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Metabolic testing partially predicts islet yield in patients undergoing total pancreatectomy and islet autotransplantation
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Abstracts / Pancreatology 13 (2013) e1–e94
P137. Pancreatic stellate cells promote the migration of pancreatic cancer cells J. Lu 1, S.X. Zhou 1, M. Siech 2, H. Habisch 1, M.G. Bachem 1. 1
Department of Clinical Chemistry, University of Ulm, Ulm, Germany Department of General Surgery, Ostalbklinikum Aalen, Aalen, Germany Introduction: Pancreatic stellate cells (PSCs) are the key fibrogenic cells responsible for the desmoplastic reaction in pancreatic ductal adenocarcinoma (PDAC). This study aimed to investigate the role of PSCs in the migration of pancreatic cancer cells (PCCs) and the involved mechanisms. Methods: Human PSCs were isolated from cancerous pancreas by outgrowth method. Modified Boyden chamber assay was used to evaluate the effect of PSCs supernatant (PSC-SN) on the migration of PCCs (Panc1, UlaPaCa). The effect of PSC-SN on the adhesion of PCCs was assessed, and the distribution of focal adhesion complex was examined by immunostaining. Single cell tracking assay was performed to assess the motility of PCCs. Possible signaling effectors in the PSC-SN induced migration and adhesion of PCCs were examined using anti-integrin b1 antibody (P4C10) and focal adhesion kinase (FAK) inhibitor (PF-573228). Results: PSC-SN dose-dependently induced the trans-migration of PCCs. Compared to serum free medium (SFM), 50% PSC-SN significantly facilitated the adhesion and motility of PCCs. Moreover, the adherent cells in PSC-SN showed a polarized morphology with peripheral distribution of paxillin, vinculin and phosphor-FAK. After coating culture wells with collagen I, both the adhesion and motility of PCCs in SFM were significantly increased, and the number of trans-migrated cells in SFM was equal to that in 50% PSC-SN. Pre-coated poly-L-lysine facilitated the cell adhesion, but with no influence on the motility or trans-migration of PCCs. Either P4C10 (20mg/ml) or PF-573228 (1mM) significantly inhibited the trans-migration and adhesion of PCCs. Conclusion: Collagen I secreted from PSCs, specifically promotes the migration of PCCs, partly by increasing the cell adhesion and motility. The hereby molecular mechanisms involve the activation of b1 integrin receptor and the FAK associated signaling pathway. 2
P138. Pancreatitis-associated alterations of pancreatic acinar cells in response to tobacco compared with alcohol M. Luaces-Regueira 1, M. Castiñeira-Alvariño 1, J.E. Domínguez-Muñoz 1, 2. 1 Foundation for Research in Digestive Diseases, University Hospital of Santiago de Compostela, Spain 2 Department of Gastroenterology, University Hospital of Santiago de Compostela, Spain Premature intracellular enzyme activation, alterations in intracellular calcium levels and amylase secretion, reactive oxygen species (ROS) production and acinar cell death are involved in the pathogenesis of pancreatitis. The effect of alcohol and, even more, tobacco in these events is poorly understood, despite being well accepted risk factors for chronic pancreatitis (CP). Aim: To evaluate the role of tobacco compared with alcohol in the intracellular pathophysiologic events associated with pancreatitis. Methods: Acinar cells were isolated from Swiss mice pancreas by enzymatic and mechanic degradation, and stimulated with increasing concentrations of alcohol (from 10 to 100mM) or tobacco (from 0.001 to 0.5mg/ml), and CCK as positive control. Intracellular enzyme activity, ROS production and intracellular calcium were evaluated by fluorescence using rodhamine, DCFDA and fluo-4 as substrates, respectively. Amylase secretion was evaluated using p-nitrophenyl-maltohexaoside. Cytotoxicity was measured by lactate dehydrogenase activity. Statistic analysis was performed by ANOVA. Results: Neither alcohol nor tobacco induced a significant intracellular enzyme activation. Both tobacco [11.384.89% (0.1mg/ml)-56.2613.14% (0.5mg/ml)] and alcohol [14.402.06% (10mM)-59.82.57% (75mM)] significantly increased intracellular calcium levels. Tobacco but not alcohol
induced a supraphysiological pancreatic secretion of amylase (21%, 0.4mg/ ml). Moreover, tobacco but not alcohol produced a significant cytotoxicity (p<0.05) and a dose-dependent ROS production (p<0.05). Conclusions: In pancreatic acinar cell culture, tobacco and alcohol induce a significant increase of intracellular calcium levels. Tobacco, but not alcohol, stimulates pancreatic secretion of amylase, and induces cytotoxicity and ROS production. These results support the role of smoking in the pathogenesis of CP.
P139. Rottlerin promotes apoptosis and autophagy in pancreatic stellate cells via AMPK activation A. Lugea, P. Javaherizadeh, H. Hui, R.T. Waldron, V.L.W. Go, S.J. Pandol. UCLA Center for Excellence in Pancreatic Diseases and VAGLAHS, Los Angeles, CA, USA AMP-activated kinases (AMPK) are key regulators of cellular metabolism, growth and proliferation. Activation of AMPK by low energy status inhibits anabolic processes, activates autophagy and can lead to apoptosis. Here we investigated the effects of the phytochemical rottlerin on AMPK/ mTORC1 pathways in the pancreatic stellate cell (PaSC), a key cell type involved in pancreas fibrosis and inflammation. Methods: PaSC isolated from wild-type or LC3-GFP transgenic mice were incubated for up to 72 h in 10% FBS in the absence (control) or presence of rottlerin (0.5-10 mM). Results: Rottlerin at concentrations as low as 0.5 mM induced rapid (within 3 min) mitochondrial membrane depolarization associated with AMPK activation, as determined by a significant increase in phospho-AMPKa (Thr172) levels, an effect that persisted for at least 48 h. A marked inhibition of the Akt/mTOR pathway was indicated by reduced phosphorylation of Akt (Ser473) and the mTORC1 substrates p70 S6 kinase and 4EBP1. Further, mTORC1 inhibition rapidly blocked protein translation and induced endoplasmic reticulum stress as indicated by phosphorylation of the translation initiator eIF2a (Ser51) and upregulation of proapoptotic CHOP. Rottlerin treatment altered autophagic pathways as indicated by rapid and sustained LC3-GFP puncta formation and LC3I-LC3II conversion, and accumulation of p62/SQSTM1, a protein degraded by autophagy. Interestingly, preincubation with the AMPK inhibitor compound C (20 mM) greatly reduced rottlerininduced mTORC1 inhibition, ER stress and LC3-II accumulation. Furthermore, rottlerin dose-dependently reduced cell viability and induced PaSC apoptosis, to a 6-fold increase over control at 1 mM and 36-fold at 10 mM after 72 h. Conclusions: Our data elucidate the AMPK/mTOR pathways as a key target of rottlerin in PaSC and reinforce the notion that phytochemicals are therapeutically useful to reduce pancreatic desmoplasia by eliminating active PaSC.
P140. Metabolic testing partially predicts islet yield in patients undergoing total pancreatectomy and islet autotransplantation R. Lundberg, G.J. Beilman, Ty B. Dunn, T.L. Pruett, M.L. Freeman, P. Ptacek, K.L. Berry, J.J. Wilhelm, A.N. Balamurugan, M.D. Bellin. University of Minnesota, Minneapolis, MN, USA Introduction: Prevention of diabetes following total pancreatectomy and islet autotransplantation (TP-IAT) for chronic pancreatitis (CP) is highly dependent on islet yield. However, islet yield is currently unknown prior to surgery. The objective of the current study was to investigate the potential of preoperative metabolic testing to predict isolation outcomes. Methods: 44 non-diabetic patients (38 female) with CP (mean BMI 25.4 5.9 kg/m2 and mean age 37.9 11.4) undergoing TP-IAT between 2010 and 2012 completed IV glucose tolerance testing (IVGTT) and mixed meal tolerance testing (MMTT) within 2 weeks prior to surgery. The results of these tests were correlated with islet yield quantified as total islet equivalents (IEQ) and islet equivalents/kg (IEQ/kg).
