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Importance of hedgehog signaling pathway in pancreatic cancer treatment and prognosis
Abstracts / Pancreatology 15 (2015) S1eS141
effects on murine pancreatic cancer cells 6606PDA and murine fibroblasts (Alamar-Blue-Assay). A combined treatment of TTP with gemcitabine was established. Apoptosis was analysed with activated caspases 3þ7 (fluorescence microscopy) and p38MAPK (Western Blot). Furthermore cell migration was examined with a 3D-collagen-matrix and time-lapse video microscopy. Results: The IC50TTP for 6606PDA was 100sec, for fibroblasts 535sec. The IC50gemcitabine for 6606PDA was 14nM. The combination of IC75gemcitabine and IC50TTP was significantly more effective in killing tumour cells than the monotherapies (p<0.0001 TTP; p<0.0001 gemcitabine). The fibroblasts were significantly less effected by TTP and combined treatment (p<0.001 TTP; p<0.0001 TTPþgemcitabine). The highest rate of activated caspases and P38MAPK was detected after combined treatment of 6606PDA (caspases: p<0.0001; P38MAPK: p<0.001). Also, migration was significantly inhibited after TTP and TTPþgemcitabine treatment (p<0.01). Conclusion: Pancreatic cancer cells are significantly more susceptible to TTP-induced apoptosis than non-malignant fibroblasts. The combination with gemcitabine is even more effective in killing tumour cells. The combination of TTPþgemcitabine could be a promising treatment option for pancreatic cancer in the future.
939. Smad4 deletion is associated with a distinctive exosomal microRNAs pattern Ada Aita 1, Thomas Brefort 2, Carlo-Federico Zambon 1, Dania Bozzato 1, Stefania Moz 1, Andrea Padoan 1, Hannah Schroers 2, Mario Plebani 1, Daniela Basso 1 1 2
Department of Medicine e DIMED, University of Padova, Italy Comprehensive Biomarker Center GmbH, Heidelberg, Germany
Introduction: MicroRNAs (miRNAs), emerging regulators of gene expression, might be involved in SMAD4-driven PDAC carcinogenesis and metastases. Tumor cell-derived miRNAs might be secreted in sera through exososomes and therefore the detection of PDAC and Smad4-specific exosomal miRNAs (ex-miRNAs) in blood might represent a future diagnostic challenge. Aims: To verify whether SMAD4-loss associates with a specific pattern of ex-miRNAs. Materials & methods: BxPC3 (Smad4HD) and Smad4-transfected BxPC3 (BxPC3-Smad4þ) derived exosomes were isolated from 72 hours 1%FCS-conditioned media by differential ultracentrifugation. ExmiRNAs were extracted and miRNAs expression profiles determined by Agilent SurePrintG3 Human miRNA (8x60K) microarrays. Data were analysed bioinformatically to extract significantly deregulated candidates. Results: Purified exosomes were confirmed by western blot analysis (Alix and TSG 101). All miRNAs from the Sanger miRBase release 19 were tested. The bioinformatic analyses revealed high correlation within each group (Pearson correlation coefficient- CP:0.999) and low correlation between two groups (CP:0.956). Specific clusters of significantly deregulated ex-miRNAs were identified between BxPC3 and BxPC3Smad4þ cells by different statistical approaches (Cluster analysis, MA Plot, Principal component analysis). Among the most deregulated 30 miRNAs, three had a log2 fold change lower than -3 (miR-494-3p, miR7641, miR-6132) and one higher than þ3 (miR-5585-3p) in BxPC3 vs BxPC3-Smad4þ. The Volcano plot included also miR-4299 as significantly reduced in BxPC3. Limma test added 5 further down-regulated miRNAs (miR-6785-5p, miR-1973, miR-6894-5p, miR-4485, miR-664b-5p) and 3 up-regulated (miR-1260a, miR-762, miR-328-5p) in BxPC3 vs BxPC3Smad4þ cells. Conclusion: Smad4-loss characterizes a different expression pattern of ex-miRNAs. The identification in blood of Smad4-associated ex-miRNAs might represent a challenge for PDAC diagnosis and staging.
