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ethanol metabolites
Abstracts / Pancreatology 13 (2013) e1–e94
structures were assessed in cryopreserved cells and their non-cryopreserved controls. Results: Cell recovery after thawing was >50% and viability >90%. Cellular amylase content was similar before and after cryopreservation. Cryopreserved cells maintained their ability to secrete amylase and respond to secretory stimuli after thawing and culture for three days. Cryopreserved cells were able to form amylase-expressing glandular structures in three-dimensional cultures in Matrigel. Conclusions: Differentiated characteristics of mouse pancreatic acinar cells obtained from long-term explant outgrowth cultures are largely retained during cryopreservation and thawing.
P21. Retrospective subgroup analysis to assess the efficacy and safety of pancrelipase/pancreatin (CREONÒ) in patients with pancreatic exocrine insufficiency (PEI) and a medical history of diabetes mellitus A. Bodhani 1, D. Whitcomb 2, K. Beckmann 3, M. Fuldeore 1, S. Sander 3, P. Pollack 1. 1
Abbott, Abbott Park, IL, USA University of Pittsburgh and University of Pittsburgh Medical Center, USA 3 Abbott, Hannover, Germany Purpose: To perform a retrospective exploratory post-hoc subgroup analysis to assess the efficacy and safety of CREONÒ in patients with pancreatic exocrine insufficiency (PEI) and medical history of diabetes mellitus (DM). Methods: This was an analysis of S245.3.124 (NCT00414908), a phaseIII, multi-center, double-blind, randomized, placebo-controlled parallelgroup trial. Patients with confirmed PEI due to chronic pancreatitis (CP) or pancreatic surgery (PS) were randomized to receive pancrelipase (PL) delayed-release (CREONÒ), 12,000 lipase units capsules (72,000 lipase units per meal, 36,000 per snack) or placebo. Fifty-two patients completed the 1-week double-blind period (24 PL, 28 placebo). DM was present in 34/ 52 (65.4%) participants (16 PL, 18 placebo). The primary endpoint was the change in coefficient of fat absorption (CFA) from baseline to end of randomized period. Secondary endpoints included safety parameters. Data was analyzed using non-parametric analysis of covariance. Results: The mean change in CFA from baseline was significantly greater in the PL+DM group compared to placebo+DM (35.218.6 vs. 6.9212.5, p<.0001). No significant difference was observed in the number of subjects with at least one treatment-emergent adverse event (TEAE) in the PL+DM group compared to placebo+DM (29.4% vs. 26.3%). The efficacy and safety results in the PL+no DM group (n¼8) compared to placebo+no DM (n¼10) were consistent with the findings for the two DM groups: change in CFA from baseline (24.217.8 vs. 11.812.4, p¼.0046), number of subjects with at least one TEAE (0.0% vs. 10.0%). The efficacy and safety results in the PL+DM group were also consistent with the overall trial results (change in CFA from baseline 32.118.5% vs. 8.812.5%, p < 0.0001, number of subjects with at least one TEAE (20.0% vs. 20.7%).1 Conclusion: PL (CREONÒ) was effective in treating fat maldigestion with a TEAE rate similar to that of placebo in patients with PEI due to CP or PS with or without medical history of DM. 2
P22. Insulin protects pancreatic acinar cells against fatty acid/ethanol metabolites J.I.E. Bruce 1, A. Samad 1, W. Patel 1, M. Alves-Simoes 1, J. Whitehouse 1, P. Mankad 1, A. Siriwardena 2. 1 Faculty of Life Sciences, The University of Manchester, United Kingdom 2 Hepatobiliary Surgery Unit, Manchester Royal Infirmary, Manchester, United Kingdom
e17
Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative fatty acid/ ethanol metabolites from excessive alcohol intake, which include palmitoleic acid (POA) and POA-ethylester (POAEE), are reported to be one of the major causes of pancreatitis. Studies have shown that these impair mitochondrial metabolism and induce cytosolic Ca2+ ([Ca2+]i) overload in pancreatic acinar cells. Metabolism and [Ca2+]i are linked in various ways, perhaps most critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA), which provides a final common path for cells to “defend” [Ca2+]i during cellular injury. Our previous study showed that insulin, which is protective in animal models and clinical studies of human pancreatitis, protected pancreatic acinar cells against Ca2+ overload, ATP depletion and inhibition of the PMCA induced by oxidative stress, which mimics many of the features of pancreatitis. The aim of the current study was to further test the protective effects of insulin on cellular injury induced by more pathophysiologically relevant pancreatitis-inducing agents, such as ethanol, POA and POAEE. Ethanol (>100 mM) and POAEE (100 mM) induced a small but irreverible Ca2+ overload response but had no significant effect on PMCA activity. POA (50-200 mM) on the other hand, which has been reported to impair mitochondrial function, induced a robust Ca2+ overload, ATP depletion and inhibited PMCA activity. Insulin pretreatment (100 nM for 30 minutes) attenuated the POA-induced Ca2+ overload, ATP depletion and inhibition of the PMCA, consistent with our previous studies. These data further corroborate the protective effects of insulin against acinar cell injury induced by other pathophysiologically relevant pancreatitis inducing agents, such as POA.
