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M1881 Regulation of Contractile Activity in Longitudinal Muscle of Rat Ileum by Hydrogen Sulfide
fibers with capsaicin (10-5 and 10-4M); E) K+ATP channels with glibenclamide (10-4 and 10M); and F) K+Ca channel activity with apamin (10-6 and 5x10-6M). Area under the contractile curve was used to compare the effects. RESULTS: NaHS exhibited a prominent dosedependent inhibitory effect on spontaneous and bethanechol-stimulated contractile activity (p<0.01) with effects starting at 5x10-4M and also decreased baseline tone. L-cysteine had no inhibitory effect. Establishing non-adrenergic and non-cholinergic conditions, blockade of all neural activity by tetrodotoxin, blocking NO production by L-NNA, blocking visceral primary afferents by capsaicin, blocking K+ATP channels by glibenclamide, or blocking K+Ca channels by apamin, had no effect on the inhibitory effect of 10-3M NaHS on contractile activity. CONCLUSION: H2S at physiologic levels inhibits contractile activity of ileal smooth muscle. The inhibitory effect of H2S in rat ileum does not appear to be mediated by intrinsic or extrinsic neural pathways, nitric oxide, or activity of visceral afferent nerve fibers, K+ATP channels, or K+Ca channels in ileal longitudinal muscle suggesting that the mechanisms of action of H2S proposed in other tissues do not appear to be operative in rat ileal longitudinal muscle.
M1879
3
Identification of Chromosomal Regions That Harbor Novel Genes Important for Pancreatic Cancer Pathogenesis by Genome-Wide Screening Methods Sabrina Thieltges, Tanya Kalinina, Ronald Simon, Maximilian Bockhorn, Emre F. Yekebas, Jakob R. Izbicki Background: Pancreatic adenocarcinoma is a genetically highly complex and heterogenous tumor type with strong genetic instability which makes it resistant to therapy. Known amplifications of oncogenes such as KRAS or MYC and deletions of tumor suppresor genes such as CDKN2A and SMAD4 have demonstrated the importance of genetic alteration in this tumor type. Methods: We report the use of an Affymetrix Genome-Wide Human single nucleotide polymorphism (SNP) Array 6.0 (906,600 SNPs) to screen for gene copy number changes and allelic imbalances in 8 microdissected primary pancreatic tumors and 7 established pancreatic cancer cell lines. The Gene Chip Human Genome U133 2.0 Array served for RNA expression profiling. Mutation analysis of KRAS and M-FISH analysis of cell lines was performed. Results: SNP arrays confirmed the presence of previously reported cytogenetic abnormalities in the cell lines and primary tumor probes, including MYC amplifikation at 8q24, gain of 17q12 (ERBB2/HER2), 7p12 (EGFR) and 12p12.1 (KRAS). KRAS mutation was found in 5 from 7 cell lines. We identified several alterations in signaling pathways such as Wnt/Notch Signaling and KRAS signaling. Approximately half of the cell line samples (7/15) showed an amplikon at 19q13.1-13.2 in which the serine/threonine kinase Mirk/ Dyrk1B is localized, a downstream effector of oncogenic KRAS. There was also strong concordance between primary tumors and cell lines with respect to gains on 8q, 12p and 18q. Analysis of gene expression was used to localize potential target genes. M-FISH analysis showed chromosome rearrangements such as 9p- and 18q-, regions that are known to harbor tumor suppressor genes (CDKN2A, SMAD4 and TP53). Conclusions: Several signaling pathways mediate tumor cell survival. Analysis of gene amplification and RNA expression profile provide molecular biological characteristics and an individual gene sinature of the tumor which allow us to choose more efficient drugs to an individualized treatment. Pathways activated by KRAS such as DYRK1B may offer new therapeutic targets. Further functional characterization is needed to provide evidence for the actual role of any putative target gene.
M1882 In-Vivo Assessment of a Biologic Occluder for NOTES Gastrotomy Closure Alejandro Nieponice, Alejandro F. Sanz, Toshitaka Hoppo, Bart P. Witteman, Thomas W. Gilbert, Bryan N. Brown, Stephen F. Badylak, Blair A. Jobe Introduction: NOTES has emerged as a conceptual framework that may change the paradigm of current surgical practice. Perhaps one of the biggest hurdles to overcome is to provide a “leak proof” closure of the viscera being transgressed. Failure to achieve this aim would unequivocally result in the failure of NOTES all together. The limitations of current approaches include technical challenges or the use of inert materials that leave a permanent foreign body within or around the port of entry.. Although currently used within the field of surgery, biological scaffolds for NOTES closure have not been attempted and may overcome several of the current limitations. The current study aimed to evaluate the closure of a transgastric NOTES access using a multilayer extracellular matrix (ECM) occluder in a survival study with a canine model. Methods: Four adult female mongrel dogs were subjected to transgastric NOTES peritoneoscopy. At procedure completion, the gastrotomy was closed by deploying a two-sided ECM occluder. The construct of the occluder was such that there was ECM coverage on both sides and within the gastrotomy. . Animals were survived for 7 days (n=2) and 8 weeks (n=2). Endoscopic follow-up was performed at 48hs post-op and immediately before sacrifice. Endpoints included clinical outcome, presence of leak, peritoneal swab for culture and histology. Results: All procedures were completed uneventfully. Deployment of the device was possible in all animals and air tightness could be observed immediately after placement under laparoscopoic visualization with endoscopic insufflation. All animals had an uneventful recovery with no clinical signs of abdominal discomfort or sepsis. Endoscopy showed no air leak at 48hs. Abdominal cultures were sterile and no signs of leak were detected at 7-day or 8-week necropsy. Histology demonstrated remodeling of the scaffold with a complete gastric mucosal lining and organized loose collagen bundles. No foreign body reaction was observed at the site of injury. Discussion: The ECM occluder has been demonstrated to be safe and effective in this preclinical model. Biological scaffolds may represent a useful application for human NOTES procedures.
M1880
SSAT Abstracts
Pterostilbene and Gemcitabine Have Additive Effects Against Pancreatic Cancer In Vitro Patrick Mannal, David W. McFadden Background: Resveratrol, a naturally occurring phenol is a potent antioxidant as well as having pro-apoptotic properties. Pterostilbene is an analogue of resveratrol with greater oral bioavailability. Our previous studies have shown Pterostilbene to be an effective growth inhibitor against multiple cancer cell lines. To further evaluate Pterostilbene's potential role as an agent against pancreatic cancer, we hypothesized that there would be additive effects when Gemcitabine and Pterostilbene were combined. Methods: Two pancreatic cancer cell lines (MIA-PACA and PANC-1) were cultured using standard techniques. Cells were pretreated with 1μM Gemcitabine for 18 hours followed by graduated doses of Pterostilbene (10 - 30μM). In addition, cells were pretreated with Pterostilbene (10 - 30μM) for 18 hours followed by 1μM Gemcitabine. Lastly, the same pancreatic cell lines were treated simultaneously with Pterostilbene and Gemcitabine (doses identical to those above) after which cell viability was assessed in all three groups by MTT assay at 24, 48 and 72 hours. Results: Gemcitabine and Pterostilbene show an additive effect on pancreatic cancer cell viability in a time and dose dependent manner. The best results were seen when pancreatic cancer cells were pre-treated with Pterostilbene followed by Gemcitabine treatment. With Pterostilbene pre-treatment, the greatest effects were seen at 48 and 72 hours for MIA cells, where cell viability was reduced to 23% and 21% of control, respectively (P<0.001). The Pterostilbene pre-treated PANC cells reached maximum inhibition of cell viability at 72 hours with a reduction to 34% of control (P<0.001). Simultaneous use of Gemcitabine and Pterostilbene in PANC cells reduced cell viability at 72 hours to 56% and 50% of control with Pterostilbene concentrations of 10μM and 20μM, respectively (P<0.01). MIA cells treated simultaneously with Gemcitabine and Pterostilbene also showed reduction to 62% and 58% of control at 48hours and 37% and 35% of control at 72 hours with Pterostilbene concentrations of 10μM and 20μM (P<0.01). Conclusion: Pterostilbene, a well tolerated natural compound found in blueberries, may have clinical utility in the treatment of pancreatic cancer. Combination treatment of Pterostilbene with Gemcitabine leads to an additive cytotoxic effect on pancreatic cancer cells In Vitro. This synergism occurs best in Pterostilbene pre-treated calls and simultaneously treated cells. Further delineation of the mechanisms of action of Pterostilbene is on-going to assess its potential usefulness in treatment of this devastating disease.
