelona, Spain E-mail: [email protected] Hypoxia occurs when the oxygen supply does not meet the cellular requirements for energy generation and it is often associated with diverse
pathologies including cancer. In fact many solid tumors develop under hypoxic conditions due to the oxygen gradient that it is established from the periphery to the centre of the tumor. Hypoxia activates a complex signaling program determined by the activation of transcription factors HIF-1 dNF-kE3 and the overstimulation of reactive oxygen species (ROS) from mitochondria. Given the complex and often controversial relation between ROS generation and carcinogenesis, the aim of this work was to examine the role of the mitochondrial ROS generation caused by hypoxia in the survival/susceptibility of human hepatoma HepG2 cells and the modulation of mitochondrial GSH (mGSH) (J. Biol. Chem. 280: 32243232, 2005). Methods: HepG2 cells were subjected to hypoxia (5%02) o normoxia (21%02) for 24-72 hrs with o without mGSH depletion, determining ROS, Src activation, NF-IcB regulation, gene expression by real-time PCR and cell viabilityideath. Results: 5%O2 stimulated a time-dependent ROS generation vs 21%02 (2.5-4.5 fold), abolished by blocking mitochondrial complex 1/11 by rotenone (Rot)/TTFA. The DNA binding of p65/p50 and the level of HIFl a increased in nuclear extracts by hypoxia, effects that were accompanied by PDKl, VEGF and CIA€’-2 induction, while RotiTTFA preincubation prevented these responses. Furthermore, although the levels of TkB-a and phospho-TkB-a at Ser 32 remained constant, hypoxia stimulated the phosphorylation of IkB-a in tyrosine residues. Moreover, hypoxia stimulated the levels of phospho-c-Src at tyrosine 416 and 319 by a mechanism dependent on the mitochondrial ROS generation because RotiTTFA abolished c-Src phosphorylation. The c-Src inhibitor, pp2, blocked the NF-kB induced by hypoxia and p65 downregulation by siRNA sensitized HepG2 cells to 5%02-induced cell death (40-60‘%). Finally, mGSH depletion by DEM/BSO enhanced the susceptibility of HepG2 cells to 5%02 (55-70%) due to the overgeneration of ROS (5-7 fold), being both effects antagonized by RotiTTFA. In conclusion, a moderate ROS generation by hypoxia protects hepatoma cells due to c-Src-NF-kE3 activation. However, these beneficial effects are antagonized if ROS are overgenerated following mGSH depletion, suggesting that this strategy may be of relevance in hepatocarcinogenesis.
12871 TNP-470 INDUCTION OF THE INTRINSIC APOPTOSIS PATHWAY BY IN AN IN VlVO MODEL OF HEPATOCARCINOMA J.L..Mauriz’, P. Gonzalez’, M.C. Duran’, J. Martin-Renedo’, J.P. Barrio’, J.M. Culebras2, J. Gonzalez-Gallego’ . ’Institute of’ Bionzedicine, Uniuersity of Leon, Leon; ’Hospital of Leon, Leon, SIiain E-mail: [email protected] Background: TNP-470 is a semi-synthetic derivative of fumagillin, a metabolite of Aspergillus fumigatus, with antiagiogenic and antineoplasic effects, and it is able to reduce hepatocarcinoma (HCC) progression in animal models. Neoplasia involves different processes, but in almost all instances deregulated cell proliferation and suppressed cell death provide the underlying platform for neoplasic progression. Imbalance between cell proliferation and apoptosis is a critical key in HCC. Previously we have described that TNP-470 was able to reduce cellular proliferation in HCC, however currently its effects in hepatic apoptotic mechanism are poor known. Aims: To study if the antineoplasic effect of TNP-470 is related to apoptotic mechanisms in an animal model of HCC. Methodology: Male Wistar rats weighing 120g at the beginning of experiment were used. Hepatocarcinogenesis was induced according to the Solt-Farber protocol, animals received a single i.p. injection of diethylnitrosamine (200 mgilcg bw) to initiate hepatocarcinogenesis, and were injected with 2-acetilaminofluorene (5mg/kg bw) from days 14 to 35 (twice a week). On day 2 I the animals underwent two-thirds hepatectomy. TNP-470 was injected s.c (30mgikg bw) three times per week from weeks 20 to 28. Animals were sacrificed 28 weeks after initiating the treatment. Results: Carcinomatous tissue growing outside the dysplasic nodules with enlargement of liver sinosoids and sprouting neovessels at the tumour-
