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HGF Attenuates Taurocholate Damage to Pancreatic Acinar Cells Through Apoptosis
(Capan1 and AsPc1). There was a steady increase in the percentage of proliferating cancer cells as the proportion of stellate cells increased with a median of 33% (15-40 IQR), when only cancer cells (Capan1) were present, to 54% (46-60 IQR) when the ratio was 1:5 (Friedman's test, p<0.001). Conversely, there was a decrease in the percentage of apoptotic cancer cells (Capan1) from a median of 4.5% (3.8-5.1 IQR) to 2.4% (2.0- 2.8 IQR, Friedman's test p<0.0001) when the ratio was 1:2. There was no invasion seen (either single cell or cohort) in the gels with cancer cells only. However, as the proportion of PS cells increased, the number of invading cancer cells showed an increase from a median of approximately one cell per high power field (ratio 10:1) to about four cells (ratio 1:2). Similarly, the number of invading cohorts also increased from an average of one cohort (10:1) to about eight cohorts (1:5, p<0.001). Further investigations to understand the mechanisms to define the ‘tipping point’ are underway. Conclusion The ratio of cancer cells to stellate cells of 1:2 and above plays a critical role in modulating the behaviour of cancer cells and pushing them toward a more aggressive phenotype (decreased apoptosis, increased proliferation and invasion).
Mo2070 Involvement of ICOS and IL-10 Positive Regulatory T Cells in the Development of IgG4-Related Autoimmune Pancreatitis Takeo Kusuda, Kazushige Uchida, Yutaku Sakaguchi, Katsunori Yoshida, Toshiro Fukui, Akiyoshi Nishio, Kazuichi Okazaki Objectives: IgG4-related autoimmune pancreatitis (AIP) is a new clinical entity of pancreatic disorder. There are immunologic and histological abnormalities, including increased serum IgG4 levels and the infiltration of lymphocytes and IgG4-positive plasmacytes. However, the role of IgG4 is unclear. Recently, regulatory T cells (Tregs) were reported to contribute to the development of various autoimmune diseases as well as in B cell shifting to IgG4producing plasmacytes. For this report, we studied regulatory T cells in the pancreas by immunohistochemistory and ICOS+ Tregs and IL-10 producing cells in the peripheral blood lymphocytes by flowcytometry. Methods: We recruited 44 patients with IgG4-related AIP. For comparison, we recruited 37 patients with other pancreatic diseases and 27 healthy subjects as controls. We studied infiltrating cells in the pancreas by immunohistochemistry, and analyzed ICOS+ Tregs and IL-10+ Tregs in the peripheral blood by flow cytometry. Results: The ratio of Foxp3-positive cells to infiltrated mononuclear cells (Foxp3/Mono) in AIP patients (0.091 ± 0.023) was significantly higher than in patients with alcoholic chronic pancreatitis (0.012 ± 0.003; p<0.05). In AIP, Foxp3/Mono and IgG4/Mono were positively correlated (p<0.05; R =0.91). ICOS+ Tregs (%ICOS-positive Tregs of total Tregs) were significantly higher in AIP patients (3.45% ± 1.58%) than in the patients with other pancreatic diseases (alcoholic chronic pancreatitis; 1.71% ± 0.98%, idiopathic chronic pancreatitis; 1.80% ± 0.86%) and the healthy control group (1.57% ± 0.69%, p<0.05). IL-10+ Tregs were significantly higher in AIP patients than in the healthy control group. Conclusions: Increased quantities of ICOS+ Tregs may influence IgG4 production in IgG4-related AIP.
