Toxicological aspects of bile acids and human fecal water on cultured human colon carcinoma cells
Author: Glinghammar, Björn
Date: 2001-06-01
Location: Hörsalen, plan 4, Novum
Time: 13.00
Department: Institutionen för medicinsk näringslära / Department of Medical Nutrition
Abstract
Colorectal tumorigenesis involves activation of mutations in
proto-oncogenes, such as ras and c-myc, as well as mutations that
inactivate tumor suppressor genes including the APC and the p53 gene.
These genetic events, in turn, lead to epigenetic changes in signal
transduction pathways, which regulate processes such as cell
proliferation, differentiation and apoptosis. There exists also a large
body of evidence from animal carcinogenesis studies and human
epidemiology that a high fat intake drastically affects tumor incidence
in the colon. However, there has been little understanding of how the
dietary factors and genetic/epigentic events interact. It is, generally
believed that this interaction is at least in part mediated by events
occurring in the lumen of the large bowel. Bile acids have been suggested
to mediate the tumor promoter effect of a high fat diet. The mechanism of
bile acid induced tumor promoter activity is poorly understood.
The aim of this thesis was to understand more about bile acids cellular effects, in the context of their tumor promoter activity, and to study the mechanisms behind the observed effects. By using cultured human colonic cell lines, we showed that dihydroxy bile acids induced the transcription factor AP-1, while other luminal components like cholesterol and long-chain fatty acids were without effect. The bile acid, deoxycholic acid (DCA) induced cell proliferation at the same concentrations as it induced AP-1. Incubation of the extracellular fluid of feces (human fecal water), which is the stool water in contact with the epithelial cells in vivo, resulted in activation of AP-1 and induction of cell proliferation which varied between different samples. Our attention then moved to, COX-2, another early response gene which has been shown to play an important role in colon tumorigenesis. We showed that dihydroxy bile acids and human fecal waters induced COX-2 promoter activity. This resulted in an increase in COX-2 protein expression. Interestingly, the concentrations of DCA that induced highest COX- 2 expression were cytotoxic. This led us to further studies on cell death induced by bile acids. We showed that bile acids induced apoptosis in the colonic cells, in a dose and time dependent fashion. Many substances which induce apoptosis are DNA damaging agents, which led us to test if DCA induced DNA damage. We observed that shortly after exposure of cells to DCA, damage to DNA occurred. The result of this damage is activation of the apoptosis program, which results for most cells in death. However, the few cells surviving the induced apoptosis showed low levels of caspase-3 and increased activity of NF-kB and AP-1 driven reporters, as well as an increase in COX-2 promoter activity. Mapping the region of the COX-2 promoter responsible for the effect, showed that both CRE and NF-kb as well as to some extent C/EBP elements are needed for full activation of the COX-2 promoter by DCA. In a follow up study, we characterized further the genotoxic effect of bile acids, and observed dose-dependent increase of DNA damage from DCA and lithocholic acid at concentrations above 300 µM. Testing 35 human fecal water samples, showed that 30% had very high potential to induce damage in intact cells while 50% had no effect. Finally, in a human dietary intervention study, we shifted the diet of healthy volunteers from a dairy product rich to a dairy product free diet, and monitored cytotoxicity and genotoxicity of their fecal waters. The result showed that when participants went off dairy products, the cytotoxicity of their fecal waters increased significantly, while there was no change observed for the genotoxicity of fecal waters. In summary, our results have contributed to the understanding of the molecular mechanisms by which bile acids influence tumorigenesis in the colon.
The aim of this thesis was to understand more about bile acids cellular effects, in the context of their tumor promoter activity, and to study the mechanisms behind the observed effects. By using cultured human colonic cell lines, we showed that dihydroxy bile acids induced the transcription factor AP-1, while other luminal components like cholesterol and long-chain fatty acids were without effect. The bile acid, deoxycholic acid (DCA) induced cell proliferation at the same concentrations as it induced AP-1. Incubation of the extracellular fluid of feces (human fecal water), which is the stool water in contact with the epithelial cells in vivo, resulted in activation of AP-1 and induction of cell proliferation which varied between different samples. Our attention then moved to, COX-2, another early response gene which has been shown to play an important role in colon tumorigenesis. We showed that dihydroxy bile acids and human fecal waters induced COX-2 promoter activity. This resulted in an increase in COX-2 protein expression. Interestingly, the concentrations of DCA that induced highest COX- 2 expression were cytotoxic. This led us to further studies on cell death induced by bile acids. We showed that bile acids induced apoptosis in the colonic cells, in a dose and time dependent fashion. Many substances which induce apoptosis are DNA damaging agents, which led us to test if DCA induced DNA damage. We observed that shortly after exposure of cells to DCA, damage to DNA occurred. The result of this damage is activation of the apoptosis program, which results for most cells in death. However, the few cells surviving the induced apoptosis showed low levels of caspase-3 and increased activity of NF-kB and AP-1 driven reporters, as well as an increase in COX-2 promoter activity. Mapping the region of the COX-2 promoter responsible for the effect, showed that both CRE and NF-kb as well as to some extent C/EBP elements are needed for full activation of the COX-2 promoter by DCA. In a follow up study, we characterized further the genotoxic effect of bile acids, and observed dose-dependent increase of DNA damage from DCA and lithocholic acid at concentrations above 300 µM. Testing 35 human fecal water samples, showed that 30% had very high potential to induce damage in intact cells while 50% had no effect. Finally, in a human dietary intervention study, we shifted the diet of healthy volunteers from a dairy product rich to a dairy product free diet, and monitored cytotoxicity and genotoxicity of their fecal waters. The result showed that when participants went off dairy products, the cytotoxicity of their fecal waters increased significantly, while there was no change observed for the genotoxicity of fecal waters. In summary, our results have contributed to the understanding of the molecular mechanisms by which bile acids influence tumorigenesis in the colon.
List of papers:
I. Glinghammar B, Holmberg K, Rafter J (1999). "Effects of colonic lumenal components on AP-1-dependent gene transcription in cultured human colon carcinoma cells. " Carcinogenesis 20(6): 969-76
Pubmed
II. Glinghammar B, Rafter J (2001). "Colonic luminal contents induce cyclooxygenase 2 transcription in human colon carcinoma cells. " Gastroenterology 120(2): 401-10
Pubