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Mechanisms of sulindac-induced apoptosis and cell cycle arrest
GASTROENTEROLOGY Vol. 118, No.4
A246 AGA ABSTRACTS
1438 IMPLICATION OF REACTIVE OXYGEN SPECIES (ROS) IN GASTROPROTECTION AND ULCER HEALING BY NITRIC OXIDE (NO)·RELEASING ASPIRIN. Tomasz Brzozowski, Slawomir Kwiecien, Danuta Drozdowicz, Michal Pawlik , Stanislaw 1. Konturek, Dept Physiol , Univ Med Sch, Cracow , Poland . NSAIDs such as aspirin (ASA) are known to suppress cyclooxygenase (COX) activity and induce gastric mucosal damage but new class of NO-releasing NSAIDs that was also shown to inhibit COX remained without mucosal damage. ASA damage were originally attributed in part to enhanced generation of ROS and lipid peroxidation but it is unknown whether NO-ASA can affect ROS and antioxidative mechanism in the gastric mucosa . In this study we compared the effect of ASA with that of NO-AS A on gastric mucosal lesions provoked by 100% ethanol (series A) and healing of gastric ulcers induced by serosal application of acetic acid (ulcer area 28 mm 2)(Series B). Gastric injury was measured by planimetry, the gastric blood flow (GBF) was assessed by H2-gas clearance and blood was collected for measurement of ROS by luminol-dependent chernilurninence assay. Myeloperoxidase (MPO), an index of ASA-dependent neutrophil activation and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and malonyldialdehyde (MDA) concentration, an index of oxygen radical- lipid peroxidation, were measured in the gastric mucosa. The expression of SOD and GPx mRNA in the gastric mucosa were analyzed by RT-PCR. ASA (100 mg/kg i.g.) produced acute gastric lesions that were accompanied by significant fall in the GBF (by 45%) and PGE 2 generation (by 90%) and the rise in luminol-arnplified chemiluminescence and 2-3 fold increase in MPO activity and MDA levels. The deleterious effect of native ASA was accompanied by a potent suppression of SOD and GPx activity and expression of SOD and GPx mRNAs . In contrast, NO-AS A alone produced negligible gross and histologic mucosal damage but dose-dependently attenuated ethanol lesions being accompanied by the rise in GBF and luminal NO and a significant fall in the chemiluminescence. Coupl ing of ASA with NO releasing agent such as SNAP (2 mg/kg i.g.) reduced significantly the changes in lipid peroxidation and MPO, SOD and GPx activities as compared to those treated with native ASA . Treatment with ASA delayed ulcer healing and decreased GBF at ulcer margin and these effects were counteracted by NO-ASA . We conclude that: I)ASA damages the gastric mucosa due to enhancement in ROS, lipid peroxidation and suppression of antioxidizing mechanisms such as SOD and GPx activity and 2) suppression of ROS and lipid peroxidation as well as preservation of antioxidative enzyme activity play an important role in the mechanism of gastroprotection and ulcer healing by NO-ASA.
