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Cholecystokinin regulates the invasiveness of human pancreatic cancer cell lines via protein kinase C pathway
Pancreatic Disorders #_469
April 1998
reconstitute. How far the preparatory trauma influences following incubations remains to be elucidated. Nevertheless, the time dependent pattern of acinar reconstitution should influence interpretation of results from following settings. Trypan blue is inappropiate for analysis of acinar viability. • G1907 EXTRACELLULAR PHOSPHOLIPASES A2 IN SEVERE ACUTE PANCREATITIS -ASSOCIATED SYSTEMIC INFLAMMATORY RESPONSE SYNDROME (SIRS). A. Hietaranta (1), E. Kemppainen (1), P. Puolakkainen (1), V. Sainio (1), R. Haapialnen (1), E. Kivilaakso (1) and T. Nevalainen (2). From the (1) Second Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland and (2) Department of Pathology, University of Turku, Turku, Finland.
Background & Aims. The pathophysiology of severe acute pancreatitis (AP) resulting in systemic inflammatory response syndrome (SIRS) share similar mechanisms to other conditions predisposing to multiple organ failure. Since extracellular phospholipases A2 (PLA2) have been implicated in the pathophysiology of severe AP, our aim was to measure the serum concentrations of these enzymes in patients with severe acute panereatitis with or without SIRS. Methods. The serum samples were collected daily for one week from admission (days 1-8) in 57 patients with severe AP. The severity grading of AP was based on the Atlanta classification (1992). SIRS was defined according to ACCP/SCCM Consensus Conference Committee (1991). Time-resolved fluoroimmunoassay was used for group I and group II PLA2 measurements. Complications of AP as well as daily MOF-score and the association of local and systemic complications of the disease and SIRS were recorded. Results. Thirty-nine (68.4%) patients fulfilled the criteria of SIRS within first week from admission (days 1-8). Pancreatic necrosis was detected in 43 (75.4%) patients. Infected necrosis was detected preoperatively or at operation in 5 (8.8%) patients. Twenty-four (42.1%) and 7 (12.3%) patients suffered from respiratory and renal failure, respectively. All patients with renal failure had respiratory failure as well. Seven (12.3%) patients died of their disease. All patients with systemic complications fulfilled the criteria of SIRS during the first week from admission. However, only the association between respiratory failure and SIRS was statistically significant (p < 0.05). There was no association between SIRS and pancreatic necrosis. The concentrations of extracellular PLA2s in serum did not differ significantly in patients with or without SIRS. However, the concentration of group II PLA2 progressively increased (p < 0.01) during the first week after admission in patients with SIRS but remained stable in patients without. The concentration of group I PLA2 decreased (p < 0.05) in both groups during follow-up. The concentration of serum C-reactive protein (CRP) was significantly higher (p < 0.05) in patients with SIRS as compared to patients without. However, the temporal serum kinetics were similar in both groups. Conclusiom'. Systemic complications but not pancreatic necrosis are associated with SIRS in severe AP. The increasing concentration of serum group II PLA2 during the first week from admission in patients with SIRS seems to reflect the ongoing inflammatory process. The serial measurement of group II PLA2 may be useful in monitoring the progression of SIRS in severe AP. G1908
