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Changes in expression of the subunits of Ribonucleotide Reductase induce gemcitabine resistance in the Suit-2 Pancreatic Cancer Cell Line
Abstracts / Pancreatology 16 (2016) S1eS130
FRAX597 on proliferation and PAK1 activity on isolated human PSCs was examined by 3H-thymidine assay and western blot, respectively. The impact of stromal PAK1 on survival was determined in an orthotopic murine PC model using PAK1 knockout (KO) and wildtype (WT) mice. Results: PAK1 was expressed in the stroma of human specimens and in the isolated PSCs. FRAX597 inhibited human stellate cell proliferation with a reduction in PAK1 activity, stellate cell activation and activity. A significant increase in survival was observed in PAK1 KO mice compared to PAK1 WT mice with a hazard ratio of 0.42. Tumours from PAK1 KO mice had reduced PAK1 staining compared to WT mice. Conclusion: PAK1 appears to play a role in PSC activation and activity. Targeting stellate cell PAK1 to indirectly target the pancreatic tumours could be a novel strategy to increase survival.
Abstract ID: 1578. High c-MET expression defines a subset of pancreatic adenocarcinoma patients with poor prognosis following surgical resection: Exploration of mechanisms driving c-MET overexpression, transcriptomic analyses, and in vitro effects of c-MET inhibitors Jerome Cros 1, Cindy Neuzillet 2, Jerome Raffenne 3, Alice Boyez 3, Annemilai Tijeras-Raballand 4, Florent Dumont 5, Alain Sauvanet 6, Jean Baptiste Bachet 7, Pierre Bedossa 1, Pascal Hammel 2, Valerie Paradis 1, Anne Couvelard 1 1
Department of Pathology, Bichat-Beaujon University Hospital, France Department of Medical Oncology, Beaujon University Hospital, France 3 INSERM UMR1149, France 4 AAREC FILIA RESEARCH, France 5 Genomic Plateform, Institut Cochin, France 6 Department of Biliary and Pancreatic Surgery, Beaujon University Hospital, France 7 -Salpe trie re University Department of Gastroenterology, Pitie Hospital, France 2
Introduction: HGF/c-MET pathway is an hypoxia-inducible pathway involved in tumor-stroma interactions and invasion in pancreatic ductal adenocarcinoma (PDAC). c-MET inhibitors are under clinical development. Aims: We aimed to (i) assess the prognostic value of c-MET overexpression in PDAC and its underlying mechanisms, (ii) characterize the transcriptomic profile of c-MET-high tumors, (iii) study the effects of cMET inhibitors in 4 characterized PDAC cell lines. Patients & methods: 131 consecutive patients with resected PDAC were retrospectively selected. c-MET expression, hypoxia (CAIX/HIF-1a), and microvascular density were assessed by immunochemistry. MET gene copy number was assessed by FISH and copy number variation assay. c-MET signaling pathway activation was assessed by Western blot (pMET/pAKT/pERK1-2/pSTAT3). Transcriptomic profiles of c-MET-high/ low tumors were obtained and confirmed in 2 independent published datasets. Four c-MET inhibitors (tepotinib/cabozantinib/foretinib/crizotinib) were tested in 6 PDAC cell lines that were characterized for c-MET expression and signaling (HGF dependence and downstream pathway activation). Results: High c-MET expression was independently associated with shorter DFS and OS. There was no correlation between hypoxia-related markers and c-MET expression. No MET amplification was detected. cMET-high tumors displayed higher levels of p-MET and p-ERK1/2. Transcriptome analyses revealed that c-MET-high tumors were enriched for the poor prognosis “basal-like” and “activated stroma” signatures defined by Moffit et al. High c-MET expression was also associated with signatures related to matrix remodeling (TGFb signaling/axonal guidance), epithelial-to-mesenchymal transition (Wnt/SHH/Notch/migration), and increased proliferation (KRAS addiction/PI3K pathway activation). Crizotinib displayed the highest antiproliferative effects on cell lines. Conclusion: c-MET defines a poor prognosis subgroup of PDAC and may represent a new treatment target.