04A. MOLECULAR AND CELLULAR BIOLOGY liver interface was observed in HCC-rats. GST-P expression, a marker of preneoplasic and neoplasic cells, was increased in rats with hepatocarcinogenesis. HCC induced a significant reduction on the expression of pro-apoptotic p-2 1 and caspase-3 proteins (-63% and -56% respectively) and on the release of cytochrome c (-50%). Moreover, expression of anti-apoptotic protein Bcl-2 in HCC-rats was increased. No changes were observed on caspase-8 expression. All those changes were inhibited by the TNP-470 treatment. Conclusions: In OUT animal in vivo model, treatment with TNP-470 is able to reduce HCC development. This effect seems to be related, at least in part, to an induction of the intrinsic apoptosis pathway (mitochondriarelated). TNP-470 could be an interesting candidate to the chemotherapeutic arsenal for HCC treatment. Project supported by the Junta de Castilla y Leon, Spain.
12881 THE LIVER-SPECIFIC PROMOTER OF COLLAGEN XVlll IS A FUNCTIONAL TARGET OF THE ClEBPB TRANSCRIPTION FACTOR IN HUMAN LIVER L. Armelin-Correa2, A. Fautrel’, E. Lavergne’, D. Quelard’ , D. Bonnier’, N. Theret’, B. Turlin3, M.R. Passos Bueno2, B. Clement’, 0. Musso’. ‘INSERM U620, Uniuersiti de Rennes I, Rennes, fiance; ’Human Genonte Centel; Bioscience Institute, Suo Puulo Uniuersiv, Sao Puulo, Bruzil; ”Anatontic Puthology Luhoratov, Pontchaillou Hospital, Rennes, Frunce E-mail: [email protected] Collagen XVTTT (CIS) is a basement membrane component and a plasma protein secreted by hepatocytes. C18 contains two motifs of biological importance, endostatin, an endogenous inhibitor of angiogenesis in mice and Frizzled, homologous to the extracellular part of the frizzled receptors. We previously showed that tumor progression and angiogenesis are associated with low expression of C18 in human hepatocellular carcinomas (Cancer Res, 61:45) and that C18 is both a plasma protein and a major extracellular matrix component (Hepatology, 33:868). This unique feature is explained by two CI 8 variants, expressed under the control of separate promoters. Variant#l is expressed by activated hepatic stellate cells and endothelial cells under the control of a ubiquitous promoter. Variant #2 is a hepatocyte-specific plasma protein expressed under the control of a liverenriched promoter (Hepatology, 32: 1377; Matrix Biol, 24:550). By in silico analysis, we identified several putative CCAAT/enhancer binding protein (C/EBP) binding sites in the C18 liver-enriched promoter, which led us to hypothesize that C18 may be a target of CIEBPb. Then, we investigated the expression of C18 and C/EBPb mRNAs in a human liver tissue collection (n = 89; including 47 cirrhoses, 10 fibroses, 8 histologically normal livers and 24 HCCs). Real-time PCR analysis showed that the expression of the liver-specific C18 variant and that the expression of CIEBPb mRNAs were correlated (R= 0.50; p = 0.000000; n = 87). Moreover, cotransfection of a luciferase reporter cDNA containing a C I8 minimal promoter with a CiEBPb binding site and the MSVWEBPb expression vector in Hep3B or Huh-7 human HCC cell lines showed a dose-dependent increase in CI 8 promoter activity. Furthermore, a mutated C/EBPb site in the C18 minimal promoter abrogated the response to CiEBPb in Hep3B and Huh-7 cells. These findings suggest an involvement of C/EBPb in C I8 expression in liver fibrosis and cancer, providing a possible mechanism to the decrease in C18 expression during tumor progression of HCCs.
