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911 A Novel Use of Angiotensin II Receptor Blocker (ARB) Losartan to Inhibit AOM Induced Tumorigenesis and Neoangiogenesis in Experimental Colon Cancer
AGA Abstracts
vascular changes as shown in the tumor of a mouse treated with AOM and given Losartan (D). In vivo imaging with confocal laser endomicroscopy demonstrated similar changes in AOM induced tumors with (E) and without (F) Losartan.
0.0004, R=0.6774), indicating that the production of PGE2 is associated with DNMT3B expression in vivo. Moreover, methylation array analysis of vehicle- and PGE2-treated GES1, HGC27 and MKN1 cells revealed that PGE2 induced the promoter hypermethylation of 102 genes, including tumor suppressor genes that are associated with gastric cancer. Hypermethylation genes induced by PGE2 were enriched in two signaling pathways (cell cycle regulation and DNA helicase activity). In particular, CAB39L, transcriptionally silenced by PGE2induced promoter methylation, functions as a tumor suppressor gene in gastric cancer. Combinatorial treatment with COX-2 (celecoxib) and DNMT (5-aza-2'-deoxycytidine) inhibitors synergistically suppressed the growth of gastric cancer cells in vitro and HGC27 xenografts in nude mice. CONCLUSIONS: PGE2 mediates epigenetic dysregulation in gastric cancer via induction of DNMT3B activity and consequent DNA promoter hypermethylation of tumor suppressor genes. Co-targeting of PGE2 and DNMT synergistically inhibits gastric cancer growth.
912 Early Angiogenic Changes Associated With Field Carcinogenesis in Experimental Colon Cancer Sarah Ruderman, Vesta Valuckaite, Anas Almoghrabi, John Hart, Hemant K. Roy, Marc Bissonnette, Vani J. Konda, Vadim Backman Background: Our group previously demonstrated that early changes in blood supply detected in the field may increase risk of colon neoplasia. Exploiting the renin angiotensin system (RAS) pathway could provide a mechanism to alter colonic blood supply and determine the effect on tumor initiation and progression. The goal of the present study was to gain a better understanding of the role of blood supply in early tumorigenesis. Methods: 60 male A/J mice were randomly assigned to one of 3 RAS conditions (n=20 mice per group): a) Water control to serve as the baseline comparison for the other groups; b) AngII injections (4mg / kg i.p.) and c) the ARB Losartan in drinking water (0.1 µg/kg/day). Tumors were induced in a subset of each condition with a standard protocol of 6 serial AOM injections (10mg/ kg of body weight i.p) and the remainder received saline (vehicle). We performed serial colonoscopy and assessments of microvascular changes using confocal laser endomicroscopy (CLE) with FITC-Dextran for in-vivo imaging of vessels and polarization-gated spectroscopy (PGS) to quantify rectal microvascular perfusion ([Hb]) and blood vessel radius (BVR). Results: The microvascular network of the Saline-Water baseline group remained constant throughout the study, both in vessel images and perfusion quantification. Among the AOMWater tumor bearing mice, CLE images revealed abnormalities in the pericryptal capillaries as early as 12 weeks post-AOM injection and prior to tumor formation. These abnormalities include classic angiogenic features, such as vessel dilation, loss of co-planarity, irregularity, and vessel sprouting. At later time points, these vessel characteristics were pronounced throughout the colon, including development of irregular networks. PGS quantification demonstrated an increase in rectal [Hb] as early as 12 weeks post-AOM injections (p = 0.04). The BVR decreased at these early time points, correlating with the vessel sprouting observed. The AOM-Losartan mice did not exhibit the same increase in [Hb] or decrease in vessel radius, indicating that Losartan could mitigate these early angiogenic changes. The AOM-AngII mice did not have any significant trends. Conclusion: In-vivo imaging of vessels and blood supply quantification reveal colonic field carcinogenesis through angiogenic changes prior to tumor formation. PGS is an effective, non-invasive method to assess rectal colonic blood supply as an indicator of field changes. The quantified PGS parameters of blood supply correlate with the in-vivo CLE images that demonstrated sprouting, dilation, and other distortions of the capillary network. The suppression of RAS modulation with Losartan demonstrated an effective way to alter colonic blood supply during tumorigenesis and can be used in future mechanistic studies to understand the early vessel changes observed.