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Abstract ID: 1579. Values of tumor markers as predicting factors of unresectability in pancreatic adenocracinoma Ilija Pejovic 1, Dejan Radenkovic 1, Andrija Antic 1, Stefan Kmezic 1, Aleksandar Ninic 1, Nebojsa Prijovic 1, Branislav Lazic 1, Srdjan Djuranovic 2, Snezana Lukic 2, Sanja Dragasevic 2, Djordje Bajec 1 1
First Surgical Clinic, Clinical Center of Serbia, Serbia Clinic for Gastroenterology and Hepathology, Clinical Center of Serbia, Serbia 2
Introduction: The significance of preoperative carcinoembryonic antigen (CEA) levels, carbohydrate antigen 19-9 (Ca 19-9) and values of Ca 72-4, as predictive factors of treatment outcome, is still questionable. Aims: The aim of the study was to asses the relationship between serum levels of Ca 72-4; CEA and Ca19-9 and unresectability of pancreatic adenocarcinoma. Patients & methods: A retrospective single-center study was conucted at Clinic for digestive surgery, Clinical center Serbia including 50 patients with pancreatic adenocarcinoma. The values of serum tumor markers are determined by commercial radioimmunoassay kits. Results: A total of 50 patients (27 male, 23 female) with pancreatic adenocarcinoma were analyzed in this study. Mean age of study population was 63 ±11. Among all patients, 32 cases (64%) underwent curative resection, whereas 18 (36%) were found unresectable. No statistically significant association was found between unresectable pancreatic adenocarcinoma and the preoperative values of CEA (p ¼0.33), Ca19-9 (p ¼0.62) and Ca 72-4 (p¼0.5) Conclusion: Identification of prognostic factors, indicating advanced pancreatic carcinoma may fasciliate stratification of patients for future clinical trials. Use of preoperative serum levels of tumor markers as predictive factors are arguable, however further studies based on larger sample size are still needed.
Abstract ID: 1581. Changes in expression of the subunits of Ribonucleotide Reductase induce gemcitabine resistance in the Suit-2 Pancreatic Cancer Cell Line Kulbir Mann, James Melling, Eithne Costello, Chris Halloran, Paula Ghaneh, William Greenhalf University of Liverpool, United Kingdom Introduction: Inhibition of Ribonucleotide Reductase (RRM) pauses the cell cycle in G1, while incorporation of fluorinated nucleotides in DNA causes damage and cell death. We developed resistant pancreatic cancer cell lines by exposure to increasing gemcitabine concentrations. The literature suggests high RRM1 levels are associated with gemcitabine resistance, but we've identified several resistant cell lines with low RRM1 levels. The p53R2 subunit is a p53-regulated homologue of RRM2. Aims: Knocking down RRM1 will reduce gemcitabine sensitivity in sensitive but not resistant cells (Suit-2R). Resistance to the S-phase dependent cytotoxicity from low RRM expression will induce p53R2 expression, in place of other RRM2 isoforms. Materials & methods: Suit-2 and Suit-2R cells were transfected with RRM1 siRNA. After 24hrs, varying doses of gemcitabine were added for 48hrs. An MTS assay was performed and IC50 calculated. Western analyses of RRM2 and p53R2 were performed on cell lysates. Results: The initial Suit-2 and Suit-2R assays demonstrated a gemcitabine IC50 of 17nm and 83nm, but the knockdown assay demonstrated a decrease in IC50 of both cell lines; 11nm and 25nm respectively. Suit-2R Western analysis depicted the expected reduction in RRM1, no significant change to RRM2 but a significant increase in p53R2. Conclusion: Reduced RRM1 slows growth in both Suit-2 and Suit-2R cell lines, but is insufficent to protect cells from the S-phase specific cytotoxicity of gemcitabine. This may be due to the increase in p53R2
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Abstracts / Pancreatology 16 (2016) S1eS130
which was not seen in the acquired resistance to gemcitabine associated with reduction in RRM. This suggests a p53-mediated sensitivity to gemcitabine that acquired resistance overcomes.