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A) CELL CYCLE CONTROL/APOPTOSIS
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12891 HYDROPHOBIC BILE SALTS ACTIVATE NADPH OXIDASE THROUGH ENDOSOMAL ACIDIFICATION R. Reinehr’, S. Beclcer’ , S. Grether-Beck2, A. Eberle’, D. Graf’ , S. vom Dahl’ , D. Haussinger’ . ‘Clinic For Gustroenteroloa, Hepatology and Infkctiology, Heinrich-Heine-linioevsity,Diiesseldoyf,~,’lnstitut Fuer Umwrltmrdizinischr Forschung (IUF), Hrinrich-Heinr- Uniuersity, Duesseldoyf,. Germany E-mail: reinehrauni-duesseIdorf.de Introduction: Hydrophobic bile salts activate NADPH oxidase (Nox). dependent reactive oxygen species (ROS) generation through a ceramide and protein kinase C (PKC)E-dependent pathway as an important upstream event of bile salt-induced hepatocyte apoptosis (Reinehr et al., Gastroenterology 2005). Methods: In primary rat hepatocytes and hepatocytes from the p47phox knock-out mice NADPH oxidase expression was detected by PCR, western blotting and immunocytochemistry, ceramide formation by lipid extraction and high performance thin layer chromatography (HPTLC) and ROS generation by DCFDA fluorescence. Apparent vesicular pH was determined using endocytosed FTTC-dextran and cytosolic [CI-] using MQAE fluorescence. ASM protein knock-down was achieved using antisense oligonucleotides. CD95/EGFR association and membrane trafficking was visualized using EGFR-CFP and CD95-YFP cotransfection in Huh7 cells and subsequent fluorescence resonance engery transfer (FRET)microscopy. Hepatocyte shrinkage was measured in the intact perfused rat liver and calculated from the difference of washout profiles of simultaneously infused [ ‘ 4 C ] ~ e aand [3H]inulin. Results: Proapoptotic bile salts, such as taurolithocholylsulfate (TLCS), but not taurocholate (TC) lowered within seconds the apparent vesicular pH from 6.0 to 5.6 in a FITC-dextran-accessible, endosomal compartment which also contains acidic sphingomyelinase (ASM). Simultaneously, a rapid decrease of MQAE-fluorescence was observed, suggestive for an increase of cytosolic [CI-1, which is known to activate vacuolartype H+-ATPase. Inhibition of TLCS-induced endosomal acidification by bafilomycin or DlDS largely abolished the TLCS-induced ceramideformation, p47phox-serine phosphorylation and ROS generation as well as CD95-activation and apoptosis. These responses were also abolished after knock-down of ASM in rat hepatocytes or in hepatocytes from p47phox knock-out mice. Another consequence of TLCS-induced ROS-formation was hepatocyte shrinkage, which was prevented by desipramine, apocynin, bafilomycin and DlDS and in hepatocytes from p47phox knock-out mice. Conclusions: The data suggest that hydrophobic, proapoptotic bile salts activate Nox isofoms through a chloride-dependent acidification of endosomes, which augments ASM-dependent ceramide-formation and PKCre-dependent serine-phosphorylation of p47phox. The resulting ROSresponse triggers not only CD95-activation, but also hepatocyte shrinkage. Because shrinkage by itself stimulates ROS-formation, a vicious cycle ensues, which may finally result in hepatocyte apoptosis. The data link TLCS-induced alterations of ion homeostasis to apoptosis induction by hydrophobic bile salts.
12901 URSODEOXYCHOLIC ACID REDUCES BILE ACIDINDUCED HEPATOCYTE APOPTOSIS BY MODULATION OF AP-1 C. Rust, T. Vennegeerts, U. Beuers. Department of Medicine 2 Grosshadern,Uniuer~sityof Munich, Munich, Germany E-mail: [email protected] Background and Aims: Hydrophobic bile acids induce hepatocyte apoptosis thereby contributing to liver injury in cholestasis. In contrast, nontoxic bile acids like tauroursodeoxycholic acid (TUDCA) protect against bile acid-induced apoptosis. The human transcription factor AP-1 is a mediator of bile acid-induced liver cell apoptosis (BBRC 2006;340:800).