M2056 Prediction of Prognosis by Expression Profiling Analysis of Pancreatic Ductal Adenocancinoma Using DNA-Arrays Robert Grützmann, Marco Niedergethmann, Moritz Wente, Helmut Friess, Glen Kristiansen, Christof Winter, Marcus Bahra, Petra Ruemmele, Hans D. Saeger, Christian Pilarsky Prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains poor. Despite of increasing knowledge about the molecular basis of PDAC no specific marker for early diagnosis nor a target protein for a new therapeutic approach have been identified so far. Surgery is the only potentially curative option. But even after R0-resection of PDAC, the prognosis is bad and most patients suffer from recurrences and metastases. Moreover, the individual prognosis is not known. This might be of nterest for indication of adjuvant therapy. We were therefore interested in the analysis of differential gene expression between PDAC with relatively good and worse prognosis. We used fresh frozen tissue from 29 patients with pancreatic ductal adenocarcinoma. From every single patient, the clinical characteristics, pathological data as well as follow up has been collected prospectively. The tissues were obtained during surgery and freshly frozen. The type of each frozen tissue sample was re evaluated pathologically. The RNA was extracted using the RNeasy Mini Kit. The quality of the RNA was assessed using the Agilent Lab on a Chip System and only samples displaying a RIN > 4 were used. For hybridization we used 100 ng of total RNA, and samples were prepared according the Affymetrix two cycle amplification labelling protocol. Samples were hybridised to Affymetrix U133 2.0 plus GeneChips. The obtained data from the microarray were analysed using dCHIP. Median survival of the patients was 13 (2-53) months. Using this time point we classified the samples into two groups. Differentially gene expression analysis (FC > 2, difference of means > 100; p value < 0.05) revealed 21 probe sets. Hierarchical clustering using these 21 probe sets displayed two clusters of samples. One cluster contained only samples from patients with a survival time < 13 months. Sensitivity and specificity calculations based on the cluster data resulted in 100 % sensitivity and 73 % specificity for the detection of patient samples with a survival > 13 month. Several genes have been valedated using tissue microarrays. In conclusion, gene expression analysis of the tumor tissue of PDAC enables prediction of prognosis with high specificity and good sensitivity. The role of adjuvant treatment has to be elucidated. Moreover, using the set of differentially expressed genes we might identify new markers and therapeutic targets for pancreatic cancer.
