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Triptolide induces cell death in pancreatic cancer through glycosylation of the transcription factor Sp1
Abstracts / Pancreatology 13 (2013) e1–e94
has been shown to promote development of both carcinogen induced and Kras genetic models of pancreatic cancer. Both CCK and its related peptide gastrin have been associated with pancreatic cancer and gastrin has been shown to promote growth by an autocrine mechanism. The role of endogenous CCK in pancreatic cancer is unknown. Aim: It was hypothesized that pancreatic cancer is under endogenous growth regulation by CCK and the down-regulation of this peptide will decrease growth. Methods: Human pancreatic cancer cells BxPC-3, were stably transfected with anti-CCK small hairpin RNA interference (sh RNA) which targeted the CCK mRNA. Clones were selected by hygromycin resistance. RNA was extracted from AsPC-1 clones and tested for CCK knockdown using RTPCR. BxPC-3 cancer cells were also transfected with shRNA for gastrin and clones selected by neomycin resistance. A second pancreatic cancer cell line PANC-1 was also transfected with siRNA to CCK. Results: CCK mRNA expression was significantly down regulated by RNAi in both pancreatic cancer cell lines. Gastrin mRNA was also significantly down regulated. However, when the knockdown clones were grown in nude mice, no effects on tumor growth rate were detected in the CCK knockdown clones compared to nonspecific shRNA clones or controls. Tumor growth from gastrin shRNA knockdown clones was markedly decreased. Conclusions: Although CCK peptide may be present in human pancreatic cancer, this peptide does not serve to regulate growth of this cancer in an autocrine fashion. Gastrin is the predominant endogenous peptide that acts in an autocrine fashion to stimulate pancreatic cancer growth.
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Karyopharm, Boston, USA Dept of Oncology, Karmanos Cancer Institute, Wayne State Univ, Detroit, MI, USA The nuclear export, and consequent functional inactivation of nearly all major tumor suppressor proteins (TSPs) is mediated exclusively by CRM-1 protein. Elevated CRM-1 expression has been shown to be correlated with poor prognosis in pancreatic cancer (PC), and thus CRM-1 could be an attractive therapeutic target. Using a structure-based drug design, we have identified a new class of CRM-1 inhibitors (SINE), and show (a) targeted inhibition of CRM-1 could become an effective therapeutic strategy for PC, and (b) mechanistically, we identified a previously un-recognized nuclear export mediated suppressive mechanism of CRM-1 against TSP Par-4. The inhibitors bound to the cys-528 nuclear exclusion signal (NES) recognizing amino acid in CRM-1 thereby blocking its function. Our inhibitors (KPT185, KPT-127, KPT-205, KPT-227 but not inactive analog KPT-Trans) induced growth inhibition and apoptosis through PAR-4 nuclear retention in five PC cell lines (IC50 w150 nM) while sparing normal HPDE cells (>5mM). Co-immunoprecipitation studies showed that KPT-185 was able to dissociate CRM-1-PAR-4 interactions, and PAR-4 siRNA knock-down abrogated apoptosis. PC cells expressing a truncated PAR-4 construct that lacks CRM-1 recognizable NES showed induction of cell death even in the absence of CRM-1 inhibitors. Moreover, cys-528 mutant did not respond to drug treatment. Oral administration of clinical candidate SINE KPT-330 at 20 mg/kg reduced tumor size in MiaPaCa-2 and Colo-357 subcutaneous and orthotopic xenografts with no toxicity. Immunohistochemical studies on drug-treated tumors confirmed the role of PAR-4 activation as the mechanism of anti-tumor activity of CRM-1 inhibitors. We also demonstrated in-situ disruption of the Par-4-CRM-1 complex by the inhibitors. In conclusion, we showed that inhibitors of CRM-1 could be attractive novel agents for the treatment of PC. 3
Salinomycin is effective against pancreatic cancer stem cells and targets metastasis-promoting fascin B. Aykut 1, M. Schenk 1, N. Giese 1, S. Kleber 2, A. Martin-Villalba 2, T. Welsch 1. 1