CHOLECYSTOKININ REGULATES THE INVASIVENESS OF HUMAN PANCREATIC CANCER CELL LINES VIA PROTEIN KINASE C PATHWAY. Manabu Hirata, Akira Tsuchida, Teshiyasu Iwao, Noriaki Eguchi, Tamito Sasaki, Kenji Matsubara, Shigeru Yamamoto, Kenji Morinaka, Yohsuke Kawasaki and Gore Kajiyama. First Dept. of Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan. BACKGROUNDS: In many patients with pancreatic cancer, invasive diseases are already present at the time of diagnosis. Therefore, its prognosis is poor. Matrix metalloproteinase-9 (MMP-9) has been implicated as an important enzyme in tumor invasion and metastasis. It has been reported that some growth factors regulate MMP-9. Present studies have reported that cholecystokinin (CCK) is thought to be an important growth factor in human pancreatic cancers. Recently, we have reported that CCK plays an important role in the invasiveness and the production of MMP-9 in human pancreatic cancer cell lines. In this report, we investigated the pathway of the invasiveness associated with MMP-9 of human pancreatic cancer cell lines regulated by CCK. METHODS: PANC-I, a human pancreatic cancer cell line, was treated with CCK-8 alone, CCK-8 and staurosporine (STN), or CCK-8 and indomethacine (IND). Cells were harvested and examined in the invasion assay using a Boyden chamber. The production and gelatinolytic activity of MMP-9 in supernatant from PANC-1 were analyzed by the methods of Western blot analysis using a mouse monoclonal anti-MMP-9 antibody and gelatin zymogram. RESULTS: The penetrating cell number was decreased with STN in both cell lines but not IND. The production and gelatinolytic activity of MMP-9 were also inhibited by STN but not IND. CONCLUSIONS: These results suggest that CCK may regulate the invasiveness and the invasiveness associated with MMP-9 via PKC in human pancreatic cancer cell. Therefore, CCK receptor antagonist may be useful therapeutic agents against the invasion of the human pancreatic cancer.
G1909
EGF ENHANCES THE EFFECT OF VASOACTIVE INTESTINAL HORMONE IN ELEVATING THE LEVEL OF THE PANCREATIC TUMOR ANTIGEN ND2. J.J.L. Ho, E.R. Farrelly, and Y.S. Kim. Dept. of Medicine, University of California and GI Research Laboratory, Verterans Affairs Medical Center, S. F., CA. INTRODUCTION: Monoclonal antibody Nd2 detected over 70% of the pancreatic exocrine tumors in patients (Jpn. J. Cancer Res. 88:427). Methods that elevate the cytoplasmic levels of Nd2 antigen may increase this rate of detection. Nd2 antibody recognizes a mucin-associated epitope that is tumor specific (Cancer Res. 51:372). Little is known about how the levels of mucinassociated antigens are regulated in pancreatic tumors. In the present study we examined the effects of vasoactive intestinal hormone (VIP) and epidermal growth factor (EGF) on the levels of Nd2 antigen. METHODS: The SW1990 pancreatic cancer cell line was used in these studies. Antigens from the cytoplasm and conditioned media were quantitiated by immunoslot blotting. Antigens adsorbed to nitro-cellulose membranes were reacted with Nd2, 19-9, 139H2 antibodies or a monoclonal antibody directed against EGF (that had no cross-reactivity with TGFu), antimouse second antibody and radio-iodinated protein A. Presence of EGF receptor (EGFR) was determined by flow cytometry on unfixed cells using an antibody directed against the surface domain of the receptor and a phycoerythrin conjugated second antibody. RESULTS; Treatment of cells for up to seven days with VIP produced a progressive increase in the cellular level of Nd2 antigen (up to sevenfold). Ceils grown on porous membranes increased their apical release of Nd2 antigen in response to VIP. Increased