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alpha. In all groups of patients investigated, a constant 4-8 fold increase of interferon gamma production was observed by coincubation of adenosine with interferon alpha as compared to stimulation with interferon alpha alone. This increased TFN gamma production required the presence of CD14+ cells and was independent from CD56+ cells. Conclusion: Thus, OUT data suggest that adenosine and interferon alpha may synergistically increase effector functions during acute infections but also prevent overwhelming immune responses by inhibiting proliferation and induction of apoptosis of activated lymphoid cells. The potential therapeutic role of adenosine receptor agonists in the treatment with interferon alpha-based therapies in viral hepatitis requires further investigation.
12851 UP-REGULATION OF A20, AN ANTI-APOPTOTIC PROTEIN, IS PART OF THE PROTECTIVE RESPONSE TO HYPOXIC STRESS IN THE LIVER A. Keogh, M. Gass, D. Candinas, D. Stroka. Clinic of Visceral and Transplantation Surgev, Unioersiiy of Bern, Bern, Switzerland E-mai I : adrian. [email protected] i be .ch Background and Aim: A20 was originally described as a TNF-inducible protein whose expression protects against apoptosis and limits inflammation via inhibition of NF-kB. We previously demonstrated that A20 mRNA is induced in livers of mice during surgically induced hypoxia. We identified within the A20 promoter putative binding sites for the hypoxia-inducible transcription factor (H1F)-1. The aim of this study was to determine whether hypoxia directly effects the expression of A20 and if the mechanism was dependent on HIF-1. Methods: Isolated primary human hepatocytes were cultured in William’s E medium for 24-48 hours followed by either incubated in a hypoxic (1.5% 0 2 ) or normoxic environment for 6 hours. To investigate the effects of inflammation, hepatocytes were incubated with TNF ( 1 Ong/mL) with and without hypoxia. To determine HTF dependent activation, cells were incubated with the HTF- 1 activators, dimethyloxaloylglycine (DMOG) ( I 25mM), CoC12 ( 1 00mM) or desferoxamine (DFX) ( I 00mM) for 4 hours under normal culture conditions. RNA and protein were isolated for each condition for real time PCR and western blot analysis respectively. Results: Hypoxia increased A20 mRNA 33-foldf10 (n=21) compared to normoxic hepatocytes (p < 0.001). The median fold increase by hypoxia was significantly higher than cells cultured in the presence of TNF 9-fold&3 (p < 0.001) (n=9) alone. However, the combination of hypoxia and TNF resulted in a 1 7 8 f 2 0 median fold increase. Western blot analysis confirms the respective increase of A20 protein. Stabilization of HTF-I a protein after treated with DMOG, CoC12 or DFX is confirmed by western, however there was no resulting increase of A20 mRNA 1.2-fold+0.3 (n = 6) or protein. Conclusion: Here we present the first evidence that A20 is induced by hypoxia albeit in a HTF-independent manner. Originally described as a TNF-inducible protein, hypoxic stress is a more potent stimulus of A20 expression in hepatocytes and hypoxia augments the TNF-induced response. These finding broaden the anti-apoptotic scope of A20 to include hepatoprotection under low-oxidative stress.
12861 BIFUNCTIONAL ROLE OF MITOCHONDRIAL REACTIVE OXYGEN SPECIES IN DETERMINING THE SURVlVALlSUSCEPTlBlLITY OF HEPATOMA CELLS TO HY POXlA J.M. L1uis’,2, A. Morales’,2, C. Blasco’, A. Colell’,2, M. Mari’,2, C. Garcia-Ruiz’,2, J.C. Fernandez-Checa’,2. ’Liver Unit, Instituto Mablties Digestiues, Hospital Clinic, IDIBAPS, Barcelona; ’Department of‘ Cell Death And Prolif2vrution, IIBB-CSK, Barcelona, Spain E-mail: [email protected] Hypoxia occurs when the oxygen supply does not meet the cellular requirements for energy generation and it is often associated with diverse
pathologies including cancer. In fact many solid tumors develop under hypoxic conditions due to the oxygen gradient that it is established from the periphery to the centre of the tumor. Hypoxia activates a complex signaling program determined by the activation of transcription factors HIF-1 dNF-kE3 and the overstimulation of reactive oxygen species (ROS) from mitochondria. Given the complex and often controversial relation between ROS generation and carcinogenesis, the aim of this work was to examine the role of the mitochondrial ROS generation caused by hypoxia in the survival/susceptibility of human hepatoma HepG2 cells and the modulation of mitochondrial GSH (mGSH) (J. Biol. Chem. 280: 32243232, 2005). Methods: HepG2 cells were subjected to hypoxia (5%02) o normoxia (21%02) for 24-72 hrs with o without mGSH depletion, determining ROS, Src activation, NF-IcB regulation, gene expression by real-time PCR and cell viabilityideath. Results: 5%O2 stimulated a time-dependent ROS generation vs 21%02 (2.5-4.5 fold), abolished by blocking mitochondrial complex 1/11 by rotenone (Rot)/TTFA. The DNA binding of p65/p50 and the level of HIFl a increased in nuclear extracts by hypoxia, effects that were accompanied by PDKl, VEGF and CIA€’-2 induction, while RotiTTFA preincubation prevented these responses. Furthermore, although the levels of TkB-a and phospho-TkB-a at Ser 32 remained constant, hypoxia stimulated the phosphorylation of IkB-a in tyrosine residues. Moreover, hypoxia stimulated the levels of phospho-c-Src at tyrosine 416 and 319 by a mechanism dependent on the mitochondrial ROS generation because RotiTTFA abolished c-Src phosphorylation. The c-Src inhibitor, pp2, blocked the NF-kB induced by hypoxia and p65 downregulation by siRNA sensitized HepG2 cells to 5%02-induced cell death (40-60‘%). Finally, mGSH depletion by DEM/BSO enhanced the susceptibility of HepG2 cells to 5%02 (55-70%) due to the overgeneration of ROS (5-7 fold), being both effects antagonized by RotiTTFA. In conclusion, a moderate ROS generation by hypoxia protects hepatoma cells due to c-Src-NF-kE3 activation. However, these beneficial effects are antagonized if ROS are overgenerated following mGSH depletion, suggesting that this strategy may be of relevance in hepatocarcinogenesis.
12871 TNP-470 INDUCTION OF THE INTRINSIC APOPTOSIS PATHWAY BY IN AN IN VlVO MODEL OF HEPATOCARCINOMA J.L..Mauriz’, P. Gonzalez’, M.C. Duran’, J. Martin-Renedo’, J.P. Barrio’, J.M. Culebras2, J. Gonzalez-Gallego’ . ’Institute of’ Bionzedicine, Uniuersity of Leon, Leon; ’Hospital of Leon, Leon, SIiain E-mail: [email protected] Background: TNP-470 is a semi-synthetic derivative of fumagillin, a metabolite of Aspergillus fumigatus, with antiagiogenic and antineoplasic effects, and it is able to reduce hepatocarcinoma (HCC) progression in animal models. Neoplasia involves different processes, but in almost all instances deregulated cell proliferation and suppressed cell death provide the underlying platform for neoplasic progression. Imbalance between cell proliferation and apoptosis is a critical key in HCC. Previously we have described that TNP-470 was able to reduce cellular proliferation in HCC, however currently its effects in hepatic apoptotic mechanism are poor known. Aims: To study if the antineoplasic effect of TNP-470 is related to apoptotic mechanisms in an animal model of HCC. Methodology: Male Wistar rats weighing 120g at the beginning of experiment were used. Hepatocarcinogenesis was induced according to the Solt-Farber protocol, animals received a single i.p. injection of diethylnitrosamine (200 mgilcg bw) to initiate hepatocarcinogenesis, and were injected with 2-acetilaminofluorene (5mg/kg bw) from days 14 to 35 (twice a week). On day 2 I the animals underwent two-thirds hepatectomy. TNP-470 was injected s.c (30mgikg bw) three times per week from weeks 20 to 28. Animals were sacrificed 28 weeks after initiating the treatment. Results: Carcinomatous tissue growing outside the dysplasic nodules with enlargement of liver sinosoids and sprouting neovessels at the tumour-
04A. MOLECULAR AND CELLULAR BIOLOGY liver interface was observed in HCC-rats. GST-P expression, a marker of preneoplasic and neoplasic cells, was increased in rats with hepatocarcinogenesis. HCC induced a significant reduction on the expression of pro-apoptotic p-2 1 and caspase-3 proteins (-63% and -56% respectively) and on the release of cytochrome c (-50%). Moreover, expression of anti-apoptotic protein Bcl-2 in HCC-rats was increased. No changes were observed on caspase-8 expression. All those changes were inhibited by the TNP-470 treatment. Conclusions: In OUT animal in vivo model, treatment with TNP-470 is able to reduce HCC development. This effect seems to be related, at least in part, to an induction of the intrinsic apoptosis pathway (mitochondriarelated). TNP-470 could be an interesting candidate to the chemotherapeutic arsenal for HCC treatment. Project supported by the Junta de Castilla y Leon, Spain.