Department of General, Visceral and Transplant Surgery, University of Heidelberg, Germany 2 Molecular Neurobiology (A290), German Cancer Research Center, Heidelberg, Germany Background: The monocarboxylic polyether salinomycin has been shown to selectively eliminate cancer stem cells. The present study investigated the effect of salinomycin on pancreatic cancer stem cells (PCSC). Methods: PCSC were established from patients diagnosed with primary pancreatic ductal adenocarcinoma (PDAC) and cultured in the presence of bFGF and EGF resulting in sphere formation. Annexin/PI staining was used to measure cell death. Immunohistochemistry analysis was performed using PDAC and liver metastasis samples. Results: Salinomycin potently caused PCSC death in micromolar concentrations and inhibited PCSC growth; both effects were significantly less in PANC-1 cells. Salinomycin further suppressed the Wnt-signaling effector beta-catenin. This resulted in downregulation of fascin mRNA and protein expression and reduced cell migration in vitro. Fascin is a key molecule for cancer migration and metastasis. In line with this, fascin was overexpressed in primary PDAC tissue samples, but significantly higher expressed in PDAC liver metastasis, predominantly at the invasive front. Conclusions: Salinomycin is an effective agent eliminating PCSC and impeding Wnt signaling. It inhibits the beta-catenin-TCF target fascin and thus is a promising novel drug to prevent PDAC progression and metastasis.
P15. Targeting nuclear export machinery for pancreatic cancer therapy A.S. Azmi 1, S. Shacham 2, A. Aboukameel 3, B. Bao 1, M. Kauffman 2, F.H. Sarkar 1, 3, R.M. Mohammad 3. 1
Dept of Pathology, Karmanos Cancer Institute, Wayne State Univ, Detroit, MI, USA
P16. Triptolide induces cell death in pancreatic cancer through glycosylation of the transcription factor Sp1 S. Banerjee, V. Sangwan, O. McGinn, N. Mujumdar, V. Dudeja, Z. Chen, T. Mackenzie, S.M. Vickers, A. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN, USA Background: Sp1 protein is a sequence-specific, DNA-binding protein that is a part of the Specificity protein/Kruppel-like factor family of transcription factors which is critical in the regulation of cell growth, differentiation and apoptosis. Sp1 activity is tightly regulated by several posttranslational modifications, one of which is glycosylation. The enzyme responsible for glycosylation of Sp1 is O-GlcNAc transferase (OGT) which adds a single GlcNAc. We have previously shown that Triptolide, a diterpene tri-epoxide induces pancreatic cancer cell death in vivo and in vitro through downregulation of Heat Shock proteins. However, the underlying mechanisms of triptolide action are not fully understood. In the current study we have evaluated the mechanism by which triptolide affect glycosylation of Sp1, which in turn affects downstream pathways controlling survival in pancreatic cancer cells. Results: Sp1 was found to be 5-15 fold overexpressed in pancreatic cancer cell lines over non-tumorigenic ductal cells and between 5-10 fold over normal pancreas in xenotransplanted patient tumors in mice. Inhibition of Sp1 by Mithramycin (an inhibitor for Sp1), or by siRNA, resulted in a loss in viability (30% of control). Similar loss is viability was also seen following inhibition of the glycosyltransferase (OGT) by siRNA (32% of control). Inhibition of OGT by siRNA, specific inhibitors or triptolide also resulted in downregulation of HSP70 and HSF1 protein, RNA and HSF1 activity (40% of control), and downregulation of other pro-proliferative pathways like NF-kB (30% of control) and STAT3 (40% of control) as assayed by reporter activity assay. Inhibition of OGT by siRNA, inhibitors and triptolide also resulted in blocking nuclear translocation of Sp1 and lowered transcriptional activity. This indicated that triptolide mediated downregulation of HSP70 expression was controlled by O-GlcNAc status of the transcription factor Sp1.
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Abstracts / Pancreatology 13 (2013) e1–e94
Conclusion: This study showed downregulation of either Sp1 expression or activity led to pancreatic cancer cell death. Furthermore, glycosylation of Sp1 by OGT plays a vital role in regulating proliferation, migration and survival of pancreatic cancer cells.