911 A Novel Use of Angiotensin II Receptor Blocker (ARB) Losartan to Inhibit AOM Induced Tumorigenesis and Neoangiogenesis in Experimental Colon Cancer Vesta Valuckaite, Sarah Ruderman, Anas Almoghrabi, John Hart, Almaz Abdyrakov, Hemant K. Roy, Vadim Backman, Marc Bissonnette, Vani J. Konda Background: Colon cancer development is influenced by a number of factors including genetics, environment, and diet. The formation of a new vascular network from pre-existing blood vessels (neoangiogenesis) is required for the rapid growth and metastasis of solid tumors. The renin-angiotensin system (RAS) regulates blood vessel formation and flow. Angiotensin 1 receptors (AT1) control RAS signaling and receptor blockers (ATRB) such as losartan, a widely used antihypertensive, have been suggested to inhibit neoplastic angiogenesis. In this regard, recent studies have shown that losartan can decrease pancreatic and breast cancer progression, but its effects on colonic tumorigenesis have not been examined. The aim of this study was to characterize the VEGF-A changes and blood vessel effects of losartan in AOM induced and Western diet (WD) promoted colon cancer. Methods: For tumor studies, 40 male AJ mice were treated with azoxymethane (AOM) or PBS (AOM vehicle). Following 6 injections, mice were fed WD that included 20% mixed lipids and a subset received losartan (0.1 µg/kg/day) in the drinking water. Blood vessels were serially imaged in vivo with confocal laser endomicroscopy (CLE) and at sacrifice mice were injected with Dill (fluorescent lipophilic cationic indocarbocyanine dye which labels the endothelial membrane). Microvascular perfusion was quantified with polarized gated spectroscopy (PGS) (22-24 weeks after AOM). After sacrifice, colons were harvested and analyzed for VEGF-A by Western blotting (WB) and real time PCR (qPCR). Results: Among vehicle treated controls fed WD, losartan significantly down-regulated VEGF-A protein levels (1 vs. 0.13 fold difference, p<0.01). In AOM treated mice, losartan significantly suppressed tumor incidence (50% v.100%, p<0.05), tumor multiplicity (5.6 v. 3.0, p=0.07). Compared to AOM treatment alone, losartan concomitantly suppressed VEGF-A mRNA levels (3.7 versus 1.7 fold difference over controls, NS) and VEFG-A protein levels (1.2 versus 0.5 fold difference over controls, NS). AOM induced abnormalities including vessel dilation, loss of co-planarity, irregularity, and vessel sprouting as assessed by CLE and Dill staining. Losartan suppressed these derangements in vessel architecture induced by AOM. Preliminary PGS analysis of blood vessel radius correlates with the microvascular imaging and demonstrates that Losartan mitigates the effects of AOM during tumorigenesis. Conclusion: Angiotensin II receptor antagonist losartan inhibits AOM-induced tumorigenesis and suppresses derangements in blood vessel growth factors, architecture and flow. These results suggest that losartan might reduce colon cancer risk in the setting of Western diet consumption.