12881 THE LIVER-SPECIFIC PROMOTER OF COLLAGEN XVlll IS A FUNCTIONAL TARGET OF THE ClEBPB TRANSCRIPTION FACTOR IN HUMAN LIVER L. Armelin-Correa2, A. Fautrel’, E. Lavergne’, D. Quelard’ , D. Bonnier’, N. Theret’, B. Turlin3, M.R. Passos Bueno2, B. Clement’, 0. Musso’. ‘INSERM U620, Uniuersiti de Rennes I, Rennes, fiance; ’Human Genonte Centel; Bioscience Institute, Suo Puulo Uniuersiv, Sao Puulo, Bruzil; ”Anatontic Puthology Luhoratov, Pontchaillou Hospital, Rennes, Frunce E-mail: [email protected] Collagen XVTTT (CIS) is a basement membrane component and a plasma protein secreted by hepatocytes. C18 contains two motifs of biological importance, endostatin, an endogenous inhibitor of angiogenesis in mice and Frizzled, homologous to the extracellular part of the frizzled receptors. We previously showed that tumor progression and angiogenesis are associated with low expression of C18 in human hepatocellular carcinomas (Cancer Res, 61:45) and that C18 is both a plasma protein and a major extracellular matrix component (Hepatology, 33:868). This unique feature is explained by two CI 8 variants, expressed under the control of separate promoters. Variant#l is expressed by activated hepatic stellate cells and endothelial cells under the control of a ubiquitous promoter. Variant #2 is a hepatocyte-specific plasma protein expressed under the control of a liverenriched promoter (Hepatology, 32: 1377; Matrix Biol, 24:550). By in silico analysis, we identified several putative CCAAT/enhancer binding protein (C/EBP) binding sites in the C18 liver-enriched promoter, which led us to hypothesize that C18 may be a target of CIEBPb. Then, we investigated the expression of C18 and C/EBPb mRNAs in a human liver tissue collection (n = 89; including 47 cirrhoses, 10 fibroses, 8 histologically normal livers and 24 HCCs). Real-time PCR analysis showed that the expression of the liver-specific C18 variant and that the expression of CIEBPb mRNAs were correlated (R= 0.50; p = 0.000000; n = 87). Moreover, cotransfection of a luciferase reporter cDNA containing a C I8 minimal promoter with a CiEBPb binding site and the MSVWEBPb expression vector in Hep3B or Huh-7 human HCC cell lines showed a dose-dependent increase in CI 8 promoter activity. Furthermore, a mutated C/EBPb site in the C18 minimal promoter abrogated the response to CiEBPb in Hep3B and Huh-7 cells. These findings suggest an involvement of C/EBPb in C I8 expression in liver fibrosis and cancer, providing a possible mechanism to the decrease in C18 expression during tumor progression of HCCs.