Abstracts / Pancreatology 13 (2013) e1–e94
Results: Etiologies of CP included idiopathic disease (n¼16), pancreatic divisum (11), sphincter of Oddi dysfunction (5), genetic /familial (10), and other causes (2). Mean IEQ isolated was 286,228 153,619, and mean IEQ/ kg 4,286 2,244. Higher recipient BMI correlated with greater IEQ (r¼0.41 p¼0.006) but not IEQ/kg. The area under the curve (AUC) for C-peptide and the peak C-peptide from the IVGTT correlated with higher IEQ isolated (r¼0.44, p¼0.004 and r¼0.42, p¼0.005, respectively). Similarly, total IEQ positively correlated with AUC C-peptide (r¼0.39, p¼0.01) and peak Cpeptide (r¼0.34, p¼0.03) from the MMTT. Peak C-peptide from MMTT was particularly predictive in this cohort: 91% of patients with peak C peptide 4 ng/mL received 200,000 IEQ, while 89% of those with peak C-peptide <4 ng/mL received <200,000 IEQ. Glucose levels, glucagon secretion, and HbA1c did not predict islet yield. Discussion: These preliminary results suggest that C-peptide secretion determined from simple metabolic testing may aid in predicting islet yield before TP-IAT.
P141. Spleen preserving laparoscopic distal pancreatectomy H.G. Lyu, M.A. Cooper, B.H. Edil, N. Rezaee, C.L. Wolfgang, J.L. Cameron, M.A. Makary. Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: Splenectomy has been associated with increased rates of postoperative infections, thrombocytosis, thrombosis, and hypogammaglobulinemia. For benign pancreatic lesions in the body and tail, preservation of the spleen during distal pancreatectomy is rarely perfomed and the feasibility of routine spleen preservation has not been well described. Methods: We identified candidates for spleen preserving distal pancreatectomy based on a benign appearing tail lesion presenting to a single surgeon over a five year period (2007-2012). Laparoscopy was utilized with a high-definition camera to enable a 10 times magnification of the splenic vessels and operative field. We collected data on blood loss, hospital length of stay, operative time, and mortality. Results: We identified 50 candidates for spleen preserving laparoscopic distal pancreatectomy. Of these, 34 (68%) patients underwent spleen preserving laparoscopic distal pancreatectomy and 16 (32%) patients underwent a splenectomy as part of the procedure (12 for technical reasons and 4 for malignancy identified on frozen section). Spleen preservation was associated with decreased blood loss compared to splenectomy (mean¼187 vs 745 cc, p¼0.002), however spleen preservation had a similar length of stay and operative time compared to splenectomy (mean¼5.2 vs 6.8 days, p¼0.08; mean¼177 vs 215 minutes, p¼0.09 respectively). There was no mortality in either group. One patient developed postoperative splenic infarction which resolved after 6 months of pain management. Conclusions: This series describes the highest rate of spleen preservation with laparoscopic distal pancreatectomy in the literature. Our experience suggests that spleen preservation with distal pancreatectomy is a feasible and safe procedure in patients with benign pancreatic lesions. The magnification of laparoscopy may enable increased rates of spleen preservation compared to the traditional open approach.
P142. Triptolide causes global changes in the microRNAome and transcriptome of pancreatic cancer cells T.N. MacKenzie 1, A. Sarver 3, Z. Chen 2, N. Mujumdar 2, S. Banerjee 2, V. Sangwan 2, V. Dudeja 2, S. Subramanian 1,2,3, S. Vickers 2,3, A.K. Saluja 1,2,3. 1 Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA 2 Department of Surgery, University of Minnesota, Minneapolis, MN, USA 3 Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
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Triptolide inhibits cancer cell growth in vitro and blocks growth and metastatic spread in vivo. As miRNAs are becoming increasingly recognized as important negative regulators of gene expression in cancer, we sought to test whether triptolide induces changes in the microRNAome of pancreatic cancer cells. We have shown that triptolide induces expression of miR-142-3p, a novel regulator of HSP70 expression, but we have not yet evalutated to the global changes in the microRNAome of pancreatic cancer cells in response to triptolide. We hypothesize that triptolide affects miRNAs involved in not only heat shock pathways but also those involved in inflammation and cell proliferation. Methods: We performed miRNA and mRNA microarray analysis of human pancreatic cancer cells (MiaPaCa-2 and S2-013) prior to and following different time points of triptolide treatment. We have validated the observed changes in miRNA and mRNA expression by qPCR. Using Ingenuity Pathway Analysis, we evaluated which cellular pathways which were most affected by triptolide in pancreatic cancer. Results: We found that triptolide induces miR-142-3p, this upregulation inversely correlates with levels of its validated target, the HSPA1B isoform of HSP70; therefore, triptolide inhibits the heat shock protein pathway via a miRNA-mediated mechanism.. Triptolide also induces the expression of other tumor suppressive microRNAs such as miR-204, which is predicted to target the autophagy-related gene MCL1. Conclusions: Using IPA analysis allows us to gain a better understanding of the global microRNAome and transcriptome changes induced by triptolide in pancreatic cancer. Understanding how triptolide affects microRNA and mRNA expression allows us to elucidate the molecular mechanism of action of triptolide so that we may maximize triptolide's effectiveness.