913 Significant Risk of Post-Colonoscopy Colorectal Cancer Due to Incomplete Adenoma Resection - Results of a Nation-Wide Population-Based Cohort Study Tim D. Belderbos, Hendrikus J. Pullens, Max Leenders, Marguerite E. Schipper, Martijn G. van Oijen, Peter D. Siersema Background & Aims Resection of adenomas detected during colonoscopy decreases the risk of subsequent colorectal cancer (CRC). It does however not preclude the occurrence of CRC within a few years after colonoscopy. Many of these post-colonoscopy CRCs (PCCRCs) are preventable, as they are thought to be due to missed or incompletely resected adenomas. The aim of this study was to determine the rate of PC-CRC due to incomplete adenoma resection and to identify adenoma characteristics associated with these PC-CRCs. Methods We performed a population-based cohort study identifying all patients with a first colorectal adenoma between 2000 and 2009 in PALGA, the nation-wide Dutch Pathology Registry. The primary outcome was the incidence rate of PC-CRC due to incomplete adenoma resection, which was defined as the occurrence of CRC between 6 months and 5 years after adenoma removal in the same colon segment (per adenoma analysis). Colon segments were divided into cecum, ascending, transverse and descending colon, sigmoid, recto-sigmoid and rectum. We performed a multivariable cox regression analysis to assess whether location, histology type, grade of dysplasia, number of synchronous adenomas and year of incidence were associated with PC-CRC due to incomplete adenoma resection. Results During the study period, 119,233 patients were diagnosed with a first adenoma. Mean age was 64.0 years (standard deviation 12.8) and 53.9% were male. We excluded 11,489 patients in whom prevalent CRC was found (CRC before or within 6 months after the first adenoma). Of the remaining 107,744 patients, 1543 (1.4%) developed CRC anywhere in the colon. PC-CRC due to incomplete adenoma resection occurred in 469 of 133,519 adenomas (0.4%, or 1 in 285 resected adenomas). Mean follow up per adenoma was 4.4 years (SD 1.1). The incidence rate of CRC due to incomplete adenoma resection was 0.81 per 1000 years of follow up. High-grade dysplasia and villous components were both risk factors for PC-CRC after incomplete adenoma removal (OR 2.48, 95% confidence interval (95%-CI) 1.60-3.83, and OR 1.66, 95%-CI 1.10-2.49, respectively). Adenomas removed between 2007 and 2009 were less likely to result in PC-CRC (OR 0.66, 95%-CI 0.52-0.83) than those removed in the period between 2000 and 2003. Proximal localization and adenoma multiplicity were not significantly associated with PC-CRC due to incomplete adenoma resection (OR for proximal localization 1.17, 95%-CI 0.96-1.43, OR for 2 adenomas 1.02, 95%-CI 0.72-1.46, and OR for ‡3 adenomas 1.14, 95%-CI 0.43-3.07, respectively). Conclusion In this nationwide cohort, post-colonoscopy CRC due to incomplete endoscopic resection occurred in more than one in three hundred adenomas. Our results suggest that enhanced surveillance is indicated after removal of adenomas with high grade dysplasia or villous components.
Figure 1. Ex-vivo confocal microscopy with Dil staining of vasculature demonstrates a regular honeycomb pattern in normal colon from control mice (A) and similar pattern in control mice given Losaratan (B). In contrast, imaging of tumors in mice treated with AOM demonstrate ectatic vessels and irregular network with extravasation (C). Losartan mitigated these
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vascular changes as shown in the tumor of a mouse treated with AOM and given Losartan (D). In vivo imaging with confocal laser endomicroscopy demonstrated similar changes in AOM induced tumors with (E) and without (F) Losartan.
0.0004, R=0.6774), indicating that the production of PGE2 is associated with DNMT3B expression in vivo. Moreover, methylation array analysis of vehicle- and PGE2-treated GES1, HGC27 and MKN1 cells revealed that PGE2 induced the promoter hypermethylation of 102 genes, including tumor suppressor genes that are associated with gastric cancer. Hypermethylation genes induced by PGE2 were enriched in two signaling pathways (cell cycle regulation and DNA helicase activity). In particular, CAB39L, transcriptionally silenced by PGE2induced promoter methylation, functions as a tumor suppressor gene in gastric cancer. Combinatorial treatment with COX-2 (celecoxib) and DNMT (5-aza-2'-deoxycytidine) inhibitors synergistically suppressed the growth of gastric cancer cells in vitro and HGC27 xenografts in nude mice. CONCLUSIONS: PGE2 mediates epigenetic dysregulation in gastric cancer via induction of DNMT3B activity and consequent DNA promoter hypermethylation of tumor suppressor genes. Co-targeting of PGE2 and DNMT synergistically inhibits gastric cancer growth.
