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Serotonin is a critical signalling molecule in the local and metastatic small intestinal neuroendocrine tumour microenvironment
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Abstracts / Regulatory Peptides 164 (2010) 35–51
p < 0.03) with an ~IC50 = 0.1 nM which was associated with a 25–45% (p < 0.05) decrease in intracellular cAMP and MAPK phosphorylation and could be reversed by the GLP1 antagonist (GLP9-39). Somatostatin analogs, Octreotide, had similar effects but did not potentiate GLP-1 mediated 5-HT secretory inhibition. These effects were confirmed in NCI-H716:KRJ-I co-cultures. 5-HT treatment has no significant effect on GLP-1 secretion from L cells. Conclusion: GLP-1 inhibits EC cell 5-HT secretion via cAMP/MAPKmediated pathways. Given the central role of EC cells in regulating GI function (motility, secretion, and nociception), it is possible that the L cell plays a key up-stream regulatory role in these phenomena. doi:10.1016/j.regpep.2010.07.093
A010 Serotonin is a critical signalling molecule in the local and metastatic small intestinal neuroendocrine tumour microenvironment M. Kidda, B. Svejdaa, R. Pfragnerb, I.M. Modlina a Gastrointestinal Pathobiology Research Group, Yale University School of Medicine, USA b Institute of Molecular Medicine, Medical University of Graz, Austria Introduction: Serotonin (5-HT) secretion from small intestinal neuroendocrine tumours is associated with development of distal disease such as carcinoid heart disease. This amine, however, may also regulate the tumour microenvironment, through local cell modulatory effects e.g. development of fibrosis when tumours are localized or angiogenesis/hepatocyte regeneration when they metastasize to the liver. Aim: To examine the role of 5-HT in regulating fibroblast activation (localized response) and hepatocyte growth and secretion (metastatic response) using novel co-culture systems. Methods: We studied the effect of 5-HT on fibroblastic HEK293 cells, isolated hepatocytes or co-culture systems of the small intestinal NET (KRJ-I) and either of these cells using viability assays, RT-PCR for growth factor synthesis and ELISAs for signal pathway activation and growth factor secretion. Results: 5-HT stimulated HEK293 proliferation (25%) and synthesis of TGFβ1, CTGF and FGF2, effects inhibited by targeting the 5HT2B receptor. Viability, PKA activity, cAMP signalling and TGFα1 and IGF-I synthesis and secretion were stimulated by 5-HT in hepatocytes, effects reversed by targeting the 5-HT2A/B receptor. Growth factor production and secretion were increased in each co-culture model and could be reduced 20–30% by targeting 5-HT receptors. Conclusion: Serotonin plays a critical signalling role in both the local (associated with fibrosis) and metastatic (associated with angiogenesis and elevated tumour growth) microenvironment. Targeting 5-HT receptors may be an effective antiproliferative and antifibrotic strategy for these tumours since it inhibits both tumour microenvironment fibroblasts and hepatocytes. Fibrosis and proliferation appear to be biologically interfaced neuroendocrine neoplasia domains. doi:10.1016/j.regpep.2010.07.094
A011 Somatostatin and somatostatin receptors 1, 2 and 5 selective agonists inhibit C6 glioma cell growth in vitro and in vivo: Analysis of activated intracellular pathways Monica Gatti, Alessandra Pattarozzi, Roberto Würth, Federica Barbieri, Tullio Florio Lab. Pharmacology, Dept. of Oncology, Biology and Genetics University of Genova, Italy
Introduction: Somatostatin receptors (SSTR) occur on several tumor cells, including glioma cells. Multiple intracellular mechanisms mediate these effects but the activation of phosphotyrosine phospatase (PTP) eta was reported to represent a key event in the induction of growth arrest. Aims: This study focused on the identification of and the role of individual SSTRs in the control of C6 rat glioma cell proliferation, in vitro and in vivo, and the intracellular pathways activated in response to SSTR agonists. Methods: DNA synthesis was measured by [3H]-thymdine uptake. Intracellular signalling was analyzed by PTP assay and ERK1/2 activation by western blot in the in vitro studies and by immunohistochemistry in vivo. Results: We analyzed the SSTR mRNA expression pattern in C6 cells, detecting SSTR1, 2, 3, and 5 transcripts. The activation of SSTR1, 2 and 5 by selective agonists resulted in C6 cell growth arrest. All SSTRs activated the same intracellular pathway (PTPeta dependent dephosphorylation of ERK1/2). SSTR1 and 2 agonist co-treatment showed partial synergism. In vivo, SSTR1, 2, and 5 mediated growth inhibition of C6 gliomas, through cytostatic and antiangiogenic activity, via inhibition of ERK1/2 and upregulation of p27Kip1. SSTR5 agonist displayed the highest antitumoral effects while SSTR1 and 2 were more effective in reducing tumor neovasculatrization. Discussion: SSTR1, 2, and 5 agonists are all effective in reducing C6 glioma cells in vitro and in vivo, acting through the same intracellular mechanisms although with different efficacy and showing partial synergism. Since tumor cells express multiple SSTRs, the activation of all the subtypes may grant a better control of cancer growth. doi:10.1016/j.regpep.2010.07.095