1439 GASTROPROTECTIVE EFFECTS OF AMTOLMETIN GUACYL: A NEW NON STEROIDAL ANTIINFLAMMATORY DRUG THAT ACTIVATES INDUCIBLE GASTRIC NITRIC OXIDE SYNTHASE. Gabriella Coruzzi, Gabriella Coppelli, Giulia M. Cavestro, Lajos Okolicsanyi , Claudio Pisano, Barry Tepperman, Inst Pharmacology, Parma Univ, Parma , Italy; Chair of Gastroenterology, Parma Univ, Parma, Italy; SigmaTau, Pomezia, Italy; Institute of Physiology, Univ of Western Ontario, London, Canada. The novel non steroidal antiinflammatory drug (NSAID) amtolmetin guacyl (AMG) was recently shown to be characterized by a lack of gastric toxicity in the rat and to exert protective effects against ethanol (Dig Dis Sci, 44 ; 713, 1998). The present study was undertaken to investigate the involvement of endogenous nitric oxide (NO) in the protective action of AMG. The effects of AMG on gastric mucosal potential difference (PO), gastric mucosal blood flow and neutrophil infiltration were determined in the anaesthetized rat. Moreover, the effect of AMG on NO production was assessed by citrulline formation and by evaluating protein expression of inducible NO synthase (iNOS) in the gastric mucosa by Western irnmunoblotting. The effect of AMG was compared to that of tolmetin , which is the active metabolite of AMG, responsible for the antiinflammatory effect. AMG , administered intragastrically (i.g.) at 50-300 mg/kg, did not modify basal gastric PD and significantly reduced (approximately by 50%) the fall in PO and the increase in neutrophil infiltration caused by intragastric ethanol (EtOH 50% v/v) instillation. In the same experimental model tolmctin (30-100 mg/kg i.g.) was without any effect. The protective effect induced by AMG was maximum when the compound was administered 4-6 h before EtOH . The gastroprotective effect of AMG was eliminated by pretreating rats with the non selective NOS inhibitor L-NAME (10 mg/kg i.v.), the selective iNOS inhibitor 1400w (I mg/kg i.v.) and dexamethasone (I mg/kg i.v.). Pretreatment of animals with 1400w also resulted in a significant decline in gastric mucosal blood flow in AMG (100 mg/kg)pretreated animals. Furthermore, AMG treatment resulted in an increase in
calcium-independent iNOS activity as assessed by citrulline formation and Western immunoblotting revealed iNOS protein in mucosal samples excised from AMG (100 mg/kg)-treated animals. These data suggest that AMG protects the gastric mucosa from EtOH-induced challenge via an increase in inducible NOS activity . This protective effect may be mediated via increases in gastric mucosal blood flow and a reduction in neutrophil infiltration.
1440 NSAID·INDUCED APOPTOSIS AND DOWN-REGULATION OF BCL-2 EXPRESSION, Katja Ganz, Vladan Milovic, Wolfgang F. Caspary, Juergen Stein, 2nd Dept Medicine, JW Goethe Univ, Frankfurt, Germany. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit colonic tumourigenesis and are among the few agents with an established usefulnes s in cancer prevention. There is an increasing evidence that induction of apoptosis by NSAIDs occurs via a COX-2-independent pathway , by a variety of mechanisms proposed . To elucidate this further , we evaluated the effect of non-selective COX-I/COX-2 inhibitor (indomethacin) and a selective COX-2 inhibitor (SC-58125), in conditionally immortalised intestinal epithelial cell line (2141A1). These cells undergo apoptosis after switch to non-permissive temperature. 214/AI cells express only minute amounts of COX-2 after 6h-stimulation with phorbol esters. HCT-116 human colon cancer cells, known not to express COX-2, were used as control. Methods . 214/AI cells were stably transfected with human bcl-2. Overexpression of bcl-2 in 2/4/AI-CI clone prevented apoptosis after temperature switch . Proliferation was evaluated by cell counts , cytotoxicity by assessing membrane damage (LDH release) , apoptosis by DNA fragmentation, and expression of apoptosis-related proteins by Western blot. Results. Despite variations of COX-2 expression, all cell lines showed similar sensitivity to growth inhibition and induction of apoptosis by NSAIDs. SC-58125 was more potent inhihitor of cell growth than indomethacin (IC so : 2/4/AI indomethacin 94±4 p.M, SC-58125 15±3; 2/4/AI-CI : 101±7 and 21 ± I ; HCT-116: 11O±8 and 24±3). DNA fragmentation indicated that both indomethacin and SC-581 25 induced apoptosis in all cell lines. There was no NSAID-dependent change in p53 expression in 214/AI cells (original, and -CI clone) at either permissive or non-permissive temperature . However, treatment with SC-58125 caused down-regulation in bcl-2 protein expression a time- and dose-dependent fashion, followed by rapid enhancement of changes in nuclear morphology typical for apoptosis. Conclu sion . SC-58125 causes growth inhibition and induces apoptosis in human colon cancer cells by a COX-2 independent mechanism , and is more potent growth inhibitor than indomethacin. Induction of apoptosis is associated with down-regulation of bcl-2 protein levels. In addition to its antiinflammatory actions, SC-58125 may be useful in the prevention and treatment of human colon cancer .