P143. Effects of ethanol on pancreatic developmental factors K.J. Mahan Schneider, M.A. Scheer, E.P. Maloney, D.L. Clemens. Nebraska and Western Iowa Veterans Administration Medical Center and Department of Internal Medicine, University of Nebraska Medical Center Omaha, NE, USA Introduction: Alcohol abuse is one of the most common factors associated with acute and chronic pancreatitis. Although alcohol abuse can have an important role in the development of pancreatitis, it does not appear that alcohol abuse alone is responsible for this disease. Rather, it appears that ethanol sensitizes the pancreas to damage and that other factors are required to develop alcoholic pancreatitis. Many studies have identified mechanisms by which ethanol sensitizes the pancreas to injury, but few have investigated the effects of ethanol on repair of the damaged pancreas. Under many circumstances pancreatic repair recapitulates aspects of pancreatic development. Because of this, we investigated the effects of ethanol on factors important in pancreatic development and regeneration. Methods: Using a biologically relevant model of alcoholic pancreatitis, combining chronic ethanol consumption and coxsackievirus infection, and an in vitro model consisting of the pancreatic tumor cell line 266-6, we investigated the effects of ethanol and its metabolites on the expression of important regulators of pancreatic development and regeneration. Results: The results of these studies demonstrate that chronic ethanol consumption in mice causes alterations in the expression of the critical pancreatic transcription factors, PDX1 and PTF1, and the mediator of Notch signaling, HES1. Additionally, treatment of 266-6 cells with byproducts of ethanol metabolism altered the expression of these critical factors. Conclusions: Chronic ethanol consumption impairs the expression of developmental factors important in the regeneration of the pancreas. The by-products of ethanol metabolism may be responsible for the altered expression of these factors. Impaired pancreatic regeneration may have a role in the pathogenesis of alcoholic pancreatitis.
Abstracts / Pancreatology 13 (2013) e1–e94
P137. Pancreatic stellate cells promote the migration of pancreatic cancer cells J. Lu 1, S.X. Zhou 1, M. Siech 2, H. Habisch 1, M.G. Bachem 1. 1
Department of Clinical Chemistry, University of Ulm, Ulm, Germany Department of General Surgery, Ostalbklinikum Aalen, Aalen, Germany Introduction: Pancreatic stellate cells (PSCs) are the key fibrogenic cells responsible for the desmoplastic reaction in pancreatic ductal adenocarcinoma (PDAC). This study aimed to investigate the role of PSCs in the migration of pancreatic cancer cells (PCCs) and the involved mechanisms. Methods: Human PSCs were isolated from cancerous pancreas by outgrowth method. Modified Boyden chamber assay was used to evaluate the effect of PSCs supernatant (PSC-SN) on the migration of PCCs (Panc1, UlaPaCa). The effect of PSC-SN on the adhesion of PCCs was assessed, and the distribution of focal adhesion complex was examined by immunostaining. Single cell tracking assay was performed to assess the motility of PCCs. Possible signaling effectors in the PSC-SN induced migration and adhesion of PCCs were examined using anti-integrin b1 antibody (P4C10) and focal adhesion kinase (FAK) inhibitor (PF-573228). Results: PSC-SN dose-dependently induced the trans-migration of PCCs. Compared to serum free medium (SFM), 50% PSC-SN significantly facilitated the adhesion and motility of PCCs. Moreover, the adherent cells in PSC-SN showed a polarized morphology with peripheral distribution of paxillin, vinculin and phosphor-FAK. After coating culture wells with collagen I, both the adhesion and motility of PCCs in SFM were significantly increased, and the number of trans-migrated cells in SFM was equal to that in 50% PSC-SN. Pre-coated poly-L-lysine facilitated the cell adhesion, but with no influence on the motility or trans-migration of PCCs. Either P4C10 (20mg/ml) or PF-573228 (1mM) significantly inhibited the trans-migration and adhesion of PCCs. Conclusion: Collagen I secreted from PSCs, specifically promotes the migration of PCCs, partly by increasing the cell adhesion and motility. The hereby molecular mechanisms involve the activation of b1 integrin receptor and the FAK associated signaling pathway. 2