Abstract ID: 1582. Kynurenic acid and its novel analogue SZR-72 reduce the severity of experimental acute necrotizing pancreatitis zs Kui 2, Eszter Korma nyos 2, Emese R lint 1, Lo nd ra Zsolt Balla 1, Bala eka Ba nyi 3, La szlo V €p 5, Gabriella Varga 6, ela Iva ecsei 4, Ferenc Fülo Kiss 1, B ria A. Deli 7, P n Rakonczay, Jr. 1 s Harazin 7, Ma eter Hegyi 8, Zolta Andra 1
Department of Pathophysiology, University of Szeged, Hungary First Department of Medicine, University of Szeged, Hungary 3 Department of Pathology, University of Szeged, Hungary 4 Department of Neurology, University of Szeged, Hungary 5 Institute of Pharmaceutical Chemistry, University of Szeged, Hungary 6 Institute of Surgical Research Center, University of Szeged, Hungary 7 Molecular Neurobiology Research Unit, Biological Research Center, Szeged, Hungary 8 cs, Institute for Translational Medicine & Department University of Pe of Translational Medicine/1st Department of Medicine, MTA-SZTE Lendület Translational Gastroenterology Research Group, University of Szeged, Hungary 2
Introduction: The pathogenesis of acute pancreatitis (AP) is not well understood, the disease has no specific therapy. L-kynurenic acid (KYNA) and its analogue SZR-72 have immune modulatory roles in several inflammatory diseases. KYNA and SZR-72 function as antagonists on the endogenous glutamate receptors (NMDA). Aims: We investigated the effects of KYNA and SZR-72 on experimental AP. Materials & methods: In the AP groups, SPRD rats (n¼6-8) were injected i.p. with 3 g/kg L-ornithine 1 hour after the administration of physiological saline (PS) or 30-300 mg/kg SZR-72 or KYNA. Control animals were injected i.p. with PS instead of L-ornithine and/or 30-300 mg/kg SZR72 or KYNA. Laboratory and histological parameters were measured to evaluate disease severity. We used propidium-iodide fluorescence measurement to evaluate the viability of isolated pancreatic acini in response to L-ornithine, KYNA or SZR-72 administration. Results: Pancreatic NMDA-1 receptor expression was detected by RTPCR. The injection of L-ornithine resulted in necrotizing AP in rats. Pretreatment of AP rats with 30 mg/kg SZR-72 or KYNA did not effect disease severity. All laboratory and histological parameters in AP rats were significantly reduced in response to treatment with 300 mg/kg SZR-72 or KYNA. Incubation of L-ornithine-treated (10-20 mM) isolated acini with 250 mM/l KYNA or SZR-72 significantly increased cell viability. Conclusion: Our experiments demonstrated that SZR-72 and KYNA have a dose-dependent protective effect on L-ornithine induced AP. Both KYNA and SZR-72 exerted beneficial effects on AP severity. We assume that NMDA receptors are responsible for this effect.
Abstract ID: 1585. Correlation of NQO1 and AKR1C1 abundance with patient survival in pancreatic cancer Dylan Williams, Claire Jenkinson, Thompson Gana, William Greenhalf, John P. Neoptolemos, Eithne Costello Department of Molecular and Clinical Cancer Medicine, NIHR Liverpool Pancreas Biomedical Research Unit, United Kingdom Introduction: The Aldo-Keto Reductase (AKR) superfamily of enzymes catalyses redox reactions and is a component of the Nrf2-mediated antioxidant pathway. Functioning of the antioxidant pathway has been shown to contribute to pancreatic cancer development and resistance to
chemotherapy. We therefore hypothesise that the abundance of AKR enzymes such as AKR1C1 and other Nrf2 downstream proteins such as NQO1 in tumour cells may correlate with Nrf2 activity and hence cancer severity. Aims: To optimise a method to detect AKR1C1 and NQO1 proteins and to investigate their correlation with patient survival. Materials & methods: Antibody optimisation for NQO1 and AKR1C1 was performed via immunoblotting using MiaPaCa-2 and PANC-1 cell lines respectively. siRNA knockdown was performed to separately deplete AKR1C1 and NQO1 proteins, and analysed by immunoblotting of lysates and immunocytochemistry of fixed cells. Immunohistochemistry will be performed on patient tissue microarrays (TMAs) to detect abundance of AKR1C1 and NQO1 in patient samples and to correlate with patient clinicopathologic parameters, including response to treatment. Results: The expression of AKR1C1 and NQO1 was detected using immunoblot; the signal was diminished in both immunoblot and immunocytochemistry analysis of depleted cells relative to controls. Immunohistochemistry data of patient TMAs will be gathered. Conclusion: A method to reliably detect AKR1C1 and NQO1 has been developed, and respective antibodies validated. Adaptations to this method will enable us to perform immunohistochemistry on patient TMAs to determine the relationship of AKR1C1 and NQO1 with patient characteristics. Our ultimate goal is to explore the potential clinical use of these proteins as markers predictive of response to treatment.
