- Email: [email protected]
250 GS-9450: A NOVEL LIVER-TARGETED CASPASE INHIBITOR THAT BLOCKS APOPTOTIC SIGNALING IN HEPATOCYTES
S100
Poster Session − Thursday, April 23
point of view, it is interesting to study whether DDB could protect against ischemia-reperfusion injury induced liver damage and to clarify its role in apoptotic cell death and identify at which level it could interfere with the different apoptotic cascade pathways. Methods: Rats were divided into five groups: Negative control (sham operated), Positive control upon which IRI was performed and three pretreated groups that received different doses of DDB: 100, 300 and 500 mg/kg by oral feeding 10 days prior to IRI.The following parameters were measured: activity of signaling Caspases 8 & 9 and effector Caspase 3 were measured in cell lysate. Expression of Nuclear factor kappa B, BCL2 and BAX were measured by reverse transcriptase Polymerase Chain Reaction in liver tissue.Apoptosis was detected by electrophoresis for analysis of DNA fragmentation. Results: At 500 mg/kg DDB dose level, activity of Caspases 8 and 9 was significantly decreased showing complete protection, while there was no effect on the activity of Caspase 3. At doses of 100 mg/kg and 300 mg/kg, expression of nuclear factor-úB were significantly decreased showing Partial protection, where at the higher dose of 500 mg/kg there was no protection.At doses of 100 mg/kg and 300 mg/kg expression of the antiapoptotic protein BCL2 were elevated, and levels of the protein BAX were decreased showing partial protection, also the higher dose showed no protection. Significant inhibition of nuclear DNA fragmentation was detected. Conclusion: In conclusion DDB inhibited hepatic IRI induced apoptosis through inhibiting signaling Caspases 8 & 9 rather than effector Caspase 3 also by regulating the balance between the antiapoptotic BCL2 and the proapoptotic BAX, and downregulating NF-úB expression in liver tissue. 249 DOWNSTREAM EFFECTS OF OXIDATIVE STRESS IN FERRITIN MEDIATED APOPTOSIS − ACTIVATION OF PROAPOPTOTIC FAS I. Ohlenschl¨ager, H. Jaksch, H. Lacher, P.M. Eckl, N. Bresgen. Department of Cell Biology, University of Salzburg, Salzburg, Austria E-mail: [email protected] Background and Aims: Among a variety of apoptotic stimuli, acidic isoferritins sharing homology to immunomodulatory ferritins have recently been identified as inducers of apoptosis in primary hepatocyte cultures. Ferritin treatment triggers the upregulation of p53 and stimulates proapoptotic Fas signalling. In order to investigate the underlying mechanisms, the role of Fe2+ /Fenton reaction mediated oxidative stress and its effects on FasL expression were analysed. Methods: Primary cultures of rat hepatocytes were treated with acidic isoferritins purified from 3 h hepatocyte conditioned media as well as commercially available ferritin. Assessment of hepatocyte proliferation, apoptosis, necrosis and the formation of micronuclei was based on nuclear morphology using DAPI stained cells. Immunocytochemistry was used to visualize 4-hydroxynonenal (HNE)-modified proteins (measure of lipid peroxidation) and the cellular localization of FasL. Expression of p53, FasL and Bid was analysed by immunoblotting. Results: The radical scavenging vitamin E analogue trolox and the iron chelator desferrioxamin substantially suppress ferritin mediated apoptosis. Furthermore, protein modification by HNE occurs in ferritin treated hepatocytes, supporting the hypothesis that ferritin acts via induction of oxidative stress/lipid peroxidation in the target cells. Since this mechanism also enhances damage to DNA, the observed upregulation of p53 is the expected consequence. Experiments employing neutralizing anti-FasL antibodies in addition revealed a central role of proapototic Fas in ferritin mediated apoptosis: FasL appears to be localized close to the nucleus in untreated hepatocytes but becomes redistributed to the cytosolic compartment upon ferritin exposure. Last, we demonstrated that ferritin mediated apoptosis also involves intrinsic, proapoptotic mitochondrial signalling by Bid. Conclusions: It can be concluded from these observations, that isoferritins stimulate apoptosis in primary rat hepatocytes via induction of oxidative stress, and that the stimulation of Fas appears to be transmitted by microvesicular-FasL in ferritin treated cells. In a physiological context, these findings may account for a role of distinct ferritin species as an
acute phase reactant, playing a role in liver/tissue regeneration. In addition, ferritins may also be responsible for the limited proliferative capacity of human hepatocytes in vitro and the inappropriate expansion of donor cells in the recipient liver upon cell transplantation.