P138. Pancreatitis-associated alterations of pancreatic acinar cells in response to tobacco compared with alcohol M. Luaces-Regueira 1, M. Castiñeira-Alvariño 1, J.E. Domínguez-Muñoz 1, 2. 1 Foundation for Research in Digestive Diseases, University Hospital of Santiago de Compostela, Spain 2 Department of Gastroenterology, University Hospital of Santiago de Compostela, Spain Premature intracellular enzyme activation, alterations in intracellular calcium levels and amylase secretion, reactive oxygen species (ROS) production and acinar cell death are involved in the pathogenesis of pancreatitis. The effect of alcohol and, even more, tobacco in these events is poorly understood, despite being well accepted risk factors for chronic pancreatitis (CP). Aim: To evaluate the role of tobacco compared with alcohol in the intracellular pathophysiologic events associated with pancreatitis. Methods: Acinar cells were isolated from Swiss mice pancreas by enzymatic and mechanic degradation, and stimulated with increasing concentrations of alcohol (from 10 to 100mM) or tobacco (from 0.001 to 0.5mg/ml), and CCK as positive control. Intracellular enzyme activity, ROS production and intracellular calcium were evaluated by fluorescence using rodhamine, DCFDA and fluo-4 as substrates, respectively. Amylase secretion was evaluated using p-nitrophenyl-maltohexaoside. Cytotoxicity was measured by lactate dehydrogenase activity. Statistic analysis was performed by ANOVA. Results: Neither alcohol nor tobacco induced a significant intracellular enzyme activation. Both tobacco [11.384.89% (0.1mg/ml)-56.2613.14% (0.5mg/ml)] and alcohol [14.402.06% (10mM)-59.82.57% (75mM)] significantly increased intracellular calcium levels. Tobacco but not alcohol
induced a supraphysiological pancreatic secretion of amylase (21%, 0.4mg/ ml). Moreover, tobacco but not alcohol produced a significant cytotoxicity (p<0.05) and a dose-dependent ROS production (p<0.05). Conclusions: In pancreatic acinar cell culture, tobacco and alcohol induce a significant increase of intracellular calcium levels. Tobacco, but not alcohol, stimulates pancreatic secretion of amylase, and induces cytotoxicity and ROS production. These results support the role of smoking in the pathogenesis of CP.
P139. Rottlerin promotes apoptosis and autophagy in pancreatic stellate cells via AMPK activation A. Lugea, P. Javaherizadeh, H. Hui, R.T. Waldron, V.L.W. Go, S.J. Pandol. UCLA Center for Excellence in Pancreatic Diseases and VAGLAHS, Los Angeles, CA, USA AMP-activated kinases (AMPK) are key regulators of cellular metabolism, growth and proliferation. Activation of AMPK by low energy status inhibits anabolic processes, activates autophagy and can lead to apoptosis. Here we investigated the effects of the phytochemical rottlerin on AMPK/ mTORC1 pathways in the pancreatic stellate cell (PaSC), a key cell type involved in pancreas fibrosis and inflammation. Methods: PaSC isolated from wild-type or LC3-GFP transgenic mice were incubated for up to 72 h in 10% FBS in the absence (control) or presence of rottlerin (0.5-10 mM). Results: Rottlerin at concentrations as low as 0.5 mM induced rapid (within 3 min) mitochondrial membrane depolarization associated with AMPK activation, as determined by a significant increase in phospho-AMPKa (Thr172) levels, an effect that persisted for at least 48 h. A marked inhibition of the Akt/mTOR pathway was indicated by reduced phosphorylation of Akt (Ser473) and the mTORC1 substrates p70 S6 kinase and 4EBP1. Further, mTORC1 inhibition rapidly blocked protein translation and induced endoplasmic reticulum stress as indicated by phosphorylation of the translation initiator eIF2a (Ser51) and upregulation of proapoptotic CHOP. Rottlerin treatment altered autophagic pathways as indicated by rapid and sustained LC3-GFP puncta formation and LC3I-LC3II conversion, and accumulation of p62/SQSTM1, a protein degraded by autophagy. Interestingly, preincubation with the AMPK inhibitor compound C (20 mM) greatly reduced rottlerininduced mTORC1 inhibition, ER stress and LC3-II accumulation. Furthermore, rottlerin dose-dependently reduced cell viability and induced PaSC apoptosis, to a 6-fold increase over control at 1 mM and 36-fold at 10 mM after 72 h. Conclusions: Our data elucidate the AMPK/mTOR pathways as a key target of rottlerin in PaSC and reinforce the notion that phytochemicals are therapeutically useful to reduce pancreatic desmoplasia by eliminating active PaSC.