Mo2073 Protein Kinase D1 Mediates Mitogenic Signaling in Human Pancreatic Carcinoma PANC-1 Cells James Sinnett-Smith, Sushovan Guha, Krisztina Kisfalvi, Enrique Rozengurt Background: Protein kinase D1 (PKD1) is the founding member of a new protein kinase family, separate from the previously identified PKCs. PKD family members are rapidly activated by G protein-coupled receptor (GPCR) agonists which are increasingly implicated as autocrine/paracrine growth factors for multiple solid tumors, including pancreatic ductal adenocarcinoma, a devastating disease with overall 5 year survival of only 3-5%. A variety of GPCR agonists, including neurotensin (NT), stimulate DNA synthesis in human pancreatic adenocarcinoma cell lines. We used inducible stable expression of wild-type (WT) and kinase-dead (K618N) forms of PKD1 in PANC-1 cells to elucidate its function in the regulation of NT-induced MAP kinases, DNA synthesis and cell proliferation. Results: Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. PKD1 overexpression markedly increased the duration of NT-induced ERK activation and stimulated DNA synthesis and proliferation of PANC-1 cell. In order to elucidate the mechanism by which PKD1 prolongs ERK signaling, we determined its effect on RIN1 (Ras and Rab interactor 1), a multidomain protein that binds GTP-bound RAS with high affinity and inhibits the activating interaction between Ras and Raf. RIN1is expressed by PDAC cells, including PANC-1, MIA PaCa-2, AsPC-1, BxPC-3, and Panc-28. In line with this hypothesis, we show that stimulation of PANC-1 cells with NT induces co-immunoprecipitation of RIN1 with 14-3-3 proteins and striking increase in the phosphorylation of RIN1, as probed with a PKD motif phosphoantibody. Conclusion: PKD1 signaling prolongs pro-mitogenic ERK providing a mechanism by which PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. We hypothesize that PKD-mediated RIN1 Ser351 phosphorylation leads to complex formation with 14-3-3 proteins and uncouples RIN1 from activated K-Ras, thereby allowing KRas/Raf interaction and stimulation of the MEK/ERK cascade in PDAC cells. These results suggest that PKDs could be a novel target for the development of therapeutic drugs that restrict the proliferation of pancreatic cancer cells.
Mo2071 The SRC Family Kinase, Yes, is Present in Pancreatic Acinar Cells and Activated by Numerous Gastrointestinal Hormones and Growth Factors Bernardo Nuche-Berenguer, Veronica Sancho, Robert T. Jensen Introduction: The Src Family of kinases (SFK) play a central signaling role for growth factors, cytokines, G protein coupled receptors and other stimuli. SFKs play also an important role in pancreatic acinar cell secretion, endocytosis, growth, cytoskeletal integrity and apoptosis, although the specific SFKs involved are not fully known. Aim: To determine whether the Src kinase Yes is present in rat pancreatic acinar cells and to determine whether pancreatic secretagogues or growth factors can activate it. Methods: Dispersed rat pancreatic acini were isolated. Yes protein was detected by inmunoprecipitation (IP) and detection with Yes specific antibodies in lysates. Activation was measured by Western blotting using the phospho-specific pY416 antibody, which recognizes phosphorylation of Y416 in Yes, which correlates with its activation. Results: Using Yes specific antibodies for IP and detection, which did not interact with other SFKs, Yes was identified in acini. Receptor-mediated secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post receptor stimulants directly activating PKC [TPA] or mobilizing cellular calcium [thapsigargin or the calcium ionophore (A23187)] each activated Yes. Secretagogues activating adenylate cyclase in pancreatic acini [secretin, VIP] stimulated weakly Yes activation. Pancreatic growth factors [PDGF, HGF, EGF] stimulated Yes activation but not insulin, IGF1 and bFGF. Activation of Yes caused by CCK was temporally biphasic, with a rapid maximal increment at 1 minute which then decreased. The effect of CCK was dose-dependent, causing a detectable effect at 1pM, maximal at 100 nm and half maximal at 8.5±0.4 nM. CCK-JMV, an agonist of the CCK high affinity receptor state, causing a maximal response that was 64% of the maximal stimulated by CCK. TPA stimulation of Yes activation was temporally monophasic with a maximal increase by 2.5 min and no subsequent decrease with time. TPA- and CCK-stimulated Yes activation were completely inhibited by the PKC inhibitor, GF109203X. Conclusion: In rat pancreatic acini, the SFK member Yes is not only expressed but also activated by CCK and other gastrointestinal hormones, stimulating either PLC or adenylate cyclase, as well as by pancreatic growth factors. Activation by CCK is rapid and full activation requires participation of both high and low CCK receptor affinity states. These results show Yes is activated by diverse pancreatic stimulants and suggest that the Src kinase Yes may be one of the important pancreatic SFKs mediating many of the physiological and pathophysiological roles of SFKs described in numerous studies.