apical release of CA19-9 and MUC1 mucin was also observed. Exogenous EGF (10 -9 M) greatly increased the effect of VIP (10 -7 M). EGF by itself had little effect. SW1990 cells release EGF into their media and express EGFR on their surfaces. CONCLUSIONS: Our results show that the hormone VIP elevated cytoplasmic levels of the Nd2 tumor antigen. VIP also increased the release of Nd2 antigen, CA19-9 and MUC1 mucin. Thus VIP may be useful in enhancing the sensitivity of Nd2 and other tumor antigen specific antibodies in the immunodetection and immunotherapy of pancreatic cancer. In addition, EGF enhanced the effects of VIP. Pancreatic tumor cells are known to produce high levels of EGF and other growth factors and their receptors. These factors may have autocrine actions that contribute to the observed overexpression of certain mucin-associated antigens in tumors. This study was supported by the VA Medical Research Service and by USPHS Grant CA 24321 from the National Cancer Institute. • G1910
REGULATION OF PANCREATIC MEMBRANE DIPEPTIDASE mRNA EXPRESSION AND IDENTIFICATION AS ' ZYMOGEN GRANULE MEMBRANE PROTEIN BY PEPTIDE SEQUENCING. T. H6fken*, D. Linder§, R. Kleene+, B. G6ke* and A.C.C. Wagner*; *Dept. of Gastroenterology and +Dept. of Cell Biology and Anatomy, University of Marburg, Germany; §Institute for Biochemistry, University of Giegen, Germany We are interested in the isolation and characterization of zymogen granule membrane (ZGM) proteins in order to gain further insights into the molecular mechanism of secretion in pancreatic acinar cells. Purified ZGM from pig pancreata were separated by large-scale two-dimensional gel electrophoresis using immobilized pH gradients, and several peptides were N-terminally sequenced. A 48 kDa glycoprotein is a major component in both 0.1 M Na2CO3 and 0.5 M NaCI washed membrane preparations. Sequence analysis revealed that this protein is membrane dipeptidase (mdp; EC 3.4.13.19), an enzyme involved in the degradation of glutathion (GSH) and its conjugates. While our work was in progress immunoreactivity of ZGM preparations with an antibody against mdp has recently been reported. We further investigated gene expression of mdp during stimulation of rat pancreas with FOY-305 as well as dexamethasone stimulated AR42J cells. Unstimulated AR42J ceils expressed no or only little membrane dipeptidase RNA. The RNA level increased markedly up to 72 hours after dexamethasone induction. FOY-305 stimulation had the same effect on RNA synthesis in rats. Conclusion: The presence of mdp in ZGM is highly interesting because of its involvement in GSH degradation. GSH plays a pivotal role in the physiology and pathophysiology of the pancreatic acinar cell. It has been reported that GSH depletion inhibits amylase secretion. Furthermore, it has been reported, that the decrease of GSH levels may be a key event in acute caerulein pancreatitis.
April 1998
reconstitute. How far the preparatory trauma influences following incubations remains to be elucidated. Nevertheless, the time dependent pattern of acinar reconstitution should influence interpretation of results from following settings. Trypan blue is inappropiate for analysis of acinar viability. • G1907 EXTRACELLULAR PHOSPHOLIPASES A2 IN SEVERE ACUTE PANCREATITIS -ASSOCIATED SYSTEMIC INFLAMMATORY RESPONSE SYNDROME (SIRS). A. Hietaranta (1), E. Kemppainen (1), P. Puolakkainen (1), V. Sainio (1), R. Haapialnen (1), E. Kivilaakso (1) and T. Nevalainen (2). From the (1) Second Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland and (2) Department of Pathology, University of Turku, Turku, Finland.