P17. Hypoxia-induced aggressiveness of pancreatic cancer (PC) cells is due to increased expression of VEGF, IL-6 and miR-21, which could be attenuated by CDF
higher and sustained activity in the presence of CTRC. This phenotype was essentially identical to that of mutant p.R122H. Secretion of the conversion mutant from HEK 293T cells was increased about 2-fold relative to wild type, whereas secretion of the p.R122H mutant was comparable to wild type. Conclusion: Increased activation in the presence of CTRC is the disease-relevant phenotype of the novel conversion mutant. This seems to be solely due to the p.R122H mutation. Increased secretion of the conversion mutant might represent an additional mechanism of risk, independent of the p.R122H mutation.
B. Bao, S. Ali, S. Banerjee, F. Sarkar. Department of Pathology, WSU, Detroit, MI, USA Hypoxia is commonly associated with solid tumors including PC, and is known to play critical roles in cell survival, angiogenesis, tumor invasion, and metastasis. Hypoxia and over-expression of hypoxia-inducible factor (HIF) has been shown to be associated with therapeutic resistance, leading to poor prognosis of cancer patients. Emerging evidence suggest that hypoxia and HIF signaling pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammationall which contribute to resistance to conventional therapeutics. However, the precise molecular mechanism(s) by which hypoxia/HIF drives these events are not fully understood. Here, we show, for the first time, that hypoxia leads to increased expression of VEGF, IL-6, and CSC marker genes such as Nanog, Oct4 and EZH2 consistent with increased cell migration, invasion and angiogenesis in vitro, and the formation of panreatospheres, concomitant with increased expression of miR-21 and miR-210, potent oncogenic miRNAs, in PC cells (AsPC-1 and MIaPaCa-2). The treatment of PC cells with CDF, a novel synthetic compound which previously showed anti-tumor activity in vivo, inhibited the production of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 and miR-210 under hypoxic condition. CDF treatment also led to decreased cell migration, invasion and angiogenesis in vitro, and formation of pancreatospheres under hypoxic condition. Moreover, CDF treatment decreased gene expression of miR-21, miR-210, IL-6, VEGF, and CSC markers in vivo in a mouse xenograft model of human pancreatic cancer. Taken together, these results suggest that the anti-tumor effect of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become a novel, and effective anti-tumor agent for PC therapy.
P19. A two-phase method for long-term maintenance of functionally competent human pancreatic acinar cells in vitro M. Bläuer, J. Sand, J. Laukkarinen. Tampere Pancreas Laboratory and Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland Objectives: In vitro culture models allowing long-term experimentation on human pancreatic acinar cells are currently lacking. We recently reported a long-term culture strategy for mouse pancreatic acinar cells. The aim of this study was to develop new approaches for long-term maintenance of functionally competent human pancreatic acinar cells in vitro. Methods: Tissue samples were obtained from patients undergoing pancreatic surgery. The samples were digested with collagenase and the resulting acinar clusters were collected, embedded in soft Matrigel and placed in tissue culture inserts for primary culture. After three days the clusters were dissociated and the cells were passaged onto standard cell culture plastic. The responsiveness of the cells to stimulation with 0.1 nM caerulein and 0.1 mM carbachol was determined after four days. Results: Human pancreatic acinar cells could be maintained for a minimum of seven days in vitro. Acinar clusters showed excellent morphology throughout the three-day period in primary culture and little decline in their basal amylase secretion was observed. In secondary culture acinar cells arranged into monolayers. Carbachol stimulated amylase secretion in secondary cultures but no stimulatory effect was evoked by caerulein. Conlusions: The secretory phenotype of human pancreatic acinar cells can be maintained for a minimum of seven days in the present culture conditions. The observed insensitivity of the cultivated cells to caerulein may suggest a lack of functional CCK-receptors in human pancreatic acinar cells. This novel two-phase culture system permits long-term studies on acinar clusters and isolated acinar cells in vitro.
