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P.01.2 PAF-RECEPTOR ANTAGONISTS INHIBIT HEPATOCELLULAR CARCINOMA GROWTH
Abstracts of the 20th National Congress of Digestive Diseases / Digestive and Liver Disease 46S (2014) S1–S144
POSTERS P.01.1 EFFECT OF BACILLUS CLAUSII IN THE N-METHYLNITROSUREA INDUCED MODEL OF COLONRECTAL CANCER A. Rettino 1 , V. Gerardi 1 , D. Roccarina 1 , B. Giupponi 1 , G. De Marco 1 , A. Tortora 1 , F. Scaldaferri 1 , G. Gasbarrini 2 , G. Zuccalà 2 , A. Gasbarrini 1 , F. Franceschi ∗,1 1 Gemelli
Hospital, Catholic University of Sacred Heart, Rome, Italy; Ricerca in Medicina, Bologna, Italy
2 Fondazione
Background and aim: Lactobacilli spp have been shown to reduce the risk of colorectal cancer in animal models. Whether this may also happen with Bacillus clausii has never been tested. Therefore, we designed a study aimed at assessing this issue. Material and methods: 5 mice were pre-treated with Bacillus clausii for one month before receiving intrarectal instillation of NMU twice a week for 3 months. Another group of 5 mice were only treated with intrarectal NMU. All animals were then sacrificed; samples of colon and rectum were collected and prepared for gene microarray. In order to identify an important pathway involved in the colon cancer chemically induced model, genes from the microarray analysis were functionally classified using Gene Ontology (GO) categories. The analysis was performed using the DAVID functional annotation tool. Results: Functional categorisation revealed a diverse set of GO biological processes that were statistically significant in the list of genes. Categories included cell cycle, apoptosis and response to DNA damage. Many of the genes from the microarray analysis were reported in literature to be a target of IRF1. Therefore, a RT-PCR analysis was performed to assess the abundance of IRF1 mRNA and some of its targets. IRF1 mRNA was upregulated in both the mice treated with NMU plus Bacillus clausii and those treated with NMU only. However, IRF1 target genes, such as the proapoptotic CASP7, DNA repair and cell cycle arrest Banp and Chek2 were found activated mostly in the mice treated with NMU plus Bacillus clausii. Conclusions: Bacillus clausii is able to activate the IRF1 gene pathway in an animal model of MNU-induced colorectal cancer. The transcription factor IRF1, which is involved in the response of cells to DNA damage, in the regulation of immune/inflammatory responses and in the DNA repair processes plays a pivotal role in activating genes involved in such categories. Interestingly, IRF1 can affect tumour susceptibility in mice, harbouring tumour suppressor activity, and is strictly required to prevent oncogene-induced cell transformation. Those data suggest that Bacillus Clausii may increase the functionality of this tumor suppressor gene/pathway.
P.01.2 PAF-RECEPTOR ANTAGONISTS INHIBIT HEPATOCELLULAR CARCINOMA GROWTH E. Ceni 2 , M. Tarocchi 2 , S. Polvani 2 , T. Mello 2 , G. Marroncini 2 , S. Tempesti 2 , F. Lisi 2 , S. Dei 1 , A. Galli ∗,2 1 Universita‘
di Firenze, Firenze, Italy; 2 Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Università di Firenze, Firenze, Italy Background and aim: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, there is no effective treatment for advance stage HCCs. PAF-receptor antagonists WEB-2086 and WEB-2170 (WEBs) have been previously shown to induce differentiation, growth arrest and massive apoptosis in in murine and human leukemia cells. We investigate the anti-tumor efficacy of WEB treatment in vitro and in vivo models of HCCs. Material and methods: We evaluated proliferation, apoptosis and migration of different hepatic cancer cell lines (HepG2, Hep3B, HuH7 and Hepa1-6) incubated with WEBs. We tested the in vivo efficacy of the treatment in HBV transgenic mice (TgAlb43Bri), that spontaneously develop hepatic tumors, and in a syngeneic orthotopic murine HCC model, where HCC cells were implanted directly in the animal liver. Results: WEBs were able to reduce the proliferation of cancer cells as assessed
S53
by thymidine incorporation, but no notable effects were present on apoptosis as assessed by caspase 3 activity. Furthermore pre-incubating the hepatic cancer cells with WEBs induced cell cycle arrest and impaired migration in a wound healing assay. Treatment with WEBs reduced growth and proliferation of the tumors in the transgenic and in the orthotopic murine model. Conclusions: PAF-receptor antagonists, WEB-2086 and WEB-2170, are able to reduce HCC progression in human and murine HCC models blocking cancer cell proliferation and migration.