250 GS-9450: A NOVEL LIVER-TARGETED CASPASE INHIBITOR THAT BLOCKS APOPTOTIC SIGNALING IN HEPATOCYTES C. Chung1 , K. Min1 , S. Lee1 , Y. Peng2 , G. Lundgaard2 , E. Bush2 , K. Pitts2 . 1 LG Life Sciences, Daejeon, South Korea; 2 Gilead Sciences, Inc., Westminster, Colorado, USA E-mail: [email protected] Background: Apoptosis, or programmed cell death, is characterized by distinctive morphological and biochemical changes. A biochemical hallmark of apoptosis is the activation of the cysteine aspartyl proteases known as caspases, which are responsible for cleavage of certain cellular targets leading to apoptosis. Activation of caspase activity and eventual cell apoptosis has been associated with a number of liver diseases, including non-alcoholic and alcoholic steatohepatitis, chronic hepatitis B/C virus infection, and cholestatic liver injury. This suggests that caspase inhibitors may be therapeutically useful in such diseases. Thus, we performed a screen leading to the identification of GS-9450, a novel small-molecule irreversible caspase inhibitor. Methods: GS-9450 was evaluated by protein crystallization, and tested in a variety of experiments including in vitro inhibition of recombinant enzyme activity, cell viability, and in vivo pharmacokinetics. Results: Co-crystallization of a diastereomeric mixture of GS-9450 and recombinant human caspase 8 revealed that the (R,S) diastereomer is the primary form bound to the caspase 8 active site. Kinetic experiments using recombinant human caspases, granzymes, and cathepsins showed that GS9450 is selective for caspases, with moderate selectivity for caspase 8. Jurkat cells treated with anti-Fas antibody to induce apoptosis were protected from cell death in a concentration-dependent manner when treated with GS-9450 (EC50 ~16nM). GS-9450 also inhibited caspase activity induced by TNFa/actinomycin D treatment in isolated primary rat hepatocytes. Pharmacokinetic studies in rats revealed GS-9450 hepatic exposure was much greater than systemic exposure (>100 times), suggesting the liver is the pharmacological target organ for GS-9450. Conclusions: GS-9450 is a small molecule inhibitor of caspase activity, exhibiting moderate selectivity for caspase 8. In vitro, GS-9450 potently inhibits caspase activity and apoptotic cell death induced via extrinsic pathways. In vivo, GS-9450 exhibits extensive hepatic distribution, suggesting it may serve as a therapy for apoptosis-related injury and disease in the liver.
Jurkat cell data.
Poster Session − Thursday, April 23
point of view, it is interesting to study whether DDB could protect against ischemia-reperfusion injury induced liver damage and to clarify its role in apoptotic cell death and identify at which level it could interfere with the different apoptotic cascade pathways. Methods: Rats were divided into five groups: Negative control (sham operated), Positive control upon which IRI was performed and three pretreated groups that received different doses of DDB: 100, 300 and 500 mg/kg by oral feeding 10 days prior to IRI.The following parameters were measured: activity of signaling Caspases 8 & 9 and effector Caspase 3 were measured in cell lysate. Expression of Nuclear factor kappa B, BCL2 and BAX were measured by reverse transcriptase Polymerase Chain Reaction in liver tissue.Apoptosis was detected by electrophoresis for analysis of DNA fragmentation. Results: At 500 mg/kg DDB dose level, activity of Caspases 8 and 9 was significantly decreased showing complete protection, while there was no effect on the activity of Caspase 3. At doses of 100 mg/kg and 300 mg/kg, expression of nuclear factor-úB were significantly decreased showing Partial protection, where at the higher dose of 500 mg/kg there was no protection.At doses of 100 mg/kg and 300 mg/kg expression of the antiapoptotic protein BCL2 were elevated, and levels of the protein BAX were decreased showing partial protection, also the higher dose showed no protection. Significant inhibition of nuclear DNA fragmentation was detected. Conclusion: In conclusion DDB inhibited hepatic IRI induced apoptosis through inhibiting signaling Caspases 8 & 9 rather than effector Caspase 3 also by regulating the balance between the antiapoptotic BCL2 and the proapoptotic BAX, and downregulating NF-úB expression in liver tissue. 