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S2098 Mechanisms Underlying the Beneficial Synergistic Effects of Phytochemicals On Pancreatic Cancer
Specific dietary modifications
S2098
AGA Abstracts
Mechanisms Underlying the Beneficial Synergistic Effects of Phytochemicals On Pancreatic Cancer Mouad Edderkaoui, Ling Zheng, Hongxiang Hui, Gang Li, Vay L. Go, Stephen J. Pandol Background & Aims: Pancreatic cancer is a very aggressive disease that is resistant to therapy. Evidence from population studies indicates a protective effect of fruits and vegetables in the diet on cancer including pancreatic cancer. In Vitro and In Vivo studies, including our own, suggest that several phytochemicals, such as ellagic acid, curcumin, rottlerin, embelin and lycopene may be involved in this beneficial effect. In this study we investigated the effect of selected phytochemicals, alone and in combination, on apoptosis and proliferation of pancreatic cancer cells In Vitro, and we analyzed their mechanism of action. Methods: We measured the effect of five phytochemicals: ellagic acid, curcumin, embelin, rottlerin and lycopene on DNA fragmentation using ELISA and on proliferation using thymidine incorporation in DNA in two pancreatic cancer cell lines MIA PaCa-2 and PANC-1. Effects of the compounds on p-STAT3, XIAP and survivin protein levels were measured using Western. Interaction of Bcl-xL with PUMA and Bim was analyzed by immuno-precipitation. NF-kB activity was measured using electromobility shift assay. Results: All five compounds, at different doses (0.1 to 30 uM), induced increase in DNA fragmentation and inhibited proliferation in pancreatic cancer cells. Combinations of rottlerin with curcumin or with embelin induced a synergistic effect on DNA fragmentation and proliferation. Combinations of rottlerin and ellagic acid induced a synergistic effect on proliferation alone. Ellagic acid induced apoptosis by decreasing NF-kB activity in pancreatic cancer cells. Differently, embelin decreased XIAP and survivin through a pathway involving STAT3 phosphorylation; whereas the pro-apoptotic effect of rottlerin was through decreasing the formation of the complexes Bcl-xL/PUMA and Bcl-xL/Bim. Thus, the three compounds inhibit different anti-apoptotic pathways which explains the synergistic effect of their combinations. Conclusions: Combination of the phytochemicals studied induced a synergistic effect on apoptosis and proliferation in pancreatic cancer cells. The synergistic effect is due to different mechanisms of action of these phytochemicals. Our study suggests combination of different phytochemicals for prevention and treatment strategies of pancreatic cancer.
* Modifications listed in NICE guidelines ** Fermentable Oligo, Mono and Di-saccharides and Polyols S2096 Nitrosative Stress Within Lipids Is Not Inhibited By Beta-Carotene or Vitamin E Emilie Combet, Hirotsugu Watabe, Tom Preston, Alan Crozier, Kenneth E. McColl INTRODUCTION: At the gastro-oesophageal junction, high concentration of nitrite in human saliva, derived from dietary nitrate, is converted to nitrous acid and potentially mutagenic nitrosative species on encountering the low pH of gastric juice. Vitamin C in gastric juice provides protection against nitrosation by reducing the nitrosating species to nitric oxide. However, we have recently demonstrated that the nitric oxide formed in this way may diffuse into adjacent lipids, such as luminal fat, and within it reacts with molecular oxygen to reform nitrosating species and carcinogenic N-nitrosocompounds (1). Vitamin C thus promotes nitrosation within adjacent lipid compartments. AIM & METHODS: The aim of this study was to investigate whether lipid soluble antioxidants (α-tocopherols and βcarotene) can protect against nitric oxide-induced nitrosation within lipid compartments. We employed a benchtop model of the gastro-oesophageal junction containing sodium thiocyanate and secondary amines (markers of nitrosation) in which physiological amounts of salivary nitrite were added to