Background & Aims. The pathophysiology of severe acute pancreatitis (AP) resulting in systemic inflammatory response syndrome (SIRS) share similar mechanisms to other conditions predisposing to multiple organ failure. Since extracellular phospholipases A2 (PLA2) have been implicated in the pathophysiology of severe AP, our aim was to measure the serum concentrations of these enzymes in patients with severe acute panereatitis with or without SIRS. Methods. The serum samples were collected daily for one week from admission (days 1-8) in 57 patients with severe AP. The severity grading of AP was based on the Atlanta classification (1992). SIRS was defined according to ACCP/SCCM Consensus Conference Committee (1991). Time-resolved fluoroimmunoassay was used for group I and group II PLA2 measurements. Complications of AP as well as daily MOF-score and the association of local and systemic complications of the disease and SIRS were recorded. Results. Thirty-nine (68.4%) patients fulfilled the criteria of SIRS within first week from admission (days 1-8). Pancreatic necrosis was detected in 43 (75.4%) patients. Infected necrosis was detected preoperatively or at operation in 5 (8.8%) patients. Twenty-four (42.1%) and 7 (12.3%) patients suffered from respiratory and renal failure, respectively. All patients with renal failure had respiratory failure as well. Seven (12.3%) patients died of their disease. All patients with systemic complications fulfilled the criteria of SIRS during the first week from admission. However, only the association between respiratory failure and SIRS was statistically significant (p < 0.05). There was no association between SIRS and pancreatic necrosis. The concentrations of extracellular PLA2s in serum did not differ significantly in patients with or without SIRS. However, the concentration of group II PLA2 progressively increased (p < 0.01) during the first week after admission in patients with SIRS but remained stable in patients without. The concentration of group I PLA2 decreased (p < 0.05) in both groups during follow-up. The concentration of serum C-reactive protein (CRP) was significantly higher (p < 0.05) in patients with SIRS as compared to patients without. However, the temporal serum kinetics were similar in both groups. Conclusiom'. Systemic complications but not pancreatic necrosis are associated with SIRS in severe AP. The increasing concentration of serum group II PLA2 during the first week from admission in patients with SIRS seems to reflect the ongoing inflammatory process. The serial measurement of group II PLA2 may be useful in monitoring the progression of SIRS in severe AP. G1908
CHOLECYSTOKININ REGULATES THE INVASIVENESS OF HUMAN PANCREATIC CANCER CELL LINES VIA PROTEIN KINASE C PATHWAY. Manabu Hirata, Akira Tsuchida, Teshiyasu Iwao, Noriaki Eguchi, Tamito Sasaki, Kenji Matsubara, Shigeru Yamamoto, Kenji Morinaka, Yohsuke Kawasaki and Gore Kajiyama. First Dept. of Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan. BACKGROUNDS: In many patients with pancreatic cancer, invasive diseases are already present at the time of diagnosis. Therefore, its prognosis is poor. Matrix metalloproteinase-9 (MMP-9) has been implicated as an important enzyme in tumor invasion and metastasis. It has been reported that some growth factors regulate MMP-9. Present studies have reported that cholecystokinin (CCK) is thought to be an important growth factor in human pancreatic cancers. Recently, we have reported that CCK plays an important role in the invasiveness and the production of MMP-9 in human pancreatic cancer cell lines. In this report, we investigated the pathway of the invasiveness associated with MMP-9 of human pancreatic cancer cell lines regulated by CCK. METHODS: PANC-I, a human pancreatic cancer cell line, was treated with CCK-8 alone, CCK-8 and staurosporine (STN), or CCK-8 and indomethacine (IND). Cells were harvested and examined in the invasion assay using a Boyden chamber. The production and gelatinolytic activity of MMP-9 in supernatant from PANC-1 were analyzed by the methods of Western blot analysis using a mouse monoclonal anti-MMP-9 antibody and gelatin zymogram. RESULTS: The penetrating cell number was decreased with STN in both cell lines but not IND. The production and gelatinolytic activity of MMP-9 were also inhibited by STN but not IND. CONCLUSIONS: These results suggest that CCK may regulate the invasiveness and the invasiveness associated with MMP-9 via PKC in human pancreatic cancer cell. Therefore, CCK receptor antagonist may be useful therapeutic agents against the invasion of the human pancreatic cancer.