P23. Involvement of the RNA-binding proteins Sam68 and SRSF1 in the acquisition of the resistance to gemcitabine in pancreatic adenocarcinoma cells S. Calabretta 1, 2, I. Passacantilli 1, 2, L. Adesso 1, 2, R. Geremia 2, G. Capurso 1, G. Delle Fave 1, C. Sette 2. 1 Medical Surgical Department of Tecnobiomedical Clinical Sciences and Translational Medicine, University of Rome La Sapienza, Italy 2 Department of Public Health and Cell Biology, University of Rome Tor Vergata, Italy Background: The limited effect of conventional chemotherapy in pancreatic adenocarcinoma (PDAC) urges for novel therapies, targeting more directly the molecular aberrations of this disease. The molecular characterization of the drug resistant phenotype of PDAC cells remain unexplored, even though some evidence suggests a correlation with the expression of mesenchymal markers. Notably, the epithelial-to-mesenchymal transition (EMT) is promoted by finely-tuned changes in gene expression, at both transcriptional and splicing levels. In this regard, recent observations have shown the requirement for select splicing factors during EMT. Aim: Characterization of the molecular events that lead to chemotherapeutic resistance in PDAC cells. Methods: Chronic exposure to gemcitabine to select a drugresistant PDAC subpopulation. Western blot analyses for the expression of cancer related proteins, RNA-interference of selected genes to investigate their function. Trypan blue staining, clonogenic assay, immunofluorescence against cleaved-caspase3 to analyze cell survival. Results: The chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells that display a mesenchymal phenotype and are less sensitive to drug-induced cell death. These cells express higher levels of Sam68 and SRSF1, two proto-oncogenes that encode splicing factors. Depletion of Sam68 and SRSF1 expression caused a partial recovery of drug sensitivity. Importantly, an additive effect was observed when both proteins were depleted, suggesting that they are both required for full resistance to the drug. Conclusions: Our data indicate that Sam68 and SRSF1 may represent suitable molecular targets for overcome drug resistance of PDAC.
structures were assessed in cryopreserved cells and their non-cryopreserved controls. Results: Cell recovery after thawing was >50% and viability >90%. Cellular amylase content was similar before and after cryopreservation. Cryopreserved cells maintained their ability to secrete amylase and respond to secretory stimuli after thawing and culture for three days. Cryopreserved cells were able to form amylase-expressing glandular structures in three-dimensional cultures in Matrigel. Conclusions: Differentiated characteristics of mouse pancreatic acinar cells obtained from long-term explant outgrowth cultures are largely retained during cryopreservation and thawing.
P21. Retrospective subgroup analysis to assess the efficacy and safety of pancrelipase/pancreatin (CREONÒ) in patients with pancreatic exocrine insufficiency (PEI) and a medical history of diabetes mellitus A. Bodhani 1, D. Whitcomb 2, K. Beckmann 3, M. Fuldeore 1, S. Sander 3, P. Pollack 1. 1
Abbott, Abbott Park, IL, USA University of Pittsburgh and University of Pittsburgh Medical Center, USA 3 Abbott, Hannover, Germany Purpose: To perform a retrospective exploratory post-hoc subgroup analysis to assess the efficacy and safety of CREONÒ in patients with pancreatic exocrine insufficiency (PEI) and medical history of diabetes mellitus (DM). Methods: This was an analysis of S245.3.124 (NCT00414908), a phaseIII, multi-center, double-blind, randomized, placebo-controlled parallelgroup trial. Patients with confirmed PEI due to chronic pancreatitis (CP) or pancreatic surgery (PS) were randomized to receive pancrelipase (PL) delayed-release (CREONÒ), 12,000 lipase units capsules (72,000 lipase units per meal, 36,000 per snack) or placebo. Fifty-two patients completed the 1-week double-blind period (24 PL, 28 placebo). DM was present in 34/ 52 (65.4%) participants (16 PL, 18 placebo). The primary endpoint was the change in coefficient of fat absorption (CFA) from baseline to end of randomized period. Secondary endpoints included safety parameters. Data was analyzed using non-parametric analysis of covariance. Results: The mean change in CFA from baseline was significantly greater in the PL+DM group compared to placebo+DM (35.218.6 vs. 6.9212.5, p<.0001). No significant difference was observed in the number of subjects with at least one treatment-emergent adverse event (TEAE) in the PL+DM group compared to placebo+DM (29.4% vs. 26.3%). The efficacy and safety results in the PL+no DM group (n¼8) compared to placebo+no DM (n¼10) were consistent with the findings for the two DM groups: change in CFA from baseline (24.217.8 vs. 11.812.4, p¼.0046), number of subjects with at least one TEAE (0.0% vs. 10.0%). The efficacy and safety results in the PL+DM group were also consistent with the overall trial results (change in CFA from baseline 32.118.5% vs. 8.812.5%, p < 0.0001, number of subjects with at least one TEAE (20.0% vs. 20.7%).1 Conclusion: PL (CREONÒ) was effective in treating fat maldigestion with a TEAE rate similar to that of placebo in patients with PEI due to CP or PS with or without medical history of DM. 2