acidic gastric juice containing Vitamin C and incubated at 37°C. The studies were conducted with and without lipid in the simulated gastric environment, with and without Vitamin C in the aqueous compartment and with and without the lipid anti-oxidants in the lipid compartment. After 15 minutes, aliquots from each phase were sampled and N-nitrosamine levels measured by gas chromatography-tandem mass spectrometry. RESULTS: In the absence of lipid, the Vitamin C inhibited the formation of N-nitrosoamines (130±14.2 nmoles without vs 10.1±2.3 nmoles with, p<0.001) but in the presence of lipid it promoted it (281.4±22.8 nmoles without vs 904.9±114.9 nmoles with, p<0.001). Addition of β-carotene and α-tocopherol (in concentrations ranging from 10μM to 100 mM) to the lipid compartment in experiments with Vitamin C present failed to inhibit the nitrosative stress, except at extremely high non-physiological concentrations of >100mM. CONCLUSION: Potent lipid antioxidants are unable to prevent the generation of nitrosating species and N-nitrosocompounds from the high level of nitric oxide generated at the gastro-oesophageal junction and entering adjacent lipid compartments. This may be explained by them being unable to scavenge the nitrosating species N2O3 formed by the reaction of nitric oxide with oxygen in lipid spaces. 1. Combet, E et al. Gut 2007, 56, 1678-1684.
S2099 Guggulsterone Inhibits Growth of the Human AsPc-1 Pancreatic Cancer Cell Line via Cleavage of Mcl-1 William L. Berry, Stephen M. Clayton, Rama P. Ramanujam, Courtney W. Houchen, Shrikant Anant Aim. To determine the mechanism of growth inhibition following guggulsterone [4,17(20)(cis)-pregnadiene-3,16-dione] treatment. Background. Pancreatic cancer is fourth among cancers resulting in mortality in the United States. Furthermore, there is a 5-year survival of less than 5% and there is currently no effective treatment for this devastating disease. Therefore, it is imperative that we investigate natural compounds that might be used as an effective treatment. Guggulsterone is a natural component that resides in the gum resin of the Commiphora mukul and has been used as an Indian Ayurvedic medicine for thousands of years. Myeloid cell leukemia-1 (Mcl-1) is a Bcl-2 family member that promotes cell survival. Induction of caspases can lead to Mcl-1 cleavage, thereby promoting growth inhibition. There are a sundry of mechanisms that could account for these changes, but it would be expected that different cancer cells would behave differently based on their genetic background. Methods. AsPc-1 pancreatic cancer cell line was treated with guggulsterone. Cell proliferation was determined by the hexosaminidase assay. Cell cycle analysis was performed by fluorescence activated cell sorting (FACS). Gene expression was determined by Real-time PCR and protein expression by Western blot. Results. The guggulsterone treated pancreatic cancer cell line AsPc-1 exhibited a loss in proliferating cells in a dose-dependent (0-40 μM) and time-dependent (0-48 h) manner. In flow cytometry studies, we did not observe a change in cell cycle progression at 24 h following treatment. However, we did observe an increase in the subdiploid region. Real-time PCR analyses indicated changes in the Bcl-2 family of apoptotic and anti-apoptotic genes. This resulted in no changes in the Bcl-2/Bax ratio. However, there was a significant elevation in Mcl-1 gene expression at 20 μM of guggulsterone, and a further increase at 40 μM. Western blot analyses confirmed that total Mcl1 protein levels had increased in response to guggulsterone treatment. Interestingly, however, there was also a significant increase in cleavage of Mcl-1 in the guggulsterone treated cells. In addition, activated caspase-3 protein expression increased in a dose-dependent manner. Conclusion. Guggulsterone treatment causes growth inhibition in the human AsPc-1 pancreatic cancer cell line. This inhibition of growth is due in part to the cleavage of the antiapoptotic Mcl-1 protein by activated caspase-3.