Abstract ID: 1589. Primary sensory neuron desensitization by resiniferatoxin increases the severity of L-ornithine-induced acute pancreatitis in rats lint 1, Zsolt Balla 2, Andrea Molna r 1, Chlo Emese R eka Ba e Marsollier 3, nd Kiss 2, Zsuzsanna Helyes 6, Zolta n ra eter Hegyi 5, Lo Romane Marc 4, P Rakonczay, Jr. 2 1
First Department of Medicine, University of Szeged, Hungary Department of Pathophysiology, University of Szeged, Szeged, Hungary 3 University of Angers, Angers, France 4 University of Nantes, Nantes, France 5 cs, Institute for Translational Medicine & Department University of Pe of Translational Medicine/1st Department of Medicine, MTA-SZTE Lendület Translational Gastroenterology Research Group, University of Szeged, Hungary 6 Department of Pharmacology and Pharmacotherapy, University of cs, Pe cs, Hungary Pe 2
Introduction: Transient receptor potential vanilloid 1 (TRPV1) is a nociceptor predominantly expressed by primary sensory neurons. TRPV1 participates in neurogenic inflammation and thus to have a major role in the pathogenesis of inflammatory disorders. Aims: We aimed to examine if desensitization of TRPV1 neurons affects the severity of experimental acute pancreatitis (AP) in rats. Materials & methods: To induce AP, Sprague-Dawley rats were injected intraperitoneally (i.p.) with 3 g/kg L-ornithine. 4 weeks before the induction of AP, primary neurons expressing TRPV1 were desensitized by resiniferatoxin (RTX), an agonist of TRPV1. RTX was administered by three i.p. injections (30 mg/kg, 70 mg/kg and 100 mg/kg, 30, 29 and 28 days before the induction of AP, respectively). Rats treated with L-ornithine and/or RTX were compared to their respective control groups treated with physiological saline. To determine AP severity, laboratory and histological parameters were measured. Results: L-ornithine induced necrotizing AP caused decreased pancreatic amylase activity and increased serum amylase and pancreatic myeloperoxidase activities, water content and heat-shock-protein 72 expression. RTX administration in itself did not significantly influence any of the measured parameters compared to the absolute control group (physiological saline instead of both RTX and L-ornithine). Most laboratory and histological parameters demonstrated that primary sensory neuron desensitization increased AP severity vs vehicle-pretreated L-ornithineinduced AP.
FRAX597 on proliferation and PAK1 activity on isolated human PSCs was examined by 3H-thymidine assay and western blot, respectively. The impact of stromal PAK1 on survival was determined in an orthotopic murine PC model using PAK1 knockout (KO) and wildtype (WT) mice. Results: PAK1 was expressed in the stroma of human specimens and in the isolated PSCs. FRAX597 inhibited human stellate cell proliferation with a reduction in PAK1 activity, stellate cell activation and activity. A significant increase in survival was observed in PAK1 KO mice compared to PAK1 WT mice with a hazard ratio of 0.42. Tumours from PAK1 KO mice had reduced PAK1 staining compared to WT mice. Conclusion: PAK1 appears to play a role in PSC activation and activity. Targeting stellate cell PAK1 to indirectly target the pancreatic tumours could be a novel strategy to increase survival.