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1198. Importance of hedgehog signaling pathway in pancreatic cancer treatment and prognosis Beatrice Mohelnikova-Duchonova 1, Martin Kocik 2, Barbara Duchonova 3, Veronika Brynychova 4, Martin Oliverius 2, Jan Hlavsa 5, Eva Honsova 6, Jan Mazanec 7, Zdenek Kala 5, Iwao Ojima 8, Pavel Soucek 4 1 Department of Toxicogenomics, NIPH, Prague and Department of Oncology, Palacky University Medical School and Teaching Hospital, Czech Republic 2 Department of Transplantation Surgery, Institute of Clinical and Expe, Czech Republic 3 Faculty of Science, Palacky University, Olomouc, Czech Republic 4 Department of Toxicogenomics, National Institute of Public Health, Prague, Czech Republic 5 Department of Surgery, Masaryk University Hospital and Faculty of Medicine, Brno Bohunice, Czech Republic 6 Department of Clinical and Transplantation Pathology, Institute of Clinical and Experimental Medicine, Prague, Czech Republic 7 Department of Pathology, Masaryk University Hospital and Faculty of Medicine, Brno Bohunice, Czech Republic 8 Institute of Chemical Biology and Drug Discovery, State University of New York at Stony Brook, Stony Brook, NY, United States
Aims: The aim of the present study was to investigate prognostic significance of Hedgehog (HH) signaling pathway in pancreatic cancer patients who underwent a radical resection. The effect of experimental chemotherapy on the expression of HH genes has been evaluated in vivo using mouse model. Patients & methods: Three different types of xenografts have been prepared using pancreatic cancer cell lines, namely BxPC-3, Paca-44, and MiaPaca-2. Mice have been treated by experimental taxanes (Stony Brook Taxane SBT-1214). Tumors and adjacent non-neoplastic pancreatic tissues were obtained from 50 patients with histologically verified ductal pancreatic adenocarcinoma (PDAC). The transcript profile of 36 hedgehog signaling pathway genes was assessed using qPCR. Results: HH pathway was overexpressed in PDAC when compared to non-malignant adjacent tissue. However, no association has been found between the level of HH pathway expression and overall survival of PDAC patients, or KRAS mutations status. In vivo, the expression of SHH ligand was upregulated, but the downstream genes were mostly downregulated after SBT-1214 treatment. The expression of HH pathway significantly differs among tumors derived from different cell lines. MiaPaca-2 xenografts were least sensitive to SBT-1214 with biological aggressive behavior. HH pathway was upregulated in MiaPaca-2 xenografts when compared to the other two types. Conclusion: Despite the fact that expression of HH pathway genes is upregulated in PDAC and correlates with the treatment outcome in vivo, it seems to have a limited effect on the prognosis of patients with the resectable stage of the disease.Supported by Czech Science Foundation grant P301/12/1734, and by the Palacky University projectLF_2015_015.
946. Differential impact of diabetes type II and chronic inflammation on pancreatic cancer Dietmar Zechner 1, Tobias Radecke 1, Jonas Amme 1, Florian Bürtin 1, Ann-Christin Albert 1, Lars Ivo Partecke 2, Brigitte Vollmar 1 1 Rostock University Medical Center, Institute for Experimental Surgery Rostock, Germany 2 Ernst-Moritz-Arndt-University, Department of General, Visceral, Thoracic and Vascular Surgery, Germany
Introduction: Well established risk factors for pancreatic cancer such as type II diabetes and chronic inflammation have been demonstrated to influence precancerous lesions in the pancreas. However, little is known if
effects on murine pancreatic cancer cells 6606PDA and murine fibroblasts (Alamar-Blue-Assay). A combined treatment of TTP with gemcitabine was established. Apoptosis was analysed with activated caspases 3þ7 (fluorescence microscopy) and p38MAPK (Western Blot). Furthermore cell migration was examined with a 3D-collagen-matrix and time-lapse video microscopy. Results: The IC50TTP for 6606PDA was 100sec, for fibroblasts 535sec. The IC50gemcitabine for 6606PDA was 14nM. The combination of IC75gemcitabine and IC50TTP was significantly more effective in killing tumour cells than the monotherapies (p<0.0001 TTP; p<0.0001 gemcitabine). The fibroblasts were significantly less effected by TTP and combined treatment (p<0.001 TTP; p<0.0001 TTPþgemcitabine). The highest rate of activated caspases and P38MAPK was detected after combined treatment of 6606PDA (caspases: p<0.0001; P38MAPK: p<0.001). Also, migration was significantly inhibited after TTP and TTPþgemcitabine treatment (p<0.01). Conclusion: Pancreatic cancer cells are significantly more susceptible to TTP-induced apoptosis than non-malignant fibroblasts. The combination with gemcitabine is even more effective in killing tumour cells. The combination of TTPþgemcitabine could be a promising treatment option for pancreatic cancer in the future.
939. Smad4 deletion is associated with a distinctive exosomal microRNAs pattern Ada Aita 1, Thomas Brefort 2, Carlo-Federico Zambon 1, Dania Bozzato 1, Stefania Moz 1, Andrea Padoan 1, Hannah Schroers 2, Mario Plebani 1, Daniela Basso 1 1 2
Department of Medicine e DIMED, University of Padova, Italy Comprehensive Biomarker Center GmbH, Heidelberg, Germany
Introduction: MicroRNAs (miRNAs), emerging regulators of gene expression, might be involved in SMAD4-driven PDAC carcinogenesis and metastases. Tumor cell-derived miRNAs might be secreted in sera through exososomes and therefore the detection of PDAC and Smad4-specific exosomal miRNAs (ex-miRNAs) in blood might represent a future diagnostic challenge. Aims: To verify whether SMAD4-loss associates with a specific pattern of ex-miRNAs. Materials & methods: BxPC3 (Smad4HD) and Smad4-transfected BxPC3 (BxPC3-Smad4þ) derived exosomes were isolated from 72 hours 1%FCS-conditioned media by differential ultracentrifugation. ExmiRNAs were extracted and miRNAs expression profiles determined by Agilent SurePrintG3 Human miRNA (8x60K) microarrays. Data were analysed bioinformatically to extract significantly deregulated candidates. Results: Purified exosomes were confirmed by western blot analysis (Alix and TSG 101). All miRNAs from the Sanger miRBase release 19 were tested. The bioinformatic analyses revealed high correlation within each group (Pearson correlation coefficient- CP:0.999) and low correlation between two groups (CP:0.956). Specific clusters of significantly deregulated ex-miRNAs were identified between BxPC3 and BxPC3Smad4þ cells by different statistical approaches (Cluster analysis, MA Plot, Principal component analysis). Among the most deregulated 30 miRNAs, three had a log2 fold change lower than -3 (miR-494-3p, miR7641, miR-6132) and one higher than þ3 (miR-5585-3p) in BxPC3 vs BxPC3-Smad4þ. The Volcano plot included also miR-4299 as significantly reduced in BxPC3. Limma test added 5 further down-regulated miRNAs (miR-6785-5p, miR-1973, miR-6894-5p, miR-4485, miR-664b-5p) and 3 up-regulated (miR-1260a, miR-762, miR-328-5p) in BxPC3 vs BxPC3Smad4þ cells. Conclusion: Smad4-loss characterizes a different expression pattern of ex-miRNAs. The identification in blood of Smad4-associated ex-miRNAs might represent a challenge for PDAC diagnosis and staging.
S37
1198. Importance of hedgehog signaling pathway in pancreatic cancer treatment and prognosis Beatrice Mohelnikova-Duchonova 1, Martin Kocik 2, Barbara Duchonova 3, Veronika Brynychova 4, Martin Oliverius 2, Jan Hlavsa 5, Eva Honsova 6, Jan Mazanec 7, Zdenek Kala 5, Iwao Ojima 8, Pavel Soucek 4 1 Department of Toxicogenomics, NIPH, Prague and Department of Oncology, Palacky University Medical School and Teaching Hospital, Czech Republic 2 Department of Transplantation Surgery, Institute of Clinical and Expe, Czech Republic 3 Faculty of Science, Palacky University, Olomouc, Czech Republic 4 Department of Toxicogenomics, National Institute of Public Health, Prague, Czech Republic 5 Department of Surgery, Masaryk University Hospital and Faculty of Medicine, Brno Bohunice, Czech Republic 6 Department of Clinical and Transplantation Pathology, Institute of Clinical and Experimental Medicine, Prague, Czech Republic 7 Department of Pathology, Masaryk University Hospital and Faculty of Medicine, Brno Bohunice, Czech Republic 8 Institute of Chemical Biology and Drug Discovery, State University of New York at Stony Brook, Stony Brook, NY, United States
Aims: The aim of the present study was to investigate prognostic significance of Hedgehog (HH) signaling pathway in pancreatic cancer patients who underwent a radical resection. The effect of experimental chemotherapy on the expression of HH genes has been evaluated in vivo using mouse model. Patients & methods: Three different types of xenografts have been prepared using pancreatic cancer cell lines, namely BxPC-3, Paca-44, and MiaPaca-2. Mice have been treated by experimental taxanes (Stony Brook Taxane SBT-1214). Tumors and adjacent non-neoplastic pancreatic tissues were obtained from 50 patients with histologically verified ductal pancreatic adenocarcinoma (PDAC). The transcript profile of 36 hedgehog signaling pathway genes was assessed using qPCR. Results: HH pathway was overexpressed in PDAC when compared to non-malignant adjacent tissue. However, no association has been found between the level of HH pathway expression and overall survival of PDAC patients, or KRAS mutations status. In vivo, the expression of SHH ligand was upregulated, but the downstream genes were mostly downregulated after SBT-1214 treatment. The expression of HH pathway significantly differs among tumors derived from different cell lines. MiaPaca-2 xenografts were least sensitive to SBT-1214 with biological aggressive behavior. HH pathway was upregulated in MiaPaca-2 xenografts when compared to the other two types. Conclusion: Despite the fact that expression of HH pathway genes is upregulated in PDAC and correlates with the treatment outcome in vivo, it seems to have a limited effect on the prognosis of patients with the resectable stage of the disease.Supported by Czech Science Foundation grant P301/12/1734, and by the Palacky University projectLF_2015_015.
946. Differential impact of diabetes type II and chronic inflammation on pancreatic cancer Dietmar Zechner 1, Tobias Radecke 1, Jonas Amme 1, Florian Bürtin 1, Ann-Christin Albert 1, Lars Ivo Partecke 2, Brigitte Vollmar 1 1 Rostock University Medical Center, Institute for Experimental Surgery Rostock, Germany 2 Ernst-Moritz-Arndt-University, Department of General, Visceral, Thoracic and Vascular Surgery, Germany
Introduction: Well established risk factors for pancreatic cancer such as type II diabetes and chronic inflammation have been demonstrated to influence precancerous lesions in the pancreas. However, little is known if