S2097 Inhibition of Acid-Catalysed Nitrosation: Superiority of Specific Dietary Phenolics in Both Presence and Absence of Lipid Emilie Combet, Aziza Elmesmari, Hirotsugu Watabe, Tom Preston, Alan Crozier, Kenneth E. McColl INTRODUCTION: Acid-catalysed nitrosation is focused at the gastro-oesophageal junction where salivary nitrite, derived from dietary nitrate, encounters the acidic gastric juice to produce potentially carcinogenic nitrosative species. This reaction is inhibited by Vitamin C, which converts nitrosative species to nitric oxide (NO). However, in presence of fat, NO concentrates in the lipid, where it reacts with O2 to reform nitrosative species (1). In this way, Vitamin C can promote acid nitrosation. AIM & METHODS: The aim of this study was to determine whether a range of water-soluble antioxidants other than Vitamin C could inhibit nitrosation in both absence and presence of lipid. Phenolics (caffeic, ferulic, gallic or chlorogenic acids, 2mM each) were added individually to simulated gastric juice containing sodium thiocyanate and secondary amines (markers of nitrosation), with or without lipid. Negative (no antioxidant) and positive control reactions (ascorbic acid, 2mM) were run in parallel experiments. Addition of nitrite (100μM) triggered the reaction (37°C, 15 minutes). NO and O2 levels were monitored by electrochemical detection. NOC were measured in both aqueous and lipid phases by gas chromatography-tandem mass spectrometry. RESULTS: Nitrite conversion to NO was high with ascorbic acid (62.7+1.2μM) and varied with the phenolic acids: 21.9±2.2μM with chlorogenic acid, 20.1±1.1μM with gallic acid, 0.9±0.1μM with ferulic acid and 1.44±0.1μM with caffeic acid. In absence of lipid, inhibition of nitrosation was 35.9±7.4% by gallic acid, 56.3±6.2% by chlorogenic acid, 86.6±5.6% by caffeic acid, 93+0.6% by ferulic acid and 92.8+0.8% by ascorbic acid. In the presence of lipids, the impact of each antioxidant on nitrosation was inversely correlated to the levels of NO they generated (r2=0.95, p<0.01). Compared to the negative control (100% nitrosation), gallic, chlorogenic and ascorbic acid promoted nitrosation (111.5±5.9%, 184±5.9% and 321.5±40.8%, respectively), while ferulic and caffeic acids markedly inhibited nitrosation (13.3±1.9% and 17.2±2%, respectively). CONCLUSION: Ferulic and caffeic acids, present in coffee and various fruit and vegetables, are highly efficient at inhibiting nitrosation in presence or absence of lipid in a simulated gastric environment. This can be explained by their ability to inhibit nitrosation without generating a burst of NO, which otherwise reforms nitrosating species within the lipid. 1. Combet, E et al. Gut 2007, 56, 1678-1684.
AGA Abstracts
M1012 Can a 6 Minute Withdrawal Time Be Achieved in a Large Community Practice Partnership? Jeffrey M. Rank, John I. Allen, Linda Rudquist Six minute or greater withdrawal time (WT) for colonoscopy is a quality indicator for colonoscopy. Minnesota Gastroenterology is a community-based GI group with 50 BC gastroenterologists performing >30,000 colonoscopies yearly. Because of documented individual adenoma find rate (AFR) variation within our group, we implemented a program to increase individual >6 minute WT. A 4-step program included discussion and education (Q1-2 2007), measurement of individual data (Q3 2007), reporting individual data (Q4 2007), and a pay for performance (P4P) parameter requiring 95% > 6 minute WT. WT is defined by passage beyond ileo-cecal valve to extubation, as recorded by endoscopy nurse. From Q3 2007- q2 2008, we noted an increase from 72% average >6 minute WT to 96% (Table 1) (Table 1) mean > 6 minute Table 2 adenoma find rates withdrawal time Q2 2007Q2 2008 Q2 2007-Q2 2008 The p-value associated with the Friedman test for 6 minute withdrawal percentages Q3 2007 to Q2 2008 was < 0.0001. As WT compliance improved, there was an increase in adenoma find rate (Table 2). The p-value did not reach significance for male AFR, but reached 0.0001 in the female AFR by the Friedman test. Conclusion: Our
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S2098
AGA Abstracts