P18. Gene conversion between cationic trypsinogen (PRSS1) and the pseudogene trypsinogen 6 (PRSS3P2) in a patient with chronic pancreatitis S. Beer 1, F.-U. Weiss 2, P. Simon 2, M.M. Lerch 2, M. Sahin-Tóth 1. 1
Department of Molecular and Cell Biology, Boston University, Boston, MA, USA 2 Department of Medicine A, Ernst-Moritz-Arndt-University, Greifswald, Germany Background and aim: Mutation p.R122H in cationic trypsinogen (PRSS1) is the most frequent disease-causing genetic variation found in patients with hereditary pancreatitis. Trypsinogen 6 (PRSS3P2) is a pseudogene which contains His instead of Arg122, which mimics the p.R122H mutation. We identified a gene conversion event between PRSS3P2 and PRSS1 in a patient with chronic pancreatitis. Our aim was to characterize the biochemical phenotype and cellular secretion of this novel mutant in comparison with wild-type and p.R122H mutant trypsinogen. Methods: Mutant and wild type trypsinogen was made recombinantly in E. coli and purified by affinity chromatography. Autoactivation was investigated in the absence and presence of chymotrypsin C (CTRC). Secretion was studied from transiently transfected HEK 293T cells. Results: The conversion event resulted in three alterations at the protein level, p.S115T, p.R116P and p.R122H. Autoactivation of the conversion mutant was markedly increased compared to wild type, with
P20. Cryopreserved mouse pancreatic acinar cells from long-term explant outgrowth cultures maintain their functional phenotype after thawing M. Bläuer, J. Sand, J. Laukkarinen. Tampere Pancreas Laboratory and Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland Objectives: An explant outgrowth method for obtaining functionally competent mouse pancreatic acinar cells for long-term in vitro purposes was recently developed in our laboratory. Cryopresrvation without loss of viability or phenotypic characteristics would greatly facilitate the experimental use of these cells. Our aim was to investigate the effect of cryopreservation on acinar cells obtained from primary explant cultures. Methods: Acinar cells were prepared by the explant culture method and each cell batch was divided into two aliquots. One aliquot was cryopreserved in 10% DMSO and the other represented the non-frozen control. Cell recovery, viability, cellular amylase content, basal and stimulated amylase release in secondary culture and ability to form glandular
has been shown to promote development of both carcinogen induced and Kras genetic models of pancreatic cancer. Both CCK and its related peptide gastrin have been associated with pancreatic cancer and gastrin has been shown to promote growth by an autocrine mechanism. The role of endogenous CCK in pancreatic cancer is unknown. Aim: It was hypothesized that pancreatic cancer is under endogenous growth regulation by CCK and the down-regulation of this peptide will decrease growth. Methods: Human pancreatic cancer cells BxPC-3, were stably transfected with anti-CCK small hairpin RNA interference (sh RNA) which targeted the CCK mRNA. Clones were selected by hygromycin resistance. RNA was extracted from AsPC-1 clones and tested for CCK knockdown using RTPCR. BxPC-3 cancer cells were also transfected with shRNA for gastrin and clones selected by neomycin resistance. A second pancreatic cancer cell line PANC-1 was also transfected with siRNA to CCK. Results: CCK mRNA expression was significantly down regulated by RNAi in both pancreatic cancer cell lines. Gastrin mRNA was also significantly down regulated. However, when the knockdown clones were grown in nude mice, no effects on tumor growth rate were detected in the CCK knockdown clones compared to nonspecific shRNA clones or controls. Tumor growth from gastrin shRNA knockdown clones was markedly decreased. Conclusions: Although CCK peptide may be present in human pancreatic cancer, this peptide does not serve to regulate growth of this cancer in an autocrine fashion. Gastrin is the predominant endogenous peptide that acts in an autocrine fashion to stimulate pancreatic cancer growth.