M1881 Regulation of Contractile Activity in Longitudinal Muscle of Rat Ileum by Hydrogen Sulfide Munenori Nagao, Judith A. Duenes, David R. Linden, Gianrico Farrugia, Michael G. Sarr BACKGROUND: Endogenous hydrogen sulfide (H2S) is a newly discovered modulator of GI motility; however, the mechanisms of action of H2S are not well understood. AIM: To determine effects and mechanisms of action of H2S on contractile activity in longitudinal muscle of rat ileum. METHODS: Ileal longitudinal muscle strips from 12 Lewis rats were prepared to measure contractile activity in a temperature controlled tissue chamber. Sodium hydrosulfide (NaHS; 10-5 to 10-3M) was used as an exogenous donor of H2S yielding bath solution levels of H2S of approximately 1 to 180 μM. Physiologic endogenous levels of H2S are thought to be 50-200 μM. Effects of NaHS were evaluated on spontaneous contractile activity and after pre-contraction with bethanechol (3x10-6M). L-cysteine (10-4, 10-3, and 10-2M), the substrate for production of H2S, was used as an endogenous donor of H2S. We evaluated involvement of A) Extrinsic nerves with atropine (10-7M), phentolamine (10-5M), and propranolol (5x10-6M); B) Enteric nervous system with tetrodotoxin (10-6M); C) Nitric oxide with L-NG-nitro arginine (L-NNA; 10-4 and 10-3M); D) Visceral primary afferent nerve
SSAT Abstracts
S-880
M1879
3
Identification of Chromosomal Regions That Harbor Novel Genes Important for Pancreatic Cancer Pathogenesis by Genome-Wide Screening Methods Sabrina Thieltges, Tanya Kalinina, Ronald Simon, Maximilian Bockhorn, Emre F. Yekebas, Jakob R. Izbicki Background: Pancreatic adenocarcinoma is a genetically highly complex and heterogenous tumor type with strong genetic instability which makes it resistant to therapy. Known amplifications of oncogenes such as KRAS or MYC and deletions of tumor suppresor genes such as CDKN2A and SMAD4 have demonstrated the importance of genetic alteration in this tumor type. Methods: We report the use of an Affymetrix Genome-Wide Human single nucleotide polymorphism (SNP) Array 6.0 (906,600 SNPs) to screen for gene copy number changes and allelic imbalances in 8 microdissected primary pancreatic tumors and 7 established pancreatic cancer cell lines. The Gene Chip Human Genome U133 2.0 Array served for RNA expression profiling. Mutation analysis of KRAS and M-FISH analysis of cell lines was performed. Results: SNP arrays confirmed the presence of previously reported cytogenetic abnormalities in the cell lines and primary tumor probes, including MYC amplifikation at 8q24, gain of 17q12 (ERBB2/HER2), 7p12 (EGFR) and 12p12.1 (KRAS). KRAS mutation was found in 5 from 7 cell lines. We identified several alterations in signaling pathways such as Wnt/Notch Signaling and KRAS signaling. Approximately half of the cell line samples (7/15) showed an amplikon at 19q13.1-13.2 in which the serine/threonine kinase Mirk/ Dyrk1B is localized, a downstream effector of oncogenic KRAS. There was also strong concordance between primary tumors and cell lines with respect to gains on 8q, 12p and 18q. Analysis of gene expression was used to localize potential target genes. M-FISH analysis showed chromosome rearrangements such as 9p- and 18q-, regions that are known to harbor tumor suppressor genes (CDKN2A, SMAD4 and TP53). Conclusions: Several signaling pathways mediate tumor cell survival. Analysis of gene amplification and RNA expression profile provide molecular biological characteristics and an individual gene sinature of the tumor which allow us to choose more efficient drugs to an individualized treatment. Pathways activated by KRAS such as DYRK1B may offer new therapeutic targets. Further functional characterization is needed to provide evidence for the actual role of any putative target gene.