Mo2074 HGF Attenuates Taurocholate Damage to Pancreatic Acinar Cells Through Apoptosis Jinliang Ni, Ruihua Shi, Wenbo Liu, Yaozong Yuan Aims: To investigate the protective effect of HGF on the damage of pancreatic acinar cells caused by tarurocholate. Methods: Pancreatic acinar cells primary culture were performed on plates coated by type one rat tail collagen after rat pancreatic acini were harvested. Acinar cells were divided into 4 groups: control, taurocholte, HGF and blocker. All groups had ROS, mitochondrial membrane potential(MMP) and cell apoptosis level measured after 3 hr incubation with 3% taurocholate except the control group, and the blocker group were given the blocker of the receptor of HGF. Results: HGF group had lower ROS level, higher mitochondrial potential and higher apoptosis level than damage group, while blocker group had similar ROS level, mitochondrial potential and apoptosis level as damage group, as the following tables and figures demonstrated. Conclusions: HGF could attenuate the taurocholate damage to rat pancreatic primarily cultured acinar cell and might through mitochondrial apoptosis pathway. Rat pancreatic acinar cell ROS and MMP levels 3 hr after damage (u/mg pr)
Mo2072 The Ratio of Pancreatic Stellate Cells to Pancreatic Cancer Cells Plays a Critical Role in Modulating Carcinoma Cell Behaviour Raghu Kadaba, Fieke Froeling, Erdinc Soylu, Satyajit Bhattacharya, Ian Hart, Hemant Kocher
# indicated P<0.05 as to Damage group, * indicated P<0.05 as to Control group Mo2075
Pancreatic stellate cells (PS) have been implicated as the source of the characteristic desmoplasia in pancreatic cancer. Tumours with high stellate cell number and activity exhibit a poorer prognosis. We attempted to dissect out the precise ‘tipping point’ for stromal composition upon cancer cell behaviour using our established In Vitro 3D organotypic gel model (Froeling et al, Am J Path 2009). Maintaining the total cell count constant at half million cells per gel, the ratio of cancer cells to PS cells was altered in an incremental fashion (cancer cell : PS 1:0, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 0:1). Gels were harvested after 10 days of culture and markers for proliferation (Ki67), apoptosis (Cleaved Caspase 3) and invasion of cancer cells, both single cells and cohorts (cytokeratin stain to distinguish between the epithelial cancer cell and the stromal stellate cell), were used to determine the effect of increasing stellate cell presence. All experiments were done in triplicate using two cancer cell lines
Heat Shock Protein-70 is Elevated in the Serum of Mouse Models of Acute and Chronic Pancreatitis as Well as Pancreatic Cancer Yan Bakman, Raghuwansh P. Sah, Sulagna Banerjee, Rajinder Dawra, Ashok Saluja Background: The inducible form of Heat Shock Protein-70 (HSP-70) is known to be elevated in pancreatic cancer, and likely serves a protective role in pancreatic cancer cells. Targeted decrease in HSP-70 results in enhanced tumor cell death. Increase in tissue HSP-70 levels has also been shown in acute pancreatitis, but its expression levels in tissue are unknown in chronic pancreatitis. Also, it is unknown whether the intracellular alterations in HSP-70 are reflected in the serum. We evaluated serum HSP-70 levels in mouse models of orthotopic
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AGA Abstracts
AGA Abstracts
changes in gene expression. These results provide new insight into the role of TRPs in chronic pancreatic pain and suggest that an early approach to synergistic antagonism is an effective therapy for chronic pancreatitis. Acknowledgements: Supported by T32 DK063922 and RO1 NS19912 (GFG)
Mo2070 Involvement of ICOS and IL-10 Positive Regulatory T Cells in the Development of IgG4-Related Autoimmune Pancreatitis Takeo Kusuda, Kazushige Uchida, Yutaku Sakaguchi, Katsunori Yoshida, Toshiro Fukui, Akiyoshi Nishio, Kazuichi Okazaki Objectives: IgG4-related autoimmune pancreatitis (AIP) is a new clinical entity of pancreatic disorder. There are immunologic and histological abnormalities, including increased serum IgG4 levels and the infiltration of lymphocytes and IgG4-positive plasmacytes. However, the role of IgG4 is unclear. Recently, regulatory T cells (Tregs) were reported to contribute to the development of various autoimmune diseases as well as in B cell shifting to IgG4producing plasmacytes. For this report, we studied regulatory T cells in the pancreas by immunohistochemistory and ICOS+ Tregs and IL-10 producing cells in the peripheral blood lymphocytes by flowcytometry. Methods: We recruited 44 patients with IgG4-related AIP. For comparison, we recruited 37 patients with other pancreatic diseases and 27 healthy subjects as controls. We studied infiltrating cells in the pancreas by immunohistochemistry, and analyzed ICOS+ Tregs and IL-10+ Tregs in the peripheral blood by flow cytometry. Results: The ratio of Foxp3-positive cells to infiltrated mononuclear cells (Foxp3/Mono) in AIP patients (0.091 ± 0.023) was significantly higher than in patients with alcoholic chronic pancreatitis (0.012 ± 0.003; p<0.05). In AIP, Foxp3/Mono and IgG4/Mono were positively correlated (p<0.05; R =0.91). ICOS+ Tregs (%ICOS-positive Tregs of total Tregs) were significantly higher in AIP patients (3.45% ± 1.58%) than in the patients with other pancreatic diseases (alcoholic chronic pancreatitis; 1.71% ± 0.98%, idiopathic chronic pancreatitis; 1.80% ± 0.86%) and the healthy control group (1.57% ± 0.69%, p<0.05). IL-10+ Tregs were significantly higher in AIP patients than in the healthy control group. Conclusions: Increased quantities of ICOS+ Tregs may influence IgG4 production in IgG4-related AIP.