Mechanisms Underlying the Beneficial Synergistic Effects of Phytochemicals On Pancreatic Cancer Mouad Edderkaoui, Ling Zheng, Hongxiang Hui, Gang Li, Vay L. Go, Stephen J. Pandol Background & Aims: Pancreatic cancer is a very aggressive disease that is resistant to therapy. Evidence from population studies indicates a protective effect of fruits and vegetables in the diet on cancer including pancreatic cancer. In Vitro and In Vivo studies, including our own, suggest that several phytochemicals, such as ellagic acid, curcumin, rottlerin, embelin and lycopene may be involved in this beneficial effect. In this study we investigated the effect of selected phytochemicals, alone and in combination, on apoptosis and proliferation of pancreatic cancer cells In Vitro, and we analyzed their mechanism of action. Methods: We measured the effect of five phytochemicals: ellagic acid, curcumin, embelin, rottlerin and lycopene on DNA fragmentation using ELISA and on proliferation using thymidine incorporation in DNA in two pancreatic cancer cell lines MIA PaCa-2 and PANC-1. Effects of the compounds on p-STAT3, XIAP and survivin protein levels were measured using Western. Interaction of Bcl-xL with PUMA and Bim was analyzed by immuno-precipitation. NF-kB activity was measured using electromobility shift assay. Results: All five compounds, at different doses (0.1 to 30 uM), induced increase in DNA fragmentation and inhibited proliferation in pancreatic cancer cells. Combinations of rottlerin with curcumin or with embelin induced a synergistic effect on DNA fragmentation and proliferation. Combinations of rottlerin and ellagic acid induced a synergistic effect on proliferation alone. Ellagic acid induced apoptosis by decreasing NF-kB activity in pancreatic cancer cells. Differently, embelin decreased XIAP and survivin through a pathway involving STAT3 phosphorylation; whereas the pro-apoptotic effect of rottlerin was through decreasing the formation of the complexes Bcl-xL/PUMA and Bcl-xL/Bim. Thus, the three compounds inhibit different anti-apoptotic pathways which explains the synergistic effect of their combinations. Conclusions: Combination of the phytochemicals studied induced a synergistic effect on apoptosis and proliferation in pancreatic cancer cells. The synergistic effect is due to different mechanisms of action of these phytochemicals. Our study suggests combination of different phytochemicals for prevention and treatment strategies of pancreatic cancer.
* Modifications listed in NICE guidelines ** Fermentable Oligo, Mono and Di-saccharides and Polyols S2096 Nitrosative Stress Within Lipids Is Not Inhibited By Beta-Carotene or Vitamin E Emilie Combet, Hirotsugu Watabe, Tom Preston, Alan Crozier, Kenneth E. McColl INTRODUCTION: At the gastro-oesophageal junction, high concentration of nitrite in human saliva, derived from dietary nitrate, is converted to nitrous acid and potentially mutagenic nitrosative species on encountering the low pH of gastric juice. Vitamin C in gastric juice provides protection against nitrosation by reducing the nitrosating species to nitric oxide. However, we have recently demonstrated that the nitric oxide formed in this way may diffuse into adjacent lipids, such as luminal fat, and within it reacts with molecular oxygen to reform nitrosating species and carcinogenic N-nitrosocompounds (1). Vitamin C thus promotes nitrosation within adjacent lipid compartments. AIM & METHODS: The aim of this study was to investigate whether lipid soluble antioxidants (α-tocopherols and βcarotene) can protect against nitric oxide-induced nitrosation within lipid compartments. We employed a benchtop model of the gastro-oesophageal junction containing sodium thiocyanate and secondary amines (markers of nitrosation) in which physiological amounts of salivary nitrite were added to acidic gastric juice containing Vitamin C and incubated at 37°C. The studies were conducted with and without lipid in the simulated gastric environment, with and without Vitamin C in the aqueous compartment and with and without the lipid anti-oxidants in the lipid compartment. After 15 minutes, aliquots from each phase were sampled and N-nitrosamine levels measured by gas chromatography-tandem mass spectrometry. RESULTS: In the absence of lipid, the Vitamin C inhibited the formation of N-nitrosoamines (130±14.2 nmoles without vs 10.1±2.3 nmoles with, p<0.001) but in the presence of lipid it promoted it (281.4±22.8 nmoles without vs 904.9±114.9 nmoles with, p<0.001). Addition of β-carotene and α-tocopherol (in concentrations ranging from 10μM to 100 mM) to the lipid compartment in experiments with Vitamin C present failed to inhibit the nitrosative stress, except at extremely high non-physiological concentrations of >100mM. CONCLUSION: Potent lipid antioxidants are unable to prevent the generation of nitrosating species and N-nitrosocompounds from the high level of nitric oxide generated at the gastro-oesophageal junction and entering adjacent lipid compartments. This may be explained by them being unable to scavenge the nitrosating species N2O3 formed by the reaction of nitric oxide with oxygen in lipid spaces. 1. Combet, E et al. Gut 2007, 56, 1678-1684.