P140. Metabolic testing partially predicts islet yield in patients undergoing total pancreatectomy and islet autotransplantation R. Lundberg, G.J. Beilman, Ty B. Dunn, T.L. Pruett, M.L. Freeman, P. Ptacek, K.L. Berry, J.J. Wilhelm, A.N. Balamurugan, M.D. Bellin. University of Minnesota, Minneapolis, MN, USA Introduction: Prevention of diabetes following total pancreatectomy and islet autotransplantation (TP-IAT) for chronic pancreatitis (CP) is highly dependent on islet yield. However, islet yield is currently unknown prior to surgery. The objective of the current study was to investigate the potential of preoperative metabolic testing to predict isolation outcomes. Methods: 44 non-diabetic patients (38 female) with CP (mean BMI 25.4 5.9 kg/m2 and mean age 37.9 11.4) undergoing TP-IAT between 2010 and 2012 completed IV glucose tolerance testing (IVGTT) and mixed meal tolerance testing (MMTT) within 2 weeks prior to surgery. The results of these tests were correlated with islet yield quantified as total islet equivalents (IEQ) and islet equivalents/kg (IEQ/kg).
Abstracts / Pancreatology 13 (2013) e1–e94
Results: Etiologies of CP included idiopathic disease (n¼16), pancreatic divisum (11), sphincter of Oddi dysfunction (5), genetic /familial (10), and other causes (2). Mean IEQ isolated was 286,228 153,619, and mean IEQ/ kg 4,286 2,244. Higher recipient BMI correlated with greater IEQ (r¼0.41 p¼0.006) but not IEQ/kg. The area under the curve (AUC) for C-peptide and the peak C-peptide from the IVGTT correlated with higher IEQ isolated (r¼0.44, p¼0.004 and r¼0.42, p¼0.005, respectively). Similarly, total IEQ positively correlated with AUC C-peptide (r¼0.39, p¼0.01) and peak Cpeptide (r¼0.34, p¼0.03) from the MMTT. Peak C-peptide from MMTT was particularly predictive in this cohort: 91% of patients with peak C peptide 4 ng/mL received 200,000 IEQ, while 89% of those with peak C-peptide <4 ng/mL received <200,000 IEQ. Glucose levels, glucagon secretion, and HbA1c did not predict islet yield. Discussion: These preliminary results suggest that C-peptide secretion determined from simple metabolic testing may aid in predicting islet yield before TP-IAT.
P141. Spleen preserving laparoscopic distal pancreatectomy H.G. Lyu, M.A. Cooper, B.H. Edil, N. Rezaee, C.L. Wolfgang, J.L. Cameron, M.A. Makary. Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: Splenectomy has been associated with increased rates of postoperative infections, thrombocytosis, thrombosis, and hypogammaglobulinemia. For benign pancreatic lesions in the body and tail, preservation of the spleen during distal pancreatectomy is rarely perfomed and the feasibility of routine spleen preservation has not been well described. Methods: We identified candidates for spleen preserving distal pancreatectomy based on a benign appearing tail lesion presenting to a single surgeon over a five year period (2007-2012). Laparoscopy was utilized with a high-definition camera to enable a 10 times magnification of the splenic vessels and operative field. We collected data on blood loss, hospital length of stay, operative time, and mortality. Results: We identified 50 candidates for spleen preserving laparoscopic distal pancreatectomy. Of these, 34 (68%) patients underwent spleen preserving laparoscopic distal pancreatectomy and 16 (32%) patients underwent a splenectomy as part of the procedure (12 for technical reasons and 4 for malignancy identified on frozen section). Spleen preservation was associated with decreased blood loss compared to splenectomy (mean¼187 vs 745 cc, p¼0.002), however spleen preservation had a similar length of stay and operative time compared to splenectomy (mean¼5.2 vs 6.8 days, p¼0.08; mean¼177 vs 215 minutes, p¼0.09 respectively). There was no mortality in either group. One patient developed postoperative splenic infarction which resolved after 6 months of pain management. Conclusions: This series describes the highest rate of spleen preservation with laparoscopic distal pancreatectomy in the literature. Our experience suggests that spleen preservation with distal pancreatectomy is a feasible and safe procedure in patients with benign pancreatic lesions. The magnification of laparoscopy may enable increased rates of spleen preservation compared to the traditional open approach.
P142. Triptolide causes global changes in the microRNAome and transcriptome of pancreatic cancer cells T.N. MacKenzie 1, A. Sarver 3, Z. Chen 2, N. Mujumdar 2, S. Banerjee 2, V. Sangwan 2, V. Dudeja 2, S. Subramanian 1,2,3, S. Vickers 2,3, A.K. Saluja 1,2,3. 1 Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA 2 Department of Surgery, University of Minnesota, Minneapolis, MN, USA 3 Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
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Triptolide inhibits cancer cell growth in vitro and blocks growth and metastatic spread in vivo. As miRNAs are becoming increasingly recognized as important negative regulators of gene expression in cancer, we sought to test whether triptolide induces changes in the microRNAome of pancreatic cancer cells. We have shown that triptolide induces expression of miR-142-3p, a novel regulator of HSP70 expression, but we have not yet evalutated to the global changes in the microRNAome of pancreatic cancer cells in response to triptolide. We hypothesize that triptolide affects miRNAs involved in not only heat shock pathways but also those involved in inflammation and cell proliferation. Methods: We performed miRNA and mRNA microarray analysis of human pancreatic cancer cells (MiaPaCa-2 and S2-013) prior to and following different time points of triptolide treatment. We have validated the observed changes in miRNA and mRNA expression by qPCR. Using Ingenuity Pathway Analysis, we evaluated which cellular pathways which were most affected by triptolide in pancreatic cancer. Results: We found that triptolide induces miR-142-3p, this upregulation inversely correlates with levels of its validated target, the HSPA1B isoform of HSP70; therefore, triptolide inhibits the heat shock protein pathway via a miRNA-mediated mechanism.. Triptolide also induces the expression of other tumor suppressive microRNAs such as miR-204, which is predicted to target the autophagy-related gene MCL1. Conclusions: Using IPA analysis allows us to gain a better understanding of the global microRNAome and transcriptome changes induced by triptolide in pancreatic cancer. Understanding how triptolide affects microRNA and mRNA expression allows us to elucidate the molecular mechanism of action of triptolide so that we may maximize triptolide's effectiveness.
P143. Effects of ethanol on pancreatic developmental factors K.J. Mahan Schneider, M.A. Scheer, E.P. Maloney, D.L. Clemens. Nebraska and Western Iowa Veterans Administration Medical Center and Department of Internal Medicine, University of Nebraska Medical Center Omaha, NE, USA Introduction: Alcohol abuse is one of the most common factors associated with acute and chronic pancreatitis. Although alcohol abuse can have an important role in the development of pancreatitis, it does not appear that alcohol abuse alone is responsible for this disease. Rather, it appears that ethanol sensitizes the pancreas to damage and that other factors are required to develop alcoholic pancreatitis. Many studies have identified mechanisms by which ethanol sensitizes the pancreas to injury, but few have investigated the effects of ethanol on repair of the damaged pancreas. Under many circumstances pancreatic repair recapitulates aspects of pancreatic development. Because of this, we investigated the effects of ethanol on factors important in pancreatic development and regeneration. Methods: Using a biologically relevant model of alcoholic pancreatitis, combining chronic ethanol consumption and coxsackievirus infection, and an in vitro model consisting of the pancreatic tumor cell line 266-6, we investigated the effects of ethanol and its metabolites on the expression of important regulators of pancreatic development and regeneration. Results: The results of these studies demonstrate that chronic ethanol consumption in mice causes alterations in the expression of the critical pancreatic transcription factors, PDX1 and PTF1, and the mediator of Notch signaling, HES1. Additionally, treatment of 266-6 cells with byproducts of ethanol metabolism altered the expression of these critical factors. Conclusions: Chronic ethanol consumption impairs the expression of developmental factors important in the regeneration of the pancreas. The by-products of ethanol metabolism may be responsible for the altered expression of these factors. Impaired pancreatic regeneration may have a role in the pathogenesis of alcoholic pancreatitis.