P.01.3 IN VITRO EVALUATION OF DNA DAMAGE IN HUMAN COLONIC EPITHELIAL CELL LINES EXPOSED TO METHYLENE BLUE AND WHITE LIGHT A. Repici ∗,1 , M. Gerloni 2 , C. Rosette 2 , C. Ciscato 1 , M. Wallace 3 , P. Sharma 5 , R. Kiesslich 4 1 Istituto
Clinico Humanitas, Rozzano, Milan, Italy; 2 Research & Development Cosmo Bioscience, San Diego, United States; 3 Mayo Clinic Florida, Jacksonville, United States; 4 Medizinische Klinik St. Marienkrankenhaus, Frankfurt, Germany; 5 Veterans Medical Center & University of Kansas School of Medicine, Kansas City, United States Background and aim: Methylene Blue (MB) is a vital dye commonly used during chromoendoscopy. Although this procedure is generally considered safe, some concerns have been raised about its potential genotoxicity. However, the results of tests with mouse micronuclei exposed to high concentration of injectd MB suggest that genotoxic effects should not occur in vivo. Aim: To support the clinical use of MB-MMX tablets as a diagnostic tool during endoscopic examinations, experiments were conducted in vitro using HT-29 cell line to ascertain the level of DNA damage in cells exposed to white light for different time periods, in order to determine if photoexcited methylene blue damages DNA. Material and methods: To simulate chromoendoscopy in vitro, a monolayer of cultured HT-29 cells was incubated with 1 mL of 0.5% MB. Thereafter, the cells were exposed to white light of the same wavelength to different durations of light exposure (from 5 to 60 minutes) and conditions (presence or absence of oxidative conditions). MB-induced DNA breaks were evaluated on histone H2A variant (H2AX) that is phosphorylated (γH2AX) in response to DNA double strand damage. In additional tests, the same exposure times were repeated with and without a redox protective factor (N-acetyl cysteine, 50 mM). γH2AX determination required fixation and permeabilization of cells, followed by incubation with a fluorescent labeled antibody to γH2AX. Fluorescence intensity was quantified by flow cytometry. Results: HT-29 cells treated with MB and continuously exposed to white light for 5 and 10 minutes did not show statistically significant levels of DNA damage. Prolonged exposure to light from 15 to 60 minutes led to a proportional increase of DNA damage. We also demonstrated that the presence of a redox agent (N-acetyl cysteine) completely prevented the DNA damage. Conclusions: These tests show that the potential genotoxic effect of MB on HT-29 cells is closely related to the duration of the light exposure, with a cut-off time of at least 10 minutes of continuous exposure. Such exposure times i.e. continuous white light for >10 minutes on gastrointestinal mucosa is not typically reached during a normal colonoscopy procedure. Moreover, in cases of longer exposure times, the presence of a reducing agent was able to prevent completely the DNA damage.