Abstract ID: 1578. High c-MET expression defines a subset of pancreatic adenocarcinoma patients with poor prognosis following surgical resection: Exploration of mechanisms driving c-MET overexpression, transcriptomic analyses, and in vitro effects of c-MET inhibitors Jerome Cros 1, Cindy Neuzillet 2, Jerome Raffenne 3, Alice Boyez 3, Annemilai Tijeras-Raballand 4, Florent Dumont 5, Alain Sauvanet 6, Jean Baptiste Bachet 7, Pierre Bedossa 1, Pascal Hammel 2, Valerie Paradis 1, Anne Couvelard 1 1
Department of Pathology, Bichat-Beaujon University Hospital, France Department of Medical Oncology, Beaujon University Hospital, France 3 INSERM UMR1149, France 4 AAREC FILIA RESEARCH, France 5 Genomic Plateform, Institut Cochin, France 6 Department of Biliary and Pancreatic Surgery, Beaujon University Hospital, France 7 -Salpe trie re University Department of Gastroenterology, Pitie Hospital, France 2
Introduction: HGF/c-MET pathway is an hypoxia-inducible pathway involved in tumor-stroma interactions and invasion in pancreatic ductal adenocarcinoma (PDAC). c-MET inhibitors are under clinical development. Aims: We aimed to (i) assess the prognostic value of c-MET overexpression in PDAC and its underlying mechanisms, (ii) characterize the transcriptomic profile of c-MET-high tumors, (iii) study the effects of cMET inhibitors in 4 characterized PDAC cell lines. Patients & methods: 131 consecutive patients with resected PDAC were retrospectively selected. c-MET expression, hypoxia (CAIX/HIF-1a), and microvascular density were assessed by immunochemistry. MET gene copy number was assessed by FISH and copy number variation assay. c-MET signaling pathway activation was assessed by Western blot (pMET/pAKT/pERK1-2/pSTAT3). Transcriptomic profiles of c-MET-high/ low tumors were obtained and confirmed in 2 independent published datasets. Four c-MET inhibitors (tepotinib/cabozantinib/foretinib/crizotinib) were tested in 6 PDAC cell lines that were characterized for c-MET expression and signaling (HGF dependence and downstream pathway activation). Results: High c-MET expression was independently associated with shorter DFS and OS. There was no correlation between hypoxia-related markers and c-MET expression. No MET amplification was detected. cMET-high tumors displayed higher levels of p-MET and p-ERK1/2. Transcriptome analyses revealed that c-MET-high tumors were enriched for the poor prognosis “basal-like” and “activated stroma” signatures defined by Moffit et al. High c-MET expression was also associated with signatures related to matrix remodeling (TGFb signaling/axonal guidance), epithelial-to-mesenchymal transition (Wnt/SHH/Notch/migration), and increased proliferation (KRAS addiction/PI3K pathway activation). Crizotinib displayed the highest antiproliferative effects on cell lines. Conclusion: c-MET defines a poor prognosis subgroup of PDAC and may represent a new treatment target.
S27
Abstract ID: 1579. Values of tumor markers as predicting factors of unresectability in pancreatic adenocracinoma Ilija Pejovic 1, Dejan Radenkovic 1, Andrija Antic 1, Stefan Kmezic 1, Aleksandar Ninic 1, Nebojsa Prijovic 1, Branislav Lazic 1, Srdjan Djuranovic 2, Snezana Lukic 2, Sanja Dragasevic 2, Djordje Bajec 1 1
First Surgical Clinic, Clinical Center of Serbia, Serbia Clinic for Gastroenterology and Hepathology, Clinical Center of Serbia, Serbia 2
Introduction: The significance of preoperative carcinoembryonic antigen (CEA) levels, carbohydrate antigen 19-9 (Ca 19-9) and values of Ca 72-4, as predictive factors of treatment outcome, is still questionable. Aims: The aim of the study was to asses the relationship between serum levels of Ca 72-4; CEA and Ca19-9 and unresectability of pancreatic adenocarcinoma. Patients & methods: A retrospective single-center study was conucted at Clinic for digestive surgery, Clinical center Serbia including 50 patients with pancreatic adenocarcinoma. The values of serum tumor markers are determined by commercial radioimmunoassay kits. Results: A total of 50 patients (27 male, 23 female) with pancreatic adenocarcinoma were analyzed in this study. Mean age of study population was 63 ±11. Among all patients, 32 cases (64%) underwent curative resection, whereas 18 (36%) were found unresectable. No statistically significant association was found between unresectable pancreatic adenocarcinoma and the preoperative values of CEA (p ¼0.33), Ca19-9 (p ¼0.62) and Ca 72-4 (p¼0.5) Conclusion: Identification of prognostic factors, indicating advanced pancreatic carcinoma may fasciliate stratification of patients for future clinical trials. Use of preoperative serum levels of tumor markers as predictive factors are arguable, however further studies based on larger sample size are still needed.