1441 MECHANISMS OF SULINDAC-INDUCED APOPTOSIS AND CELL CYCLE ARREST. Barbara Jung, Howard Brickner, Valeri Barbier, John Welsh, Arun Fotedar, Michael McClelland, Univ of CA, San Diego, La Jolla, CA; Sidney Kimmel Cancer Ctr, San Diego, CA. INTRODUCTION: Numerous epidemiologic and animal studies document the chemopreventive effects of the non-steroidal anti-inflammatory agent sulindac. In vitro, the active compound sulindac sulfide induces cell cycle arrest and apoptosis, however the involved mechanisms remain largely unknown . The following studies aim at clarifying the mechanisms of sulindac-induced cell death and growth arrest. METHODS: Using a panel of knockout mice deficient in proteins that are major player s in apopto sis, we compared death rates of knockout and wild type thymocytes using conventional cell death assays, including trypan blue exclusion , ELISA growth assays , TUNEL, annexin V expression and PI inclusion . In order to dissect sulindac-induced growth arrest , we compared BrdU-incorporation of wild type and Rb -1-, p53 -1- and p21 -1- mouse embryo fibroblasts (MEFs). In addition, sulindac-induced apoptosis was assessed in these cell lines. RESULTS: We show that sulindac-induced cell death is p53, bax, Fas and FasL independent. Further, overexpression of bcl-2 counteracts sulindac-induced apoptosis . In addition , we demonstrate that sulindacinduced cell cycle arrest is partly mediated by Rb and p21 and not by p53. Rb -1- and p21 -1- MEFs fail to undergo growth arrest following sulindac treatment. Also, those cells are more susceptible to sulindac-induced apoptosis . CONCLUSION: Sulindac-induced cell death is not dependent on p53, bax, Fas or FasL expression, indicating the involvement of a more complex than the conventional apoptosis pathways. Sulindac-induced cell cycle arrest utilizes p21 and Rb and the ability to arrest protects cells from apoptosis. This outlines a potential mechanism for the chemopreventive properties of sulindac, as its ability to target premalignant cells only might be secondary to early mutations in protective proteins such as Rb and p21.
A246 AGA ABSTRACTS
1438 IMPLICATION OF REACTIVE OXYGEN SPECIES (ROS) IN GASTROPROTECTION AND ULCER HEALING BY NITRIC OXIDE (NO)·RELEASING ASPIRIN. Tomasz Brzozowski, Slawomir Kwiecien, Danuta Drozdowicz, Michal Pawlik , Stanislaw 1. Konturek, Dept Physiol , Univ Med Sch, Cracow , Poland . NSAIDs such as aspirin (ASA) are known to suppress cyclooxygenase (COX) activity and induce gastric mucosal damage but new class of NO-releasing NSAIDs that was also shown to inhibit COX remained without mucosal damage. ASA damage were originally attributed in part to enhanced generation of ROS and lipid peroxidation but it is unknown whether NO-ASA can affect ROS and antioxidative mechanism in the gastric mucosa . In this study we compared the effect of ASA with that of NO-AS A on gastric mucosal lesions provoked by 100% ethanol (series A) and healing of gastric ulcers induced by serosal application of acetic acid (ulcer area 28 mm 2)(Series B). Gastric injury was measured by planimetry, the gastric blood flow (GBF) was assessed by H2-gas clearance and blood was collected for measurement of ROS by luminol-dependent chernilurninence assay. Myeloperoxidase (MPO), an index of ASA-dependent neutrophil activation and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and malonyldialdehyde (MDA) concentration, an index of oxygen radical- lipid peroxidation, were measured in the gastric mucosa. The expression of SOD and GPx mRNA in the gastric mucosa were analyzed by RT-PCR. ASA (100 mg/kg i.g.) produced acute gastric lesions that were accompanied by significant fall in the GBF (by 45%) and PGE 2 generation (by 90%) and the rise in luminol-arnplified chemiluminescence and 2-3 fold increase in MPO activity and MDA levels. The deleterious effect of native ASA was accompanied by a potent suppression of SOD and GPx activity and expression of SOD and GPx mRNAs . In contrast, NO-AS A alone produced negligible gross and histologic mucosal damage but dose-dependently attenuated ethanol lesions being accompanied by the rise in GBF and luminal NO and a significant fall in the chemiluminescence. Coupl ing of ASA with NO releasing agent such as SNAP (2 mg/kg i.g.) reduced significantly the changes in lipid peroxidation and MPO, SOD and GPx activities as compared to those treated with native ASA . Treatment with ASA delayed ulcer healing and decreased GBF at ulcer margin and these effects were counteracted by NO-ASA . We conclude that: I)ASA damages the gastric mucosa due to enhancement in ROS, lipid peroxidation and suppression of antioxidizing mechanisms such as SOD and GPx activity and 2) suppression of ROS and lipid peroxidation as well as preservation of antioxidative enzyme activity play an important role in the mechanism of gastroprotection and ulcer healing by NO-ASA.
1439 GASTROPROTECTIVE EFFECTS OF AMTOLMETIN GUACYL: A NEW NON STEROIDAL ANTIINFLAMMATORY DRUG THAT ACTIVATES INDUCIBLE GASTRIC NITRIC OXIDE SYNTHASE. Gabriella Coruzzi, Gabriella Coppelli, Giulia M. Cavestro, Lajos Okolicsanyi , Claudio Pisano, Barry Tepperman, Inst Pharmacology, Parma Univ, Parma , Italy; Chair of Gastroenterology, Parma Univ, Parma, Italy; SigmaTau, Pomezia, Italy; Institute of Physiology, Univ of Western Ontario, London, Canada. The novel non steroidal antiinflammatory drug (NSAID) amtolmetin guacyl (AMG) was recently shown to be characterized by a lack of gastric toxicity in the rat and to exert protective effects against ethanol (Dig Dis Sci, 44 ; 713, 1998). The present study was undertaken to investigate the involvement of endogenous nitric oxide (NO) in the protective action of AMG. The effects of AMG on gastric mucosal potential difference (PO), gastric mucosal blood flow and neutrophil infiltration were determined in the anaesthetized rat. Moreover, the effect of AMG on NO production was assessed by citrulline formation and by evaluating protein expression of inducible NO synthase (iNOS) in the gastric mucosa by Western irnmunoblotting. The effect of AMG was compared to that of tolmetin , which is the active metabolite of AMG, responsible for the antiinflammatory effect. AMG , administered intragastrically (i.g.) at 50-300 mg/kg, did not modify basal gastric PD and significantly reduced (approximately by 50%) the fall in PO and the increase in neutrophil infiltration caused by intragastric ethanol (EtOH 50% v/v) instillation. In the same experimental model tolmctin (30-100 mg/kg i.g.) was without any effect. The protective effect induced by AMG was maximum when the compound was administered 4-6 h before EtOH . The gastroprotective effect of AMG was eliminated by pretreating rats with the non selective NOS inhibitor L-NAME (10 mg/kg i.v.), the selective iNOS inhibitor 1400w (I mg/kg i.v.) and dexamethasone (I mg/kg i.v.). Pretreatment of animals with 1400w also resulted in a significant decline in gastric mucosal blood flow in AMG (100 mg/kg)pretreated animals. Furthermore, AMG treatment resulted in an increase in
calcium-independent iNOS activity as assessed by citrulline formation and Western immunoblotting revealed iNOS protein in mucosal samples excised from AMG (100 mg/kg)-treated animals. These data suggest that AMG protects the gastric mucosa from EtOH-induced challenge via an increase in inducible NOS activity . This protective effect may be mediated via increases in gastric mucosal blood flow and a reduction in neutrophil infiltration.