P.01.4 ADAM-9 EXPRESSION MEDIATES COUP-TFII-DEPENDENT INVASIVENESS OF PANCREATIC CANCER CELLS E. Ceni, T. Mello, M. Tarocchi, S. Polvani, F. Buccoliero, S. Tempesti, G. Marroncini, A. Galli ∗ Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Università degli studi di Firenze, Firenze, Italy Background and aim: Pancreatic cancer is a malignant tumor characterized by an increased incidence in western countries with poor prognosis and
POSTERS P.01.1 EFFECT OF BACILLUS CLAUSII IN THE N-METHYLNITROSUREA INDUCED MODEL OF COLONRECTAL CANCER A. Rettino 1 , V. Gerardi 1 , D. Roccarina 1 , B. Giupponi 1 , G. De Marco 1 , A. Tortora 1 , F. Scaldaferri 1 , G. Gasbarrini 2 , G. Zuccalà 2 , A. Gasbarrini 1 , F. Franceschi ∗,1 1 Gemelli
Hospital, Catholic University of Sacred Heart, Rome, Italy; Ricerca in Medicina, Bologna, Italy
2 Fondazione
Background and aim: Lactobacilli spp have been shown to reduce the risk of colorectal cancer in animal models. Whether this may also happen with Bacillus clausii has never been tested. Therefore, we designed a study aimed at assessing this issue. Material and methods: 5 mice were pre-treated with Bacillus clausii for one month before receiving intrarectal instillation of NMU twice a week for 3 months. Another group of 5 mice were only treated with intrarectal NMU. All animals were then sacrificed; samples of colon and rectum were collected and prepared for gene microarray. In order to identify an important pathway involved in the colon cancer chemically induced model, genes from the microarray analysis were functionally classified using Gene Ontology (GO) categories. The analysis was performed using the DAVID functional annotation tool. Results: Functional categorisation revealed a diverse set of GO biological processes that were statistically significant in the list of genes. Categories included cell cycle, apoptosis and response to DNA damage. Many of the genes from the microarray analysis were reported in literature to be a target of IRF1. Therefore, a RT-PCR analysis was performed to assess the abundance of IRF1 mRNA and some of its targets. IRF1 mRNA was upregulated in both the mice treated with NMU plus Bacillus clausii and those treated with NMU only. However, IRF1 target genes, such as the proapoptotic CASP7, DNA repair and cell cycle arrest Banp and Chek2 were found activated mostly in the mice treated with NMU plus Bacillus clausii. Conclusions: Bacillus clausii is able to activate the IRF1 gene pathway in an animal model of MNU-induced colorectal cancer. The transcription factor IRF1, which is involved in the response of cells to DNA damage, in the regulation of immune/inflammatory responses and in the DNA repair processes plays a pivotal role in activating genes involved in such categories. Interestingly, IRF1 can affect tumour susceptibility in mice, harbouring tumour suppressor activity, and is strictly required to prevent oncogene-induced cell transformation. Those data suggest that Bacillus Clausii may increase the functionality of this tumor suppressor gene/pathway.
P.01.2 PAF-RECEPTOR ANTAGONISTS INHIBIT HEPATOCELLULAR CARCINOMA GROWTH E. Ceni 2 , M. Tarocchi 2 , S. Polvani 2 , T. Mello 2 , G. Marroncini 2 , S. Tempesti 2 , F. Lisi 2 , S. Dei 1 , A. Galli ∗,2 1 Universita‘
di Firenze, Firenze, Italy; 2 Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Università di Firenze, Firenze, Italy Background and aim: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, there is no effective treatment for advance stage HCCs. PAF-receptor antagonists WEB-2086 and WEB-2170 (WEBs) have been previously shown to induce differentiation, growth arrest and massive apoptosis in in murine and human leukemia cells. We investigate the anti-tumor efficacy of WEB treatment in vitro and in vivo models of HCCs. Material and methods: We evaluated proliferation, apoptosis and migration of different hepatic cancer cell lines (HepG2, Hep3B, HuH7 and Hepa1-6) incubated with WEBs. We tested the in vivo efficacy of the treatment in HBV transgenic mice (TgAlb43Bri), that spontaneously develop hepatic tumors, and in a syngeneic orthotopic murine HCC model, where HCC cells were implanted directly in the animal liver. Results: WEBs were able to reduce the proliferation of cancer cells as assessed
S53
by thymidine incorporation, but no notable effects were present on apoptosis as assessed by caspase 3 activity. Furthermore pre-incubating the hepatic cancer cells with WEBs induced cell cycle arrest and impaired migration in a wound healing assay. Treatment with WEBs reduced growth and proliferation of the tumors in the transgenic and in the orthotopic murine model. Conclusions: PAF-receptor antagonists, WEB-2086 and WEB-2170, are able to reduce HCC progression in human and murine HCC models blocking cancer cell proliferation and migration.