M1882 In-Vivo Assessment of a Biologic Occluder for NOTES Gastrotomy Closure Alejandro Nieponice, Alejandro F. Sanz, Toshitaka Hoppo, Bart P. Witteman, Thomas W. Gilbert, Bryan N. Brown, Stephen F. Badylak, Blair A. Jobe Introduction: NOTES has emerged as a conceptual framework that may change the paradigm of current surgical practice. Perhaps one of the biggest hurdles to overcome is to provide a “leak proof” closure of the viscera being transgressed. Failure to achieve this aim would unequivocally result in the failure of NOTES all together. The limitations of current approaches include technical challenges or the use of inert materials that leave a permanent foreign body within or around the port of entry.. Although currently used within the field of surgery, biological scaffolds for NOTES closure have not been attempted and may overcome several of the current limitations. The current study aimed to evaluate the closure of a transgastric NOTES access using a multilayer extracellular matrix (ECM) occluder in a survival study with a canine model. Methods: Four adult female mongrel dogs were subjected to transgastric NOTES peritoneoscopy. At procedure completion, the gastrotomy was closed by deploying a two-sided ECM occluder. The construct of the occluder was such that there was ECM coverage on both sides and within the gastrotomy. . Animals were survived for 7 days (n=2) and 8 weeks (n=2). Endoscopic follow-up was performed at 48hs post-op and immediately before sacrifice. Endpoints included clinical outcome, presence of leak, peritoneal swab for culture and histology. Results: All procedures were completed uneventfully. Deployment of the device was possible in all animals and air tightness could be observed immediately after placement under laparoscopoic visualization with endoscopic insufflation. All animals had an uneventful recovery with no clinical signs of abdominal discomfort or sepsis. Endoscopy showed no air leak at 48hs. Abdominal cultures were sterile and no signs of leak were detected at 7-day or 8-week necropsy. Histology demonstrated remodeling of the scaffold with a complete gastric mucosal lining and organized loose collagen bundles. No foreign body reaction was observed at the site of injury. Discussion: The ECM occluder has been demonstrated to be safe and effective in this preclinical model. Biological scaffolds may represent a useful application for human NOTES procedures.
M1880
SSAT Abstracts
Pterostilbene and Gemcitabine Have Additive Effects Against Pancreatic Cancer In Vitro Patrick Mannal, David W. McFadden Background: Resveratrol, a naturally occurring phenol is a potent antioxidant as well as having pro-apoptotic properties. Pterostilbene is an analogue of resveratrol with greater oral bioavailability. Our previous studies have shown Pterostilbene to be an effective growth inhibitor against multiple cancer cell lines. To further evaluate Pterostilbene's potential role as an agent against pancreatic cancer, we hypothesized that there would be additive effects when Gemcitabine and Pterostilbene were combined. Methods: Two pancreatic cancer cell lines (MIA-PACA and PANC-1) were cultured using standard techniques. Cells were pretreated with 1μM Gemcitabine for 18 hours followed by graduated doses of Pterostilbene (10 - 30μM). In addition, cells were pretreated with Pterostilbene (10 - 30μM) for 18 hours followed by 1μM Gemcitabine. Lastly, the same pancreatic cell lines were treated simultaneously with Pterostilbene and Gemcitabine (doses identical to those above) after which cell viability was assessed in all three groups by MTT assay at 24, 48 and 72 hours. Results: Gemcitabine and Pterostilbene show an additive effect on pancreatic cancer cell viability in a time and dose dependent manner. The best results were seen when pancreatic cancer cells were pre-treated with Pterostilbene followed by Gemcitabine treatment. With Pterostilbene pre-treatment, the greatest effects were seen at 48 and 72 hours for MIA cells, where cell viability was reduced to 23% and 21% of control, respectively (P<0.001). The Pterostilbene pre-treated PANC cells reached maximum inhibition of cell viability at 72 hours with a reduction to 34% of control (P<0.001). Simultaneous use of Gemcitabine and Pterostilbene in PANC cells reduced cell viability at 72 hours to 56% and 50% of control with Pterostilbene concentrations of 10μM and 20μM, respectively (P<0.01). MIA cells treated simultaneously with Gemcitabine and Pterostilbene also showed reduction to 62% and 58% of control at 48hours and 37% and 35% of control at 72 hours with Pterostilbene concentrations of 10μM and 20μM (P<0.01). Conclusion: Pterostilbene, a well tolerated natural compound found in blueberries, may have clinical utility in the treatment of pancreatic cancer. Combination treatment of Pterostilbene with Gemcitabine leads to an additive cytotoxic effect on pancreatic cancer cells In Vitro. This synergism occurs best in Pterostilbene pre-treated calls and simultaneously treated cells. Further delineation of the mechanisms of action of Pterostilbene is on-going to assess its potential usefulness in treatment of this devastating disease.