249 DOWNSTREAM EFFECTS OF OXIDATIVE STRESS IN FERRITIN MEDIATED APOPTOSIS − ACTIVATION OF PROAPOPTOTIC FAS I. Ohlenschl¨ager, H. Jaksch, H. Lacher, P.M. Eckl, N. Bresgen. Department of Cell Biology, University of Salzburg, Salzburg, Austria E-mail: [email protected] Background and Aims: Among a variety of apoptotic stimuli, acidic isoferritins sharing homology to immunomodulatory ferritins have recently been identified as inducers of apoptosis in primary hepatocyte cultures. Ferritin treatment triggers the upregulation of p53 and stimulates proapoptotic Fas signalling. In order to investigate the underlying mechanisms, the role of Fe2+ /Fenton reaction mediated oxidative stress and its effects on FasL expression were analysed. Methods: Primary cultures of rat hepatocytes were treated with acidic isoferritins purified from 3 h hepatocyte conditioned media as well as commercially available ferritin. Assessment of hepatocyte proliferation, apoptosis, necrosis and the formation of micronuclei was based on nuclear morphology using DAPI stained cells. Immunocytochemistry was used to visualize 4-hydroxynonenal (HNE)-modified proteins (measure of lipid peroxidation) and the cellular localization of FasL. Expression of p53, FasL and Bid was analysed by immunoblotting. Results: The radical scavenging vitamin E analogue trolox and the iron chelator desferrioxamin substantially suppress ferritin mediated apoptosis. Furthermore, protein modification by HNE occurs in ferritin treated hepatocytes, supporting the hypothesis that ferritin acts via induction of oxidative stress/lipid peroxidation in the target cells. Since this mechanism also enhances damage to DNA, the observed upregulation of p53 is the expected consequence. Experiments employing neutralizing anti-FasL antibodies in addition revealed a central role of proapototic Fas in ferritin mediated apoptosis: FasL appears to be localized close to the nucleus in untreated hepatocytes but becomes redistributed to the cytosolic compartment upon ferritin exposure. Last, we demonstrated that ferritin mediated apoptosis also involves intrinsic, proapoptotic mitochondrial signalling by Bid. Conclusions: It can be concluded from these observations, that isoferritins stimulate apoptosis in primary rat hepatocytes via induction of oxidative stress, and that the stimulation of Fas appears to be transmitted by microvesicular-FasL in ferritin treated cells. In a physiological context, these findings may account for a role of distinct ferritin species as an
acute phase reactant, playing a role in liver/tissue regeneration. In addition, ferritins may also be responsible for the limited proliferative capacity of human hepatocytes in vitro and the inappropriate expansion of donor cells in the recipient liver upon cell transplantation.
250 GS-9450: A NOVEL LIVER-TARGETED CASPASE INHIBITOR THAT BLOCKS APOPTOTIC SIGNALING IN HEPATOCYTES C. Chung1 , K. Min1 , S. Lee1 , Y. Peng2 , G. Lundgaard2 , E. Bush2 , K. Pitts2 . 1 LG Life Sciences, Daejeon, South Korea; 2 Gilead Sciences, Inc., Westminster, Colorado, USA E-mail: [email protected] Background: Apoptosis, or programmed cell death, is characterized by distinctive morphological and biochemical changes. A biochemical hallmark of apoptosis is the activation of the cysteine aspartyl proteases known as caspases, which are responsible for cleavage of certain cellular targets leading to apoptosis. Activation of caspase activity and eventual cell apoptosis has been associated with a number of liver diseases, including non-alcoholic and alcoholic steatohepatitis, chronic hepatitis B/C virus infection, and cholestatic liver injury. This suggests that caspase inhibitors may be therapeutically useful in such diseases. Thus, we performed a screen leading to the identification of GS-9450, a novel small-molecule irreversible caspase inhibitor. Methods: GS-9450 was evaluated by protein crystallization, and tested in a variety of experiments including in vitro inhibition of recombinant enzyme activity, cell viability, and in vivo pharmacokinetics. Results: Co-crystallization of a diastereomeric mixture of GS-9450 and recombinant human caspase 8 revealed that the (R,S) diastereomer is the primary form bound to the caspase 8 active site. Kinetic experiments using recombinant human caspases, granzymes, and cathepsins showed that GS9450 is selective for caspases, with moderate selectivity for caspase 8. Jurkat cells treated with anti-Fas antibody to induce apoptosis were protected from cell death in a concentration-dependent manner when treated with GS-9450 (EC50 ~16nM). GS-9450 also inhibited caspase activity induced by TNFa/actinomycin D treatment in isolated primary rat hepatocytes. Pharmacokinetic studies in rats revealed GS-9450 hepatic exposure was much greater than systemic exposure (>100 times), suggesting the liver is the pharmacological target organ for GS-9450. Conclusions: GS-9450 is a small molecule inhibitor of caspase activity, exhibiting moderate selectivity for caspase 8. In vitro, GS-9450 potently inhibits caspase activity and apoptotic cell death induced via extrinsic pathways. In vivo, GS-9450 exhibits extensive hepatic distribution, suggesting it may serve as a therapy for apoptosis-related injury and disease in the liver.
Jurkat cell data.