Mo2073 Protein Kinase D1 Mediates Mitogenic Signaling in Human Pancreatic Carcinoma PANC-1 Cells James Sinnett-Smith, Sushovan Guha, Krisztina Kisfalvi, Enrique Rozengurt Background: Protein kinase D1 (PKD1) is the founding member of a new protein kinase family, separate from the previously identified PKCs. PKD family members are rapidly activated by G protein-coupled receptor (GPCR) agonists which are increasingly implicated as autocrine/paracrine growth factors for multiple solid tumors, including pancreatic ductal adenocarcinoma, a devastating disease with overall 5 year survival of only 3-5%. A variety of GPCR agonists, including neurotensin (NT), stimulate DNA synthesis in human pancreatic adenocarcinoma cell lines. We used inducible stable expression of wild-type (WT) and kinase-dead (K618N) forms of PKD1 in PANC-1 cells to elucidate its function in the regulation of NT-induced MAP kinases, DNA synthesis and cell proliferation. Results: Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. PKD1 overexpression markedly increased the duration of NT-induced ERK activation and stimulated DNA synthesis and proliferation of PANC-1 cell. In order to elucidate the mechanism by which PKD1 prolongs ERK signaling, we determined its effect on RIN1 (Ras and Rab interactor 1), a multidomain protein that binds GTP-bound RAS with high affinity and inhibits the activating interaction between Ras and Raf. RIN1is expressed by PDAC cells, including PANC-1, MIA PaCa-2, AsPC-1, BxPC-3, and Panc-28. In line with this hypothesis, we show that stimulation of PANC-1 cells with NT induces co-immunoprecipitation of RIN1 with 14-3-3 proteins and striking increase in the phosphorylation of RIN1, as probed with a PKD motif phosphoantibody. Conclusion: PKD1 signaling prolongs pro-mitogenic ERK providing a mechanism by which PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. We hypothesize that PKD-mediated RIN1 Ser351 phosphorylation leads to complex formation with 14-3-3 proteins and uncouples RIN1 from activated K-Ras, thereby allowing KRas/Raf interaction and stimulation of the MEK/ERK cascade in PDAC cells. These results suggest that PKDs could be a novel target for the development of therapeutic drugs that restrict the proliferation of pancreatic cancer cells.
Mo2071 The SRC Family Kinase, Yes, is Present in Pancreatic Acinar Cells and Activated by Numerous Gastrointestinal Hormones and Growth Factors Bernardo Nuche-Berenguer, Veronica Sancho, Robert T. Jensen Introduction: The Src Family of kinases (SFK) play a central signaling role for growth factors, cytokines, G protein coupled receptors and other stimuli. SFKs play also an important role in pancreatic acinar cell secretion, endocytosis, growth, cytoskeletal integrity and apoptosis, although the specific SFKs involved are not fully known. Aim: To determine whether the Src kinase Yes is present in rat pancreatic acinar cells and to determine whether pancreatic secretagogues or growth factors can activate it. Methods: Dispersed rat pancreatic acini were isolated. Yes protein was detected by inmunoprecipitation (IP) and detection with Yes specific antibodies in lysates. Activation was measured by Western blotting using the phospho-specific pY416 antibody, which recognizes phosphorylation of Y416 in Yes, which correlates with its activation. Results: Using Yes specific antibodies for IP and detection, which did not interact with other SFKs, Yes was identified in acini. Receptor-mediated secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post receptor stimulants directly activating PKC [TPA] or mobilizing cellular calcium [thapsigargin or the calcium ionophore (A23187)] each activated Yes. Secretagogues activating adenylate cyclase in pancreatic acini [secretin, VIP] stimulated weakly Yes activation. Pancreatic growth factors [PDGF, HGF, EGF] stimulated Yes activation but not insulin, IGF1 and bFGF. Activation of Yes caused by CCK was temporally biphasic, with a rapid maximal increment at 1 minute which then decreased. The effect of CCK was dose-dependent, causing a detectable effect at 1pM, maximal at 100 nm and half maximal at 8.5±0.4 nM. CCK-JMV, an agonist of the CCK high affinity receptor state, causing a maximal response that was 64% of the maximal stimulated by CCK. TPA stimulation of Yes activation was temporally monophasic with a maximal increase by 2.5 min and no subsequent decrease with time. TPA- and CCK-stimulated Yes activation were completely inhibited by the PKC inhibitor, GF109203X. Conclusion: In rat pancreatic acini, the SFK member Yes is not only expressed but also activated by CCK and other gastrointestinal hormones, stimulating either PLC or adenylate cyclase, as well as by pancreatic growth factors. Activation by CCK is rapid and full activation requires participation of both high and low CCK receptor affinity states. These results show Yes is activated by diverse pancreatic stimulants and suggest that the Src kinase Yes may be one of the important pancreatic SFKs mediating many of the physiological and pathophysiological roles of SFKs described in numerous studies.