1440 NSAID·INDUCED APOPTOSIS AND DOWN-REGULATION OF BCL-2 EXPRESSION, Katja Ganz, Vladan Milovic, Wolfgang F. Caspary, Juergen Stein, 2nd Dept Medicine, JW Goethe Univ, Frankfurt, Germany. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit colonic tumourigenesis and are among the few agents with an established usefulnes s in cancer prevention. There is an increasing evidence that induction of apoptosis by NSAIDs occurs via a COX-2-independent pathway , by a variety of mechanisms proposed . To elucidate this further , we evaluated the effect of non-selective COX-I/COX-2 inhibitor (indomethacin) and a selective COX-2 inhibitor (SC-58125), in conditionally immortalised intestinal epithelial cell line (2141A1). These cells undergo apoptosis after switch to non-permissive temperature. 214/AI cells express only minute amounts of COX-2 after 6h-stimulation with phorbol esters. HCT-116 human colon cancer cells, known not to express COX-2, were used as control. Methods . 214/AI cells were stably transfected with human bcl-2. Overexpression of bcl-2 in 2/4/AI-CI clone prevented apoptosis after temperature switch . Proliferation was evaluated by cell counts , cytotoxicity by assessing membrane damage (LDH release) , apoptosis by DNA fragmentation, and expression of apoptosis-related proteins by Western blot. Results. Despite variations of COX-2 expression, all cell lines showed similar sensitivity to growth inhibition and induction of apoptosis by NSAIDs. SC-58125 was more potent inhihitor of cell growth than indomethacin (IC so : 2/4/AI indomethacin 94±4 p.M, SC-58125 15±3; 2/4/AI-CI : 101±7 and 21 ± I ; HCT-116: 11O±8 and 24±3). DNA fragmentation indicated that both indomethacin and SC-581 25 induced apoptosis in all cell lines. There was no NSAID-dependent change in p53 expression in 214/AI cells (original, and -CI clone) at either permissive or non-permissive temperature . However, treatment with SC-58125 caused down-regulation in bcl-2 protein expression a time- and dose-dependent fashion, followed by rapid enhancement of changes in nuclear morphology typical for apoptosis. Conclu sion . SC-58125 causes growth inhibition and induces apoptosis in human colon cancer cells by a COX-2 independent mechanism , and is more potent growth inhibitor than indomethacin. Induction of apoptosis is associated with down-regulation of bcl-2 protein levels. In addition to its antiinflammatory actions, SC-58125 may be useful in the prevention and treatment of human colon cancer .
1441 MECHANISMS OF SULINDAC-INDUCED APOPTOSIS AND CELL CYCLE ARREST. Barbara Jung, Howard Brickner, Valeri Barbier, John Welsh, Arun Fotedar, Michael McClelland, Univ of CA, San Diego, La Jolla, CA; Sidney Kimmel Cancer Ctr, San Diego, CA. INTRODUCTION: Numerous epidemiologic and animal studies document the chemopreventive effects of the non-steroidal anti-inflammatory agent sulindac. In vitro, the active compound sulindac sulfide induces cell cycle arrest and apoptosis, however the involved mechanisms remain largely unknown . The following studies aim at clarifying the mechanisms of sulindac-induced cell death and growth arrest. METHODS: Using a panel of knockout mice deficient in proteins that are major player s in apopto sis, we compared death rates of knockout and wild type thymocytes using conventional cell death assays, including trypan blue exclusion , ELISA growth assays , TUNEL, annexin V expression and PI inclusion . In order to dissect sulindac-induced growth arrest , we compared BrdU-incorporation of wild type and Rb -1-, p53 -1- and p21 -1- mouse embryo fibroblasts (MEFs). In addition, sulindac-induced apoptosis was assessed in these cell lines. RESULTS: We show that sulindac-induced cell death is p53, bax, Fas and FasL independent. Further, overexpression of bcl-2 counteracts sulindac-induced apoptosis . In addition , we demonstrate that sulindacinduced cell cycle arrest is partly mediated by Rb and p21 and not by p53. Rb -1- and p21 -1- MEFs fail to undergo growth arrest following sulindac treatment. Also, those cells are more susceptible to sulindac-induced apoptosis . CONCLUSION: Sulindac-induced cell death is not dependent on p53, bax, Fas or FasL expression, indicating the involvement of a more complex than the conventional apoptosis pathways. Sulindac-induced cell cycle arrest utilizes p21 and Rb and the ability to arrest protects cells from apoptosis. This outlines a potential mechanism for the chemopreventive properties of sulindac, as its ability to target premalignant cells only might be secondary to early mutations in protective proteins such as Rb and p21.