912 Early Angiogenic Changes Associated With Field Carcinogenesis in Experimental Colon Cancer Sarah Ruderman, Vesta Valuckaite, Anas Almoghrabi, John Hart, Hemant K. Roy, Marc Bissonnette, Vani J. Konda, Vadim Backman Background: Our group previously demonstrated that early changes in blood supply detected in the field may increase risk of colon neoplasia. Exploiting the renin angiotensin system (RAS) pathway could provide a mechanism to alter colonic blood supply and determine the effect on tumor initiation and progression. The goal of the present study was to gain a better understanding of the role of blood supply in early tumorigenesis. Methods: 60 male A/J mice were randomly assigned to one of 3 RAS conditions (n=20 mice per group): a) Water control to serve as the baseline comparison for the other groups; b) AngII injections (4mg / kg i.p.) and c) the ARB Losartan in drinking water (0.1 µg/kg/day). Tumors were induced in a subset of each condition with a standard protocol of 6 serial AOM injections (10mg/ kg of body weight i.p) and the remainder received saline (vehicle). We performed serial colonoscopy and assessments of microvascular changes using confocal laser endomicroscopy (CLE) with FITC-Dextran for in-vivo imaging of vessels and polarization-gated spectroscopy (PGS) to quantify rectal microvascular perfusion ([Hb]) and blood vessel radius (BVR). Results: The microvascular network of the Saline-Water baseline group remained constant throughout the study, both in vessel images and perfusion quantification. Among the AOMWater tumor bearing mice, CLE images revealed abnormalities in the pericryptal capillaries as early as 12 weeks post-AOM injection and prior to tumor formation. These abnormalities include classic angiogenic features, such as vessel dilation, loss of co-planarity, irregularity, and vessel sprouting. At later time points, these vessel characteristics were pronounced throughout the colon, including development of irregular networks. PGS quantification demonstrated an increase in rectal [Hb] as early as 12 weeks post-AOM injections (p = 0.04). The BVR decreased at these early time points, correlating with the vessel sprouting observed. The AOM-Losartan mice did not exhibit the same increase in [Hb] or decrease in vessel radius, indicating that Losartan could mitigate these early angiogenic changes. The AOM-AngII mice did not have any significant trends. Conclusion: In-vivo imaging of vessels and blood supply quantification reveal colonic field carcinogenesis through angiogenic changes prior to tumor formation. PGS is an effective, non-invasive method to assess rectal colonic blood supply as an indicator of field changes. The quantified PGS parameters of blood supply correlate with the in-vivo CLE images that demonstrated sprouting, dilation, and other distortions of the capillary network. The suppression of RAS modulation with Losartan demonstrated an effective way to alter colonic blood supply during tumorigenesis and can be used in future mechanistic studies to understand the early vessel changes observed.
911 A Novel Use of Angiotensin II Receptor Blocker (ARB) Losartan to Inhibit AOM Induced Tumorigenesis and Neoangiogenesis in Experimental Colon Cancer Vesta Valuckaite, Sarah Ruderman, Anas Almoghrabi, John Hart, Almaz Abdyrakov, Hemant K. Roy, Vadim Backman, Marc Bissonnette, Vani J. Konda Background: Colon cancer development is influenced by a number of factors including genetics, environment, and diet. The formation of a new vascular network from pre-existing blood vessels (neoangiogenesis) is required for the rapid growth and metastasis of solid tumors. The renin-angiotensin system (RAS) regulates blood vessel formation and flow. Angiotensin 1 receptors (AT1) control RAS signaling and receptor blockers (ATRB) such as losartan, a widely used antihypertensive, have been suggested to inhibit neoplastic angiogenesis. In this regard, recent studies have shown that losartan can decrease pancreatic and breast cancer progression, but its effects on colonic tumorigenesis have not been examined. The aim of this study was to characterize the VEGF-A changes and blood vessel effects of losartan in AOM induced and Western diet (WD) promoted colon cancer. Methods: For tumor studies, 40 male AJ mice were treated with azoxymethane (AOM) or PBS (AOM vehicle). Following 6 injections, mice were fed WD that included 20% mixed lipids and a subset received losartan (0.1 µg/kg/day) in the drinking water. Blood vessels were serially imaged in vivo with confocal laser endomicroscopy (CLE) and at sacrifice mice were injected with Dill (fluorescent lipophilic cationic indocarbocyanine dye which labels the endothelial membrane). Microvascular perfusion was quantified with polarized gated spectroscopy (PGS) (22-24 weeks after AOM). After sacrifice, colons were harvested and analyzed for VEGF-A by Western blotting (WB) and real time PCR (qPCR). Results: Among vehicle treated controls fed WD, losartan significantly down-regulated VEGF-A protein levels (1 vs. 0.13 fold difference, p<0.01). In AOM treated mice, losartan significantly suppressed tumor incidence (50% v.100%, p<0.05), tumor multiplicity (5.6 v. 3.0, p=0.07). Compared to AOM treatment alone, losartan concomitantly suppressed VEGF-A mRNA levels (3.7 versus 1.7 fold difference over controls, NS) and VEFG-A protein levels (1.2 versus 0.5 fold difference over controls, NS). AOM induced abnormalities including vessel dilation, loss of co-planarity, irregularity, and vessel sprouting as assessed by CLE and Dill staining. Losartan suppressed these derangements in vessel architecture induced by AOM. Preliminary PGS analysis of blood vessel radius correlates with the microvascular imaging and demonstrates that Losartan mitigates the effects of AOM during tumorigenesis. Conclusion: Angiotensin II receptor antagonist losartan inhibits AOM-induced tumorigenesis and suppresses derangements in blood vessel growth factors, architecture and flow. These results suggest that losartan might reduce colon cancer risk in the setting of Western diet consumption.