Mo2074 HGF Attenuates Taurocholate Damage to Pancreatic Acinar Cells Through Apoptosis Jinliang Ni, Ruihua Shi, Wenbo Liu, Yaozong Yuan Aims: To investigate the protective effect of HGF on the damage of pancreatic acinar cells caused by tarurocholate. Methods: Pancreatic acinar cells primary culture were performed on plates coated by type one rat tail collagen after rat pancreatic acini were harvested. Acinar cells were divided into 4 groups: control, taurocholte, HGF and blocker. All groups had ROS, mitochondrial membrane potential(MMP) and cell apoptosis level measured after 3 hr incubation with 3% taurocholate except the control group, and the blocker group were given the blocker of the receptor of HGF. Results: HGF group had lower ROS level, higher mitochondrial potential and higher apoptosis level than damage group, while blocker group had similar ROS level, mitochondrial potential and apoptosis level as damage group, as the following tables and figures demonstrated. Conclusions: HGF could attenuate the taurocholate damage to rat pancreatic primarily cultured acinar cell and might through mitochondrial apoptosis pathway. Rat pancreatic acinar cell ROS and MMP levels 3 hr after damage (u/mg pr)
Mo2072 The Ratio of Pancreatic Stellate Cells to Pancreatic Cancer Cells Plays a Critical Role in Modulating Carcinoma Cell Behaviour Raghu Kadaba, Fieke Froeling, Erdinc Soylu, Satyajit Bhattacharya, Ian Hart, Hemant Kocher
# indicated P<0.05 as to Damage group, * indicated P<0.05 as to Control group Mo2075
Pancreatic stellate cells (PS) have been implicated as the source of the characteristic desmoplasia in pancreatic cancer. Tumours with high stellate cell number and activity exhibit a poorer prognosis. We attempted to dissect out the precise ‘tipping point’ for stromal composition upon cancer cell behaviour using our established In Vitro 3D organotypic gel model (Froeling et al, Am J Path 2009). Maintaining the total cell count constant at half million cells per gel, the ratio of cancer cells to PS cells was altered in an incremental fashion (cancer cell : PS 1:0, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 0:1). Gels were harvested after 10 days of culture and markers for proliferation (Ki67), apoptosis (Cleaved Caspase 3) and invasion of cancer cells, both single cells and cohorts (cytokeratin stain to distinguish between the epithelial cancer cell and the stromal stellate cell), were used to determine the effect of increasing stellate cell presence. All experiments were done in triplicate using two cancer cell lines
Heat Shock Protein-70 is Elevated in the Serum of Mouse Models of Acute and Chronic Pancreatitis as Well as Pancreatic Cancer Yan Bakman, Raghuwansh P. Sah, Sulagna Banerjee, Rajinder Dawra, Ashok Saluja Background: The inducible form of Heat Shock Protein-70 (HSP-70) is known to be elevated in pancreatic cancer, and likely serves a protective role in pancreatic cancer cells. Targeted decrease in HSP-70 results in enhanced tumor cell death. Increase in tissue HSP-70 levels has also been shown in acute pancreatitis, but its expression levels in tissue are unknown in chronic pancreatitis. Also, it is unknown whether the intracellular alterations in HSP-70 are reflected in the serum. We evaluated serum HSP-70 levels in mouse models of orthotopic
S-713
AGA Abstracts
AGA Abstracts
changes in gene expression. These results provide new insight into the role of TRPs in chronic pancreatic pain and suggest that an early approach to synergistic antagonism is an effective therapy for chronic pancreatitis. Acknowledgements: Supported by T32 DK063922 and RO1 NS19912 (GFG)