A012 Application of cholecystokinin (CCK) in a pancreatic tumour cell line to investigate nicotine-induced pancreatic function changes P. Chowdhury Department of Physiology & Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA Introduction: Smoking related pancreatic disorders, in particular, the role of nicotine, have been reported in recent years. However, the mechanism of this effect still remains uncertain. Aim: We have used AR42J cells, a stable functionally active pancreatic tumor cell line, to evaluate the mechanism of the effect of nicotine in this process. Methods: Cultured AR42J cells were incubated in a dose and time related manner with nicotine, hydrogen peroxide, mitogen activated protein kinase signal inhibitors, xanthine oxidase inhibitors and iNOS inhibitors to determine cell signal activation, cell proliferation and cell function alterations employing Western blotting, immunohistochemistry, fluorimetric assay and bioassay. The status of cell function was by determined by the percent of amylase released in response to maximal stimulating dose of CCK-8 (10− 10 M) at 37 °C incubated for 30 min. Lipid peroxidation assay was conducted employing malondialdehyde as a standard biomarker. Results: The data show that in AR42J cells, mitogen activated protein kinase signaling and an increase in lipid peroxidation were induced by hydrogen peroxide in concert with nicotine treatment. A decrease in these parameters occurred with inhibitors with the exception of iNOS inhibitors. The stimulated cell function remained unaffected by MAPK signal inhibitors and iNOS inhibitors. Activation of cell proliferative signal and its localization was confirmed by immunofluorescence. Discussion: The data implies a plausible mechanism by which induced oxidative stress triggered by nicotine plays a leading role in
Abstracts / Regulatory Peptides 164 (2010) 35–51
p < 0.03) with an ~IC50 = 0.1 nM which was associated with a 25–45% (p < 0.05) decrease in intracellular cAMP and MAPK phosphorylation and could be reversed by the GLP1 antagonist (GLP9-39). Somatostatin analogs, Octreotide, had similar effects but did not potentiate GLP-1 mediated 5-HT secretory inhibition. These effects were confirmed in NCI-H716:KRJ-I co-cultures. 5-HT treatment has no significant effect on GLP-1 secretion from L cells. Conclusion: GLP-1 inhibits EC cell 5-HT secretion via cAMP/MAPKmediated pathways. Given the central role of EC cells in regulating GI function (motility, secretion, and nociception), it is possible that the L cell plays a key up-stream regulatory role in these phenomena. doi:10.1016/j.regpep.2010.07.093
A010 Serotonin is a critical signalling molecule in the local and metastatic small intestinal neuroendocrine tumour microenvironment M. Kidda, B. Svejdaa, R. Pfragnerb, I.M. Modlina a Gastrointestinal Pathobiology Research Group, Yale University School of Medicine, USA b Institute of Molecular Medicine, Medical University of Graz, Austria Introduction: Serotonin (5-HT) secretion from small intestinal neuroendocrine tumours is associated with development of distal disease such as carcinoid heart disease. This amine, however, may also regulate the tumour microenvironment, through local cell modulatory effects e.g. development of fibrosis when tumours are localized or angiogenesis/hepatocyte regeneration when they metastasize to the liver. Aim: To examine the role of 5-HT in regulating fibroblast activation (localized response) and hepatocyte growth and secretion (metastatic response) using novel co-culture systems. Methods: We studied the effect of 5-HT on fibroblastic HEK293 cells, isolated hepatocytes or co-culture systems of the small intestinal NET (KRJ-I) and either of these cells using viability assays, RT-PCR for growth factor synthesis and ELISAs for signal pathway activation and growth factor secretion. Results: 5-HT stimulated HEK293 proliferation (25%) and synthesis of TGFβ1, CTGF and FGF2, effects inhibited by targeting the 5HT2B receptor. Viability, PKA activity, cAMP signalling and TGFα1 and IGF-I