P22. Insulin protects pancreatic acinar cells against fatty acid/ethanol metabolites J.I.E. Bruce 1, A. Samad 1, W. Patel 1, M. Alves-Simoes 1, J. Whitehouse 1, P. Mankad 1, A. Siriwardena 2. 1 Faculty of Life Sciences, The University of Manchester, United Kingdom 2 Hepatobiliary Surgery Unit, Manchester Royal Infirmary, Manchester, United Kingdom
e17
Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative fatty acid/ ethanol metabolites from excessive alcohol intake, which include palmitoleic acid (POA) and POA-ethylester (POAEE), are reported to be one of the major causes of pancreatitis. Studies have shown that these impair mitochondrial metabolism and induce cytosolic Ca2+ ([Ca2+]i) overload in pancreatic acinar cells. Metabolism and [Ca2+]i are linked in various ways, perhaps most critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA), which provides a final common path for cells to “defend” [Ca2+]i during cellular injury. Our previous study showed that insulin, which is protective in animal models and clinical studies of human pancreatitis, protected pancreatic acinar cells against Ca2+ overload, ATP depletion and inhibition of the PMCA induced by oxidative stress, which mimics many of the features of pancreatitis. The aim of the current study was to further test the protective effects of insulin on cellular injury induced by more pathophysiologically relevant pancreatitis-inducing agents, such as ethanol, POA and POAEE. Ethanol (>100 mM) and POAEE (100 mM) induced a small but irreverible Ca2+ overload response but had no significant effect on PMCA activity. POA (50-200 mM) on the other hand, which has been reported to impair mitochondrial function, induced a robust Ca2+ overload, ATP depletion and inhibited PMCA activity. Insulin pretreatment (100 nM for 30 minutes) attenuated the POA-induced Ca2+ overload, ATP depletion and inhibition of the PMCA, consistent with our previous studies. These data further corroborate the protective effects of insulin against acinar cell injury induced by other pathophysiologically relevant pancreatitis inducing agents, such as POA.
P23. Involvement of the RNA-binding proteins Sam68 and SRSF1 in the acquisition of the resistance to gemcitabine in pancreatic adenocarcinoma cells S. Calabretta 1, 2, I. Passacantilli 1, 2, L. Adesso 1, 2, R. Geremia 2, G. Capurso 1, G. Delle Fave 1, C. Sette 2. 1 Medical Surgical Department of Tecnobiomedical Clinical Sciences and Translational Medicine, University of Rome La Sapienza, Italy 2 Department of Public Health and Cell Biology, University of Rome Tor Vergata, Italy Background: The limited effect of conventional chemotherapy in pancreatic adenocarcinoma (PDAC) urges for novel therapies, targeting more directly the molecular aberrations of this disease. The molecular characterization of the drug resistant phenotype of PDAC cells remain unexplored, even though some evidence suggests a correlation with the expression of mesenchymal markers. Notably, the epithelial-to-mesenchymal transition (EMT) is promoted by finely-tuned changes in gene expression, at both transcriptional and splicing levels. In this regard, recent observations have shown the requirement for select splicing factors during EMT. Aim: Characterization of the molecular events that lead to chemotherapeutic resistance in PDAC cells. Methods: Chronic exposure to gemcitabine to select a drugresistant PDAC subpopulation. Western blot analyses for the expression of cancer related proteins, RNA-interference of selected genes to investigate their function. Trypan blue staining, clonogenic assay, immunofluorescence against cleaved-caspase3 to analyze cell survival. Results: The chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells that display a mesenchymal phenotype and are less sensitive to drug-induced cell death. These cells express higher levels of Sam68 and SRSF1, two proto-oncogenes that encode splicing factors. Depletion of Sam68 and SRSF1 expression caused a partial recovery of drug sensitivity. Importantly, an additive effect was observed when both proteins were depleted, suggesting that they are both required for full resistance to the drug. Conclusions: Our data indicate that Sam68 and SRSF1 may represent suitable molecular targets for overcome drug resistance of PDAC.