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A) CELL CYCLE CONTROL/APOPTOSIS
s115
12891 HYDROPHOBIC BILE SALTS ACTIVATE NADPH OXIDASE THROUGH ENDOSOMAL ACIDIFICATION R. Reinehr’, S. Beclcer’ , S. Grether-Beck2, A. Eberle’, D. Graf’ , S. vom Dahl’ , D. Haussinger’ . ‘Clinic For Gustroenteroloa, Hepatology and Infkctiology, Heinrich-Heine-linioevsity,Diiesseldoyf,~,’lnstitut Fuer Umwrltmrdizinischr Forschung (IUF), Hrinrich-Heinr- Uniuersity, Duesseldoyf,. Germany E-mail: reinehrauni-duesseIdorf.de Introduction: Hydrophobic bile salts activate NADPH oxidase (Nox). dependent reactive oxygen species (ROS) generation through a ceramide and protein kinase C (PKC)E-dependent pathway as an important upstream event of bile salt-induced hepatocyte apoptosis (Reinehr et al., Gastroenterology 2005). Methods: In primary rat hepatocytes and hepatocytes from the p47phox knock-out mice NADPH oxidase expression was detected by PCR, western blotting and immunocytochemistry, ceramide formation by lipid extraction and high performance thin layer chromatography (HPTLC) and ROS generation by DCFDA fluorescence. Apparent vesicular pH was determined using endocytosed FTTC-dextran and cytosolic [CI-] using MQAE fluorescence. ASM protein knock-down was achieved using antisense oligonucleotides. CD95/EGFR association and membrane trafficking was visualized using EGFR-CFP and CD95-YFP cotransfection in Huh7 cells and subsequent fluorescence resonance engery transfer (FRET)microscopy. Hepatocyte shrinkage was measured in the intact perfused rat liver and calculated from the difference of washout profiles of simultaneously infused [ ‘ 4 C ] ~ e aand [3H]inulin. Results: Proapoptotic bile salts, such as taurolithocholylsulfate (TLCS), but not taurocholate (TC) lowered within seconds the apparent vesicular pH from 6.0 to 5.6 in a FITC-dextran-accessible, endosomal compartment which also contains acidic sphingomyelinase (ASM). Simultaneously, a rapid decrease of MQAE-fluorescence was observed, suggestive for an increase of cytosolic [CI-1, which is known to activate vacuolartype H+-ATPase. Inhibition of TLCS-induced endosomal acidification by bafilomycin or DlDS largely abolished the TLCS-induced ceramideformation, p47phox-serine phosphorylation and ROS generation as well as CD95-activation and apoptosis. These responses were also abolished after knock-down of ASM in rat hepatocytes or in hepatocytes from p47phox knock-out mice. Another consequence of TLCS-induced ROS-formation was hepatocyte shrinkage, which was prevented by desipramine, apocynin, bafilomycin and DlDS and in hepatocytes from p47phox knock-out mice. Conclusions: The data suggest that hydrophobic, proapoptotic bile salts activate Nox isofoms through a chloride-dependent acidification of endosomes, which augments ASM-dependent ceramide-formation and PKCre-dependent serine-phosphorylation of p47phox. The resulting ROSresponse triggers not only CD95-activation, but also hepatocyte shrinkage. Because shrinkage by itself stimulates ROS-formation, a vicious cycle ensues, which may finally result in hepatocyte apoptosis. The data link TLCS-induced alterations of ion homeostasis to apoptosis induction by hydrophobic bile salts.
12901 URSODEOXYCHOLIC ACID REDUCES BILE ACIDINDUCED HEPATOCYTE APOPTOSIS BY MODULATION OF AP-1 C. Rust, T. Vennegeerts, U. Beuers. Department of Medicine 2 Grosshadern,Uniuer~sityof Munich, Munich, Germany E-mail: [email protected] Background and Aims: Hydrophobic bile acids induce hepatocyte apoptosis thereby contributing to liver injury in cholestasis. In contrast, nontoxic bile acids like tauroursodeoxycholic acid (TUDCA) protect against bile acid-induced apoptosis. The human transcription factor AP-1 is a mediator of bile acid-induced liver cell apoptosis (BBRC 2006;340:800).