S2099 Guggulsterone Inhibits Growth of the Human AsPc-1 Pancreatic Cancer Cell Line via Cleavage of Mcl-1 William L. Berry, Stephen M. Clayton, Rama P. Ramanujam, Courtney W. Houchen, Shrikant Anant Aim. To determine the mechanism of growth inhibition following guggulsterone [4,17(20)(cis)-pregnadiene-3,16-dione] treatment. Background. Pancreatic cancer is fourth among cancers resulting in mortality in the United States. Furthermore, there is a 5-year survival of less than 5% and there is currently no effective treatment for this devastating disease. Therefore, it is imperative that we investigate natural compounds that might be used as an effective treatment. Guggulsterone is a natural component that resides in the gum resin of the Commiphora mukul and has been used as an Indian Ayurvedic medicine for thousands of years. Myeloid cell leukemia-1 (Mcl-1) is a Bcl-2 family member that promotes cell survival. Induction of caspases can lead to Mcl-1 cleavage, thereby promoting growth inhibition. There are a sundry of mechanisms that could account for these changes, but it would be expected that different cancer cells would behave differently based on their genetic background. Methods. AsPc-1 pancreatic cancer cell line was treated with guggulsterone. Cell proliferation was determined by the hexosaminidase assay. Cell cycle analysis was performed by fluorescence activated cell sorting (FACS). Gene expression was determined by Real-time PCR and protein expression by Western blot. Results. The guggulsterone treated pancreatic cancer cell line AsPc-1 exhibited a loss in proliferating cells in a dose-dependent (0-40 μM) and time-dependent (0-48 h) manner. In flow cytometry studies, we did not observe a change in cell cycle progression at 24 h following treatment. However, we did observe an increase in the subdiploid region. Real-time PCR analyses indicated changes in the Bcl-2 family of apoptotic and anti-apoptotic genes. This resulted in no changes in the Bcl-2/Bax ratio. However, there was a significant elevation in Mcl-1 gene expression at 20 μM of guggulsterone, and a further increase at 40 μM. Western blot analyses confirmed that total Mcl1 protein levels had increased in response to guggulsterone treatment. Interestingly, however, there was also a significant increase in cleavage of Mcl-1 in the guggulsterone treated cells. In addition, activated caspase-3 protein expression increased in a dose-dependent manner. Conclusion. Guggulsterone treatment causes growth inhibition in the human AsPc-1 pancreatic cancer cell line. This inhibition of growth is due in part to the cleavage of the antiapoptotic Mcl-1 protein by activated caspase-3.