G1909
EGF ENHANCES THE EFFECT OF VASOACTIVE INTESTINAL HORMONE IN ELEVATING THE LEVEL OF THE PANCREATIC TUMOR ANTIGEN ND2. J.J.L. Ho, E.R. Farrelly, and Y.S. Kim. Dept. of Medicine, University of California and GI Research Laboratory, Verterans Affairs Medical Center, S. F., CA. INTRODUCTION: Monoclonal antibody Nd2 detected over 70% of the pancreatic exocrine tumors in patients (Jpn. J. Cancer Res. 88:427). Methods that elevate the cytoplasmic levels of Nd2 antigen may increase this rate of detection. Nd2 antibody recognizes a mucin-associated epitope that is tumor specific (Cancer Res. 51:372). Little is known about how the levels of mucinassociated antigens are regulated in pancreatic tumors. In the present study we examined the effects of vasoactive intestinal hormone (VIP) and epidermal growth factor (EGF) on the levels of Nd2 antigen. METHODS: The SW1990 pancreatic cancer cell line was used in these studies. Antigens from the cytoplasm and conditioned media were quantitiated by immunoslot blotting. Antigens adsorbed to nitro-cellulose membranes were reacted with Nd2, 19-9, 139H2 antibodies or a monoclonal antibody directed against EGF (that had no cross-reactivity with TGFu), antimouse second antibody and radio-iodinated protein A. Presence of EGF receptor (EGFR) was determined by flow cytometry on unfixed cells using an antibody directed against the surface domain of the receptor and a phycoerythrin conjugated second antibody. RESULTS; Treatment of cells for up to seven days with VIP produced a progressive increase in the cellular level of Nd2 antigen (up to sevenfold). Ceils grown on porous membranes increased their apical release of Nd2 antigen in response to VIP. Increased apical release of CA19-9 and MUC1 mucin was also observed. Exogenous EGF (10 -9 M) greatly increased the effect of VIP (10 -7 M). EGF by itself had little effect. SW1990 cells release EGF into their media and express EGFR on their surfaces. CONCLUSIONS: Our results show that the hormone VIP elevated cytoplasmic levels of the Nd2 tumor antigen. VIP also increased the release of Nd2 antigen, CA19-9 and MUC1 mucin. Thus VIP may be useful in enhancing the sensitivity of Nd2 and other tumor antigen specific antibodies in the immunodetection and immunotherapy of pancreatic cancer. In addition, EGF enhanced the effects of VIP. Pancreatic tumor cells are known to produce high levels of EGF and other growth factors and their receptors. These factors may have autocrine actions that contribute to the observed overexpression of certain mucin-associated antigens in tumors. This study was supported by the VA Medical Research Service and by USPHS Grant CA 24321 from the National Cancer Institute. • G1910
REGULATION OF PANCREATIC MEMBRANE DIPEPTIDASE mRNA EXPRESSION AND IDENTIFICATION AS ' ZYMOGEN GRANULE MEMBRANE PROTEIN BY PEPTIDE SEQUENCING. T. H6fken*, D. Linder§, R. Kleene+, B. G6ke* and A.C.C. Wagner*; *Dept. of Gastroenterology and +Dept. of Cell Biology and Anatomy, University of Marburg, Germany; §Institute for Biochemistry, University of Giegen, Germany We are interested in the isolation and characterization of zymogen granule membrane (ZGM) proteins in order to gain further insights into the molecular mechanism of secretion in pancreatic acinar cells. Purified ZGM from pig pancreata were separated by large-scale two-dimensional gel electrophoresis using immobilized pH gradients, and several peptides were N-terminally sequenced. A 48 kDa glycoprotein is a major component in both 0.1 M Na2CO3 and 0.5 M NaCI washed membrane preparations. Sequence analysis revealed that this protein is membrane dipeptidase (mdp; EC 3.4.13.19), an enzyme involved in the degradation of glutathion (GSH) and its conjugates. While our work was in progress immunoreactivity of ZGM preparations with an antibody against mdp has recently been reported. We further investigated gene expression of mdp during stimulation of rat pancreas with FOY-305 as well as dexamethasone stimulated AR42J cells. Unstimulated AR42J ceils expressed no or only little membrane dipeptidase RNA. The RNA level increased markedly up to 72 hours after dexamethasone induction. FOY-305 stimulation had the same effect on RNA synthesis in rats. Conclusion: The presence of mdp in ZGM is highly interesting because of its involvement in GSH degradation. GSH plays a pivotal role in the physiology and pathophysiology of the pancreatic acinar cell. It has been reported that GSH depletion inhibits amylase secretion. Furthermore, it has been reported, that the decrease of GSH levels may be a key event in acute caerulein pancreatitis.