M2056 Prediction of Prognosis by Expression Profiling Analysis of Pancreatic Ductal Adenocancinoma Using DNA-Arrays Robert Grützmann, Marco Niedergethmann, Moritz Wente, Helmut Friess, Glen Kristiansen, Christof Winter, Marcus Bahra, Petra Ruemmele, Hans D. Saeger, Christian Pilarsky Prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains poor. Despite of increasing knowledge about the molecular basis of PDAC no specific marker for early diagnosis nor a target protein for a new therapeutic approach have been identified so far. Surgery is the only potentially curative option. But even after R0-resection of PDAC, the prognosis is bad and most patients suffer from recurrences and metastases. Moreover, the individual prognosis is not known. This might be of nterest for indication of adjuvant therapy. We were therefore interested in the analysis of differential gene expression between PDAC with relatively good and worse prognosis. We used fresh frozen tissue from 29 patients with pancreatic ductal adenocarcinoma. From every single patient, the clinical characteristics, pathological data as well as follow up has been collected prospectively. The tissues were obtained during surgery and freshly frozen. The type of each frozen tissue sample was re evaluated pathologically. The RNA was extracted using the RNeasy Mini Kit. The quality of the RNA was assessed using the Agilent Lab on a Chip System and only samples displaying a RIN > 4 were used. For hybridization we used 100 ng of total RNA, and samples were prepared according the Affymetrix two cycle amplification labelling protocol. Samples were hybridised to Affymetrix U133 2.0 plus GeneChips. The obtained data from the microarray were analysed using dCHIP. Median survival of the patients was 13 (2-53) months. Using this time point we classified the samples into two groups. Differentially gene expression analysis (FC > 2, difference of means > 100; p value < 0.05) revealed 21 probe sets. Hierarchical clustering using these 21 probe sets displayed two clusters of samples. One cluster contained only samples from patients with a survival time < 13 months. Sensitivity and specificity calculations based on the cluster data resulted in 100 % sensitivity and 73 % specificity for the detection of patient samples with a survival > 13 month. Several genes have been valedated using tissue microarrays. In conclusion, gene expression analysis of the tumor tissue of PDAC enables prediction of prognosis with high specificity and good sensitivity. The role of adjuvant treatment has to be elucidated. Moreover, using the set of differentially expressed genes we might identify new markers and therapeutic targets for pancreatic cancer.
M1881 Regulation of Contractile Activity in Longitudinal Muscle of Rat Ileum by Hydrogen Sulfide Munenori Nagao, Judith A. Duenes, David R. Linden, Gianrico Farrugia, Michael G. Sarr BACKGROUND: Endogenous hydrogen sulfide (H2S) is a newly discovered modulator of GI motility; however, the mechanisms of action of H2S are not well understood. AIM: To determine effects and mechanisms of action of H2S on contractile activity in longitudinal muscle of rat ileum. METHODS: Ileal longitudinal muscle strips from 12 Lewis rats were prepared to measure contractile activity in a temperature controlled tissue chamber. Sodium hydrosulfide (NaHS; 10-5 to 10-3M) was used as an exogenous donor of H2S yielding bath solution levels of H2S of approximately 1 to 180 μM. Physiologic endogenous levels of H2S are thought to be 50-200 μM. Effects of NaHS were evaluated on spontaneous contractile activity and after pre-contraction with bethanechol (3x10-6M). L-cysteine (10-4, 10-3, and 10-2M), the substrate for production of H2S, was used as an endogenous donor of H2S. We evaluated involvement of A) Extrinsic nerves with atropine (10-7M), phentolamine (10-5M), and propranolol (5x10-6M); B) Enteric nervous system with tetrodotoxin (10-6M); C) Nitric oxide with L-NG-nitro arginine (L-NNA; 10-4 and 10-3M); D) Visceral primary afferent nerve
SSAT Abstracts
S-880