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Karyopharm, Boston, USA Dept of Oncology, Karmanos Cancer Institute, Wayne State Univ, Detroit, MI, USA The nuclear export, and consequent functional inactivation of nearly all major tumor suppressor proteins (TSPs) is mediated exclusively by CRM-1 protein. Elevated CRM-1 expression has been shown to be correlated with poor prognosis in pancreatic cancer (PC), and thus CRM-1 could be an attractive therapeutic target. Using a structure-based drug design, we have identified a new class of CRM-1 inhibitors (SINE), and show (a) targeted inhibition of CRM-1 could become an effective therapeutic strategy for PC, and (b) mechanistically, we identified a previously un-recognized nuclear export mediated suppressive mechanism of CRM-1 against TSP Par-4. The inhibitors bound to the cys-528 nuclear exclusion signal (NES) recognizing amino acid in CRM-1 thereby blocking its function. Our inhibitors (KPT185, KPT-127, KPT-205, KPT-227 but not inactive analog KPT-Trans) induced growth inhibition and apoptosis through PAR-4 nuclear retention in five PC cell lines (IC50 w150 nM) while sparing normal HPDE cells (>5mM). Co-immunoprecipitation studies showed that KPT-185 was able to dissociate CRM-1-PAR-4 interactions, and PAR-4 siRNA knock-down abrogated apoptosis. PC cells expressing a truncated PAR-4 construct that lacks CRM-1 recognizable NES showed induction of cell death even in the absence of CRM-1 inhibitors. Moreover, cys-528 mutant did not respond to drug treatment. Oral administration of clinical candidate SINE KPT-330 at 20 mg/kg reduced tumor size in MiaPaCa-2 and Colo-357 subcutaneous and orthotopic xenografts with no toxicity. Immunohistochemical studies on drug-treated tumors confirmed the role of PAR-4 activation as the mechanism of anti-tumor activity of CRM-1 inhibitors. We also demonstrated in-situ disruption of the Par-4-CRM-1 complex by the inhibitors. In conclusion, we showed that inhibitors of CRM-1 could be attractive novel agents for the treatment of PC. 3
Salinomycin is effective against pancreatic cancer stem cells and targets metastasis-promoting fascin B. Aykut 1, M. Schenk 1, N. Giese 1, S. Kleber 2, A. Martin-Villalba 2, T. Welsch 1. 1
Department of General, Visceral and Transplant Surgery, University of Heidelberg, Germany 2 Molecular Neurobiology (A290), German Cancer Research Center, Heidelberg, Germany Background: The monocarboxylic polyether salinomycin has been shown to selectively eliminate cancer stem cells. The present study investigated the effect of salinomycin on pancreatic cancer stem cells (PCSC). Methods: PCSC were established from patients diagnosed with primary pancreatic ductal adenocarcinoma (PDAC) and cultured in the presence of bFGF and EGF resulting in sphere formation. Annexin/PI staining was used to measure cell death. Immunohistochemistry analysis was performed using PDAC and liver metastasis samples. Results: Salinomycin potently caused PCSC death in micromolar concentrations and inhibited PCSC growth; both effects were significantly less in PANC-1 cells. Salinomycin further suppressed the Wnt-signaling effector beta-catenin. This resulted in downregulation of fascin mRNA and protein expression and reduced cell migration in vitro. Fascin is a key molecule for cancer migration and metastasis. In line with this, fascin was overexpressed in primary PDAC tissue samples, but significantly higher expressed in PDAC liver metastasis, predominantly at the invasive front. Conclusions: Salinomycin is an effective agent eliminating PCSC and impeding Wnt signaling. It inhibits the beta-catenin-TCF target fascin and thus is a promising novel drug to prevent PDAC progression and metastasis.