Abstract ID: 1581. Changes in expression of the subunits of Ribonucleotide Reductase induce gemcitabine resistance in the Suit-2 Pancreatic Cancer Cell Line Kulbir Mann, James Melling, Eithne Costello, Chris Halloran, Paula Ghaneh, William Greenhalf University of Liverpool, United Kingdom Introduction: Inhibition of Ribonucleotide Reductase (RRM) pauses the cell cycle in G1, while incorporation of fluorinated nucleotides in DNA causes damage and cell death. We developed resistant pancreatic cancer cell lines by exposure to increasing gemcitabine concentrations. The literature suggests high RRM1 levels are associated with gemcitabine resistance, but we've identified several resistant cell lines with low RRM1 levels. The p53R2 subunit is a p53-regulated homologue of RRM2. Aims: Knocking down RRM1 will reduce gemcitabine sensitivity in sensitive but not resistant cells (Suit-2R). Resistance to the S-phase dependent cytotoxicity from low RRM expression will induce p53R2 expression, in place of other RRM2 isoforms. Materials & methods: Suit-2 and Suit-2R cells were transfected with RRM1 siRNA. After 24hrs, varying doses of gemcitabine were added for 48hrs. An MTS assay was performed and IC50 calculated. Western analyses of RRM2 and p53R2 were performed on cell lysates. Results: The initial Suit-2 and Suit-2R assays demonstrated a gemcitabine IC50 of 17nm and 83nm, but the knockdown assay demonstrated a decrease in IC50 of both cell lines; 11nm and 25nm respectively. Suit-2R Western analysis depicted the expected reduction in RRM1, no significant change to RRM2 but a significant increase in p53R2. Conclusion: Reduced RRM1 slows growth in both Suit-2 and Suit-2R cell lines, but is insufficent to protect cells from the S-phase specific cytotoxicity of gemcitabine. This may be due to the increase in p53R2
S28
Abstracts / Pancreatology 16 (2016) S1eS130
which was not seen in the acquired resistance to gemcitabine associated with reduction in RRM. This suggests a p53-mediated sensitivity to gemcitabine that acquired resistance overcomes.
Abstract ID: 1582. Kynurenic acid and its novel analogue SZR-72 reduce the severity of experimental acute necrotizing pancreatitis zs Kui 2, Eszter Korma nyos 2, Emese R lint 1, Lo nd ra Zsolt Balla 1, Bala eka Ba nyi 3, La szlo V €p 5, Gabriella Varga 6, ela Iva ecsei 4, Ferenc Fülo Kiss 1, B ria A. Deli 7, P n Rakonczay, Jr. 1 s Harazin 7, Ma eter Hegyi 8, Zolta Andra 1
Department of Pathophysiology, University of Szeged, Hungary First Department of Medicine, University of Szeged, Hungary 3 Department of Pathology, University of Szeged, Hungary 4 Department of Neurology, University of Szeged, Hungary 5 Institute of Pharmaceutical Chemistry, University of Szeged, Hungary 6 Institute of Surgical Research Center, University of Szeged, Hungary 7 Molecular Neurobiology Research Unit, Biological Research Center, Szeged, Hungary 8 cs, Institute for Translational Medicine & Department University of Pe of Translational Medicine/1st Department of Medicine, MTA-SZTE Lendület Translational Gastroenterology Research Group, University of Szeged, Hungary 2
Introduction: The pathogenesis of acute pancreatitis (AP) is not well understood, the disease has no specific therapy. L-kynurenic acid (KYNA) and its analogue SZR-72 have immune modulatory roles in several inflammatory diseases. KYNA and SZR-72 function as antagonists on the endogenous glutamate receptors (NMDA). Aims: We investigated the effects of KYNA and SZR-72 on experimental AP. Materials & methods: In the AP groups, SPRD rats (n¼6-8) were injected i.p. with 3 g/kg L-ornithine 1 hour after the administration of physiological saline (PS) or 30-300 mg/kg SZR-72 or KYNA. Control animals were injected i.p. with PS instead of L-ornithine and/or 30-300 mg/kg SZR72 or KYNA. Laboratory and histological parameters were measured to evaluate disease severity. We used propidium-iodide fluorescence measurement to evaluate the viability of isolated pancreatic acini in response to L-ornithine, KYNA or SZR-72 administration. Results: Pancreatic NMDA-1 receptor expression was detected by RTPCR. The injection of L-ornithine resulted in necrotizing AP in rats. Pretreatment of AP rats with 30 mg/kg SZR-72 or KYNA did not effect disease severity. All laboratory and histological parameters in AP rats were significantly reduced in response to treatment with 300 mg/kg SZR-72 or KYNA. Incubation of L-ornithine-treated (10-20 mM) isolated acini with 250 mM/l KYNA or SZR-72 significantly increased cell viability. Conclusion: Our experiments demonstrated that SZR-72 and KYNA have a dose-dependent protective effect on L-ornithine induced AP. Both KYNA and SZR-72 exerted beneficial effects on AP severity. We assume that NMDA receptors are responsible for this effect.