synthesis and secretion were stimulated by 5-HT in hepatocytes, effects reversed by targeting the 5-HT2A/B receptor. Growth factor production and secretion were increased in each co-culture model and could be reduced 20–30% by targeting 5-HT receptors. Conclusion: Serotonin plays a critical signalling role in both the local (associated with fibrosis) and metastatic (associated with angiogenesis and elevated tumour growth) microenvironment. Targeting 5-HT receptors may be an effective antiproliferative and antifibrotic strategy for these tumours since it inhibits both tumour microenvironment fibroblasts and hepatocytes. Fibrosis and proliferation appear to be biologically interfaced neuroendocrine neoplasia domains. doi:10.1016/j.regpep.2010.07.094
A011 Somatostatin and somatostatin receptors 1, 2 and 5 selective agonists inhibit C6 glioma cell growth in vitro and in vivo: Analysis of activated intracellular pathways Monica Gatti, Alessandra Pattarozzi, Roberto Würth, Federica Barbieri, Tullio Florio Lab. Pharmacology, Dept. of Oncology, Biology and Genetics University of Genova, Italy
Introduction: Somatostatin receptors (SSTR) occur on several tumor cells, including glioma cells. Multiple intracellular mechanisms mediate these effects but the activation of phosphotyrosine phospatase (PTP) eta was reported to represent a key event in the induction of growth arrest. Aims: This study focused on the identification of and the role of individual SSTRs in the control of C6 rat glioma cell proliferation, in vitro and in vivo, and the intracellular pathways activated in response to SSTR agonists. Methods: DNA synthesis was measured by [3H]-thymdine uptake. Intracellular signalling was analyzed by PTP assay and ERK1/2 activation by western blot in the in vitro studies and by immunohistochemistry in vivo. Results: We analyzed the SSTR mRNA expression pattern in C6 cells, detecting SSTR1, 2, 3, and 5 transcripts. The activation of SSTR1, 2 and 5 by selective agonists resulted in C6 cell growth arrest. All SSTRs activated the same intracellular pathway (PTPeta dependent dephosphorylation of ERK1/2). SSTR1 and 2 agonist co-treatment showed partial synergism. In vivo, SSTR1, 2, and 5 mediated growth inhibition of C6 gliomas, through cytostatic and antiangiogenic activity, via inhibition of ERK1/2 and upregulation of p27Kip1. SSTR5 agonist displayed the highest antitumoral effects while SSTR1 and 2 were more effective in reducing tumor neovasculatrization. Discussion: SSTR1, 2, and 5 agonists are all effective in reducing C6 glioma cells in vitro and in vivo, acting through the same intracellular mechanisms although with different efficacy and showing partial synergism. Since tumor cells express multiple SSTRs, the activation of all the subtypes may grant a better control of cancer growth. doi:10.1016/j.regpep.2010.07.095
A012 Application of cholecystokinin (CCK) in a pancreatic tumour cell line to investigate nicotine-induced pancreatic function changes P. Chowdhury Department of Physiology & Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA Introduction: Smoking related pancreatic disorders, in particular, the role of nicotine, have been reported in recent years. However, the mechanism of this effect still remains uncertain. Aim: We have used AR42J cells, a stable functionally active pancreatic tumor cell line, to evaluate the mechanism of the effect of nicotine in this process. Methods: Cultured AR42J cells were incubated in a dose and time related manner with nicotine, hydrogen peroxide, mitogen activated protein kinase signal inhibitors, xanthine oxidase inhibitors and iNOS inhibitors to determine cell signal activation, cell proliferation and cell function alterations employing Western blotting, immunohistochemistry, fluorimetric assay and bioassay. The status of cell function was by determined by the percent of amylase released in response to maximal stimulating dose of CCK-8 (10− 10 M) at 37 °C incubated for 30 min. Lipid peroxidation assay was conducted employing malondialdehyde as a standard biomarker. Results: The data show that in AR42J cells, mitogen activated protein kinase signaling and an increase in lipid peroxidation were induced by hydrogen peroxide in concert with nicotine treatment. A decrease in these parameters occurred with inhibitors with the exception of iNOS inhibitors. The stimulated cell function remained unaffected by MAPK signal inhibitors and iNOS inhibitors. Activation of cell proliferative signal and its localization was confirmed by immunofluorescence. Discussion: The data implies a plausible mechanism by which induced oxidative stress triggered by nicotine plays a leading role in