S2097 Inhibition of Acid-Catalysed Nitrosation: Superiority of Specific Dietary Phenolics in Both Presence and Absence of Lipid Emilie Combet, Aziza Elmesmari, Hirotsugu Watabe, Tom Preston, Alan Crozier, Kenneth E. McColl INTRODUCTION: Acid-catalysed nitrosation is focused at the gastro-oesophageal junction where salivary nitrite, derived from dietary nitrate, encounters the acidic gastric juice to produce potentially carcinogenic nitrosative species. This reaction is inhibited by Vitamin C, which converts nitrosative species to nitric oxide (NO). However, in presence of fat, NO concentrates in the lipid, where it reacts with O2 to reform nitrosative species (1). In this way, Vitamin C can promote acid nitrosation. AIM & METHODS: The aim of this study was to determine whether a range of water-soluble antioxidants other than Vitamin C could inhibit nitrosation in both absence and presence of lipid. Phenolics (caffeic, ferulic, gallic or chlorogenic acids, 2mM each) were added individually to simulated gastric juice containing sodium thiocyanate and secondary amines (markers of nitrosation), with or without lipid. Negative (no antioxidant) and positive control reactions (ascorbic acid, 2mM) were run in parallel experiments. Addition of nitrite (100μM) triggered the reaction (37°C, 15 minutes). NO and O2 levels were monitored by electrochemical detection. NOC were measured in both aqueous and lipid phases by gas chromatography-tandem mass spectrometry. RESULTS: Nitrite conversion to NO was high with ascorbic acid (62.7+1.2μM) and varied with the phenolic acids: 21.9±2.2μM with chlorogenic acid, 20.1±1.1μM with gallic acid, 0.9±0.1μM with ferulic acid and 1.44±0.1μM with caffeic acid. In absence of lipid, inhibition of nitrosation was 35.9±7.4% by gallic acid, 56.3±6.2% by chlorogenic acid, 86.6±5.6% by caffeic acid, 93+0.6% by ferulic acid and 92.8+0.8% by ascorbic acid. In the presence of lipids, the impact of each antioxidant on nitrosation was inversely correlated to the levels of NO they generated (r2=0.95, p<0.01). Compared to the negative control (100% nitrosation), gallic, chlorogenic and ascorbic acid promoted nitrosation (111.5±5.9%, 184±5.9% and 321.5±40.8%, respectively), while ferulic and caffeic acids markedly inhibited nitrosation (13.3±1.9% and 17.2±2%, respectively). CONCLUSION: Ferulic and caffeic acids, present in coffee and various fruit and vegetables, are highly efficient at inhibiting nitrosation in presence or absence of lipid in a simulated gastric environment. This can be explained by their ability to inhibit nitrosation without generating a burst of NO, which otherwise reforms nitrosating species within the lipid. 1. Combet, E et al. Gut 2007, 56, 1678-1684.
AGA Abstracts
M1012 Can a 6 Minute Withdrawal Time Be Achieved in a Large Community Practice Partnership? Jeffrey M. Rank, John I. Allen, Linda Rudquist Six minute or greater withdrawal time (WT) for colonoscopy is a quality indicator for colonoscopy. Minnesota Gastroenterology is a community-based GI group with 50 BC gastroenterologists performing >30,000 colonoscopies yearly. Because of documented individual adenoma find rate (AFR) variation within our group, we implemented a program to increase individual >6 minute WT. A 4-step program included discussion and education (Q1-2 2007), measurement of individual data (Q3 2007), reporting individual data (Q4 2007), and a pay for performance (P4P) parameter requiring 95% > 6 minute WT. WT is defined by passage beyond ileo-cecal valve to extubation, as recorded by endoscopy nurse. From Q3 2007- q2 2008, we noted an increase from 72% average >6 minute WT to 96% (Table 1) (Table 1) mean > 6 minute Table 2 adenoma find rates withdrawal time Q2 2007Q2 2008 Q2 2007-Q2 2008 The p-value associated with the Friedman test for 6 minute withdrawal percentages Q3 2007 to Q2 2008 was < 0.0001. As WT compliance improved, there was an increase in adenoma find rate (Table 2). The p-value did not reach significance for male AFR, but reached 0.0001 in the female AFR by the Friedman test. Conclusion: Our
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