P15. Targeting nuclear export machinery for pancreatic cancer therapy A.S. Azmi 1, S. Shacham 2, A. Aboukameel 3, B. Bao 1, M. Kauffman 2, F.H. Sarkar 1, 3, R.M. Mohammad 3. 1
Dept of Pathology, Karmanos Cancer Institute, Wayne State Univ, Detroit, MI, USA
P16. Triptolide induces cell death in pancreatic cancer through glycosylation of the transcription factor Sp1 S. Banerjee, V. Sangwan, O. McGinn, N. Mujumdar, V. Dudeja, Z. Chen, T. Mackenzie, S.M. Vickers, A. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN, USA Background: Sp1 protein is a sequence-specific, DNA-binding protein that is a part of the Specificity protein/Kruppel-like factor family of transcription factors which is critical in the regulation of cell growth, differentiation and apoptosis. Sp1 activity is tightly regulated by several posttranslational modifications, one of which is glycosylation. The enzyme responsible for glycosylation of Sp1 is O-GlcNAc transferase (OGT) which adds a single GlcNAc. We have previously shown that Triptolide, a diterpene tri-epoxide induces pancreatic cancer cell death in vivo and in vitro through downregulation of Heat Shock proteins. However, the underlying mechanisms of triptolide action are not fully understood. In the current study we have evaluated the mechanism by which triptolide affect glycosylation of Sp1, which in turn affects downstream pathways controlling survival in pancreatic cancer cells. Results: Sp1 was found to be 5-15 fold overexpressed in pancreatic cancer cell lines over non-tumorigenic ductal cells and between 5-10 fold over normal pancreas in xenotransplanted patient tumors in mice. Inhibition of Sp1 by Mithramycin (an inhibitor for Sp1), or by siRNA, resulted in a loss in viability (30% of control). Similar loss is viability was also seen following inhibition of the glycosyltransferase (OGT) by siRNA (32% of control). Inhibition of OGT by siRNA, specific inhibitors or triptolide also resulted in downregulation of HSP70 and HSF1 protein, RNA and HSF1 activity (40% of control), and downregulation of other pro-proliferative pathways like NF-kB (30% of control) and STAT3 (40% of control) as assayed by reporter activity assay. Inhibition of OGT by siRNA, inhibitors and triptolide also resulted in blocking nuclear translocation of Sp1 and lowered transcriptional activity. This indicated that triptolide mediated downregulation of HSP70 expression was controlled by O-GlcNAc status of the transcription factor Sp1.
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Abstracts / Pancreatology 13 (2013) e1–e94
Conclusion: This study showed downregulation of either Sp1 expression or activity led to pancreatic cancer cell death. Furthermore, glycosylation of Sp1 by OGT plays a vital role in regulating proliferation, migration and survival of pancreatic cancer cells.
P17. Hypoxia-induced aggressiveness of pancreatic cancer (PC) cells is due to increased expression of VEGF, IL-6 and miR-21, which could be attenuated by CDF
higher and sustained activity in the presence of CTRC. This phenotype was essentially identical to that of mutant p.R122H. Secretion of the conversion mutant from HEK 293T cells was increased about 2-fold relative to wild type, whereas secretion of the p.R122H mutant was comparable to wild type. Conclusion: Increased activation in the presence of CTRC is the disease-relevant phenotype of the novel conversion mutant. This seems to be solely due to the p.R122H mutation. Increased secretion of the conversion mutant might represent an additional mechanism of risk, independent of the p.R122H mutation.
B. Bao, S. Ali, S. Banerjee, F. Sarkar. Department of Pathology, WSU, Detroit, MI, USA Hypoxia is commonly associated with solid tumors including PC, and is known to play critical roles in cell survival, angiogenesis, tumor invasion, and metastasis. Hypoxia and over-expression of hypoxia-inducible factor (HIF) has been shown to be associated with therapeutic resistance, leading to poor prognosis of cancer patients. Emerging evidence suggest that hypoxia and HIF signaling pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammationall which contribute to resistance to conventional therapeutics. However, the precise molecular mechanism(s) by which hypoxia/HIF drives these events are not fully understood. Here, we show, for the first time, that hypoxia leads to increased expression of VEGF, IL-6, and CSC marker genes such as Nanog, Oct4 and EZH2 consistent with increased cell migration, invasion and angiogenesis in vitro, and the formation of panreatospheres, concomitant with increased expression of miR-21 and miR-210, potent oncogenic miRNAs, in PC cells (AsPC-1 and MIaPaCa-2). The treatment of PC cells with CDF, a novel synthetic compound which previously showed anti-tumor activity in vivo, inhibited the production of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 and miR-210 under hypoxic condition. CDF treatment also led to decreased cell migration, invasion and angiogenesis in vitro, and formation of pancreatospheres under hypoxic condition. Moreover, CDF treatment decreased gene expression of miR-21, miR-210, IL-6, VEGF, and CSC markers in vivo in a mouse xenograft model of human pancreatic cancer. Taken together, these results suggest that the anti-tumor effect of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become a novel, and effective anti-tumor agent for PC therapy.