P.01.3 IN VITRO EVALUATION OF DNA DAMAGE IN HUMAN COLONIC EPITHELIAL CELL LINES EXPOSED TO METHYLENE BLUE AND WHITE LIGHT A. Repici ∗,1 , M. Gerloni 2 , C. Rosette 2 , C. Ciscato 1 , M. Wallace 3 , P. Sharma 5 , R. Kiesslich 4 1 Istituto
Clinico Humanitas, Rozzano, Milan, Italy; 2 Research & Development Cosmo Bioscience, San Diego, United States; 3 Mayo Clinic Florida, Jacksonville, United States; 4 Medizinische Klinik St. Marienkrankenhaus, Frankfurt, Germany; 5 Veterans Medical Center & University of Kansas School of Medicine, Kansas City, United States Background and aim: Methylene Blue (MB) is a vital dye commonly used during chromoendoscopy. Although this procedure is generally considered safe, some concerns have been raised about its potential genotoxicity. However, the results of tests with mouse micronuclei exposed to high concentration of injectd MB suggest that genotoxic effects should not occur in vivo. Aim: To support the clinical use of MB-MMX tablets as a diagnostic tool during endoscopic examinations, experiments were conducted in vitro using HT-29 cell line to ascertain the level of DNA damage in cells exposed to white light for different time periods, in order to determine if photoexcited methylene blue damages DNA. Material and methods: To simulate chromoendoscopy in vitro, a monolayer of cultured HT-29 cells was incubated with 1 mL of 0.5% MB. Thereafter, the cells were exposed to white light of the same wavelength to different durations of light exposure (from 5 to 60 minutes) and conditions (presence or absence of oxidative conditions). MB-induced DNA breaks were evaluated on histone H2A variant (H2AX) that is phosphorylated (γH2AX) in response to DNA double strand damage. In additional tests, the same exposure times were repeated with and without a redox protective factor (N-acetyl cysteine, 50 mM). γH2AX determination required fixation and permeabilization of cells, followed by incubation with a fluorescent labeled antibody to γH2AX. Fluorescence intensity was quantified by flow cytometry. Results: HT-29 cells treated with MB and continuously exposed to white light for 5 and 10 minutes did not show statistically significant levels of DNA damage. Prolonged exposure to light from 15 to 60 minutes led to a proportional increase of DNA damage. We also demonstrated that the presence of a redox agent (N-acetyl cysteine) completely prevented the DNA damage. Conclusions: These tests show that the potential genotoxic effect of MB on HT-29 cells is closely related to the duration of the light exposure, with a cut-off time of at least 10 minutes of continuous exposure. Such exposure times i.e. continuous white light for >10 minutes on gastrointestinal mucosa is not typically reached during a normal colonoscopy procedure. Moreover, in cases of longer exposure times, the presence of a reducing agent was able to prevent completely the DNA damage.
P.01.4 ADAM-9 EXPRESSION MEDIATES COUP-TFII-DEPENDENT INVASIVENESS OF PANCREATIC CANCER CELLS E. Ceni, T. Mello, M. Tarocchi, S. Polvani, F. Buccoliero, S. Tempesti, G. Marroncini, A. Galli ∗ Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Università degli studi di Firenze, Firenze, Italy Background and aim: Pancreatic cancer is a malignant tumor characterized by an increased incidence in western countries with poor prognosis and