913 Significant Risk of Post-Colonoscopy Colorectal Cancer Due to Incomplete Adenoma Resection - Results of a Nation-Wide Population-Based Cohort Study Tim D. Belderbos, Hendrikus J. Pullens, Max Leenders, Marguerite E. Schipper, Martijn G. van Oijen, Peter D. Siersema Background & Aims Resection of adenomas detected during colonoscopy decreases the risk of subsequent colorectal cancer (CRC). It does however not preclude the occurrence of CRC within a few years after colonoscopy. Many of these post-colonoscopy CRCs (PCCRCs) are preventable, as they are thought to be due to missed or incompletely resected adenomas. The aim of this study was to determine the rate of PC-CRC due to incomplete adenoma resection and to identify adenoma characteristics associated with these PC-CRCs. Methods We performed a population-based cohort study identifying all patients with a first colorectal adenoma between 2000 and 2009 in PALGA, the nation-wide Dutch Pathology Registry. The primary outcome was the incidence rate of PC-CRC due to incomplete adenoma resection, which was defined as the occurrence of CRC between 6 months and 5 years after adenoma removal in the same colon segment (per adenoma analysis). Colon segments were divided into cecum, ascending, transverse and descending colon, sigmoid, recto-sigmoid and rectum. We performed a multivariable cox regression analysis to assess whether location, histology type, grade of dysplasia, number of synchronous adenomas and year of incidence were associated with PC-CRC due to incomplete adenoma resection. Results During the study period, 119,233 patients were diagnosed with a first adenoma. Mean age was 64.0 years (standard deviation 12.8) and 53.9% were male. We excluded 11,489 patients in whom prevalent CRC was found (CRC before or within 6 months after the first adenoma). Of the remaining 107,744 patients, 1543 (1.4%) developed CRC anywhere in the colon. PC-CRC due to incomplete adenoma resection occurred in 469 of 133,519 adenomas (0.4%, or 1 in 285 resected adenomas). Mean follow up per adenoma was 4.4 years (SD 1.1). The incidence rate of CRC due to incomplete adenoma resection was 0.81 per 1000 years of follow up. High-grade dysplasia and villous components were both risk factors for PC-CRC after incomplete adenoma removal (OR 2.48, 95% confidence interval (95%-CI) 1.60-3.83, and OR 1.66, 95%-CI 1.10-2.49, respectively). Adenomas removed between 2007 and 2009 were less likely to result in PC-CRC (OR 0.66, 95%-CI 0.52-0.83) than those removed in the period between 2000 and 2003. Proximal localization and adenoma multiplicity were not significantly associated with PC-CRC due to incomplete adenoma resection (OR for proximal localization 1.17, 95%-CI 0.96-1.43, OR for 2 adenomas 1.02, 95%-CI 0.72-1.46, and OR for ‡3 adenomas 1.14, 95%-CI 0.43-3.07, respectively). Conclusion In this nationwide cohort, post-colonoscopy CRC due to incomplete endoscopic resection occurred in more than one in three hundred adenomas. Our results suggest that enhanced surveillance is indicated after removal of adenomas with high grade dysplasia or villous components.
Figure 1. Ex-vivo confocal microscopy with Dil staining of vasculature demonstrates a regular honeycomb pattern in normal colon from control mice (A) and similar pattern in control mice given Losaratan (B). In contrast, imaging of tumors in mice treated with AOM demonstrate ectatic vessels and irregular network with extravasation (C). Losartan mitigated these
AGA Abstracts
X : 55624$1AGA 03-28-15 04:55:56 PDFd : 55624B : e
S-172
Page 172