P19. A two-phase method for long-term maintenance of functionally competent human pancreatic acinar cells in vitro M. Bläuer, J. Sand, J. Laukkarinen. Tampere Pancreas Laboratory and Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland Objectives: In vitro culture models allowing long-term experimentation on human pancreatic acinar cells are currently lacking. We recently reported a long-term culture strategy for mouse pancreatic acinar cells. The aim of this study was to develop new approaches for long-term maintenance of functionally competent human pancreatic acinar cells in vitro. Methods: Tissue samples were obtained from patients undergoing pancreatic surgery. The samples were digested with collagenase and the resulting acinar clusters were collected, embedded in soft Matrigel and placed in tissue culture inserts for primary culture. After three days the clusters were dissociated and the cells were passaged onto standard cell culture plastic. The responsiveness of the cells to stimulation with 0.1 nM caerulein and 0.1 mM carbachol was determined after four days. Results: Human pancreatic acinar cells could be maintained for a minimum of seven days in vitro. Acinar clusters showed excellent morphology throughout the three-day period in primary culture and little decline in their basal amylase secretion was observed. In secondary culture acinar cells arranged into monolayers. Carbachol stimulated amylase secretion in secondary cultures but no stimulatory effect was evoked by caerulein. Conlusions: The secretory phenotype of human pancreatic acinar cells can be maintained for a minimum of seven days in the present culture conditions. The observed insensitivity of the cultivated cells to caerulein may suggest a lack of functional CCK-receptors in human pancreatic acinar cells. This novel two-phase culture system permits long-term studies on acinar clusters and isolated acinar cells in vitro.
P18. Gene conversion between cationic trypsinogen (PRSS1) and the pseudogene trypsinogen 6 (PRSS3P2) in a patient with chronic pancreatitis S. Beer 1, F.-U. Weiss 2, P. Simon 2, M.M. Lerch 2, M. Sahin-Tóth 1. 1
Department of Molecular and Cell Biology, Boston University, Boston, MA, USA 2 Department of Medicine A, Ernst-Moritz-Arndt-University, Greifswald, Germany Background and aim: Mutation p.R122H in cationic trypsinogen (PRSS1) is the most frequent disease-causing genetic variation found in patients with hereditary pancreatitis. Trypsinogen 6 (PRSS3P2) is a pseudogene which contains His instead of Arg122, which mimics the p.R122H mutation. We identified a gene conversion event between PRSS3P2 and PRSS1 in a patient with chronic pancreatitis. Our aim was to characterize the biochemical phenotype and cellular secretion of this novel mutant in comparison with wild-type and p.R122H mutant trypsinogen. Methods: Mutant and wild type trypsinogen was made recombinantly in E. coli and purified by affinity chromatography. Autoactivation was investigated in the absence and presence of chymotrypsin C (CTRC). Secretion was studied from transiently transfected HEK 293T cells. Results: The conversion event resulted in three alterations at the protein level, p.S115T, p.R116P and p.R122H. Autoactivation of the conversion mutant was markedly increased compared to wild type, with
P20. Cryopreserved mouse pancreatic acinar cells from long-term explant outgrowth cultures maintain their functional phenotype after thawing M. Bläuer, J. Sand, J. Laukkarinen. Tampere Pancreas Laboratory and Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland Objectives: An explant outgrowth method for obtaining functionally competent mouse pancreatic acinar cells for long-term in vitro purposes was recently developed in our laboratory. Cryopresrvation without loss of viability or phenotypic characteristics would greatly facilitate the experimental use of these cells. Our aim was to investigate the effect of cryopreservation on acinar cells obtained from primary explant cultures. Methods: Acinar cells were prepared by the explant culture method and each cell batch was divided into two aliquots. One aliquot was cryopreserved in 10% DMSO and the other represented the non-frozen control. Cell recovery, viability, cellular amylase content, basal and stimulated amylase release in secondary culture and ability to form glandular