Abstract ID: 1585. Correlation of NQO1 and AKR1C1 abundance with patient survival in pancreatic cancer Dylan Williams, Claire Jenkinson, Thompson Gana, William Greenhalf, John P. Neoptolemos, Eithne Costello Department of Molecular and Clinical Cancer Medicine, NIHR Liverpool Pancreas Biomedical Research Unit, United Kingdom Introduction: The Aldo-Keto Reductase (AKR) superfamily of enzymes catalyses redox reactions and is a component of the Nrf2-mediated antioxidant pathway. Functioning of the antioxidant pathway has been shown to contribute to pancreatic cancer development and resistance to
chemotherapy. We therefore hypothesise that the abundance of AKR enzymes such as AKR1C1 and other Nrf2 downstream proteins such as NQO1 in tumour cells may correlate with Nrf2 activity and hence cancer severity. Aims: To optimise a method to detect AKR1C1 and NQO1 proteins and to investigate their correlation with patient survival. Materials & methods: Antibody optimisation for NQO1 and AKR1C1 was performed via immunoblotting using MiaPaCa-2 and PANC-1 cell lines respectively. siRNA knockdown was performed to separately deplete AKR1C1 and NQO1 proteins, and analysed by immunoblotting of lysates and immunocytochemistry of fixed cells. Immunohistochemistry will be performed on patient tissue microarrays (TMAs) to detect abundance of AKR1C1 and NQO1 in patient samples and to correlate with patient clinicopathologic parameters, including response to treatment. Results: The expression of AKR1C1 and NQO1 was detected using immunoblot; the signal was diminished in both immunoblot and immunocytochemistry analysis of depleted cells relative to controls. Immunohistochemistry data of patient TMAs will be gathered. Conclusion: A method to reliably detect AKR1C1 and NQO1 has been developed, and respective antibodies validated. Adaptations to this method will enable us to perform immunohistochemistry on patient TMAs to determine the relationship of AKR1C1 and NQO1 with patient characteristics. Our ultimate goal is to explore the potential clinical use of these proteins as markers predictive of response to treatment.
Abstract ID: 1589. Primary sensory neuron desensitization by resiniferatoxin increases the severity of L-ornithine-induced acute pancreatitis in rats lint 1, Zsolt Balla 2, Andrea Molna r 1, Chlo Emese R eka Ba e Marsollier 3, nd Kiss 2, Zsuzsanna Helyes 6, Zolta n ra eter Hegyi 5, Lo Romane Marc 4, P Rakonczay, Jr. 2 1
First Department of Medicine, University of Szeged, Hungary Department of Pathophysiology, University of Szeged, Szeged, Hungary 3 University of Angers, Angers, France 4 University of Nantes, Nantes, France 5 cs, Institute for Translational Medicine & Department University of Pe of Translational Medicine/1st Department of Medicine, MTA-SZTE Lendület Translational Gastroenterology Research Group, University of Szeged, Hungary 6 Department of Pharmacology and Pharmacotherapy, University of cs, Pe cs, Hungary Pe 2
Introduction: Transient receptor potential vanilloid 1 (TRPV1) is a nociceptor predominantly expressed by primary sensory neurons. TRPV1 participates in neurogenic inflammation and thus to have a major role in the pathogenesis of inflammatory disorders. Aims: We aimed to examine if desensitization of TRPV1 neurons affects the severity of experimental acute pancreatitis (AP) in rats. Materials & methods: To induce AP, Sprague-Dawley rats were injected intraperitoneally (i.p.) with 3 g/kg L-ornithine. 4 weeks before the induction of AP, primary neurons expressing TRPV1 were desensitized by resiniferatoxin (RTX), an agonist of TRPV1. RTX was administered by three i.p. injections (30 mg/kg, 70 mg/kg and 100 mg/kg, 30, 29 and 28 days before the induction of AP, respectively). Rats treated with L-ornithine and/or RTX were compared to their respective control groups treated with physiological saline. To determine AP severity, laboratory and histological parameters were measured. Results: L-ornithine induced necrotizing AP caused decreased pancreatic amylase activity and increased serum amylase and pancreatic myeloperoxidase activities, water content and heat-shock-protein 72 expression. RTX administration in itself did not significantly influence any of the measured parameters compared to the absolute control group (physiological saline instead of both RTX and L-ornithine). Most laboratory and histological parameters demonstrated that primary sensory neuron desensitization increased AP severity vs vehicle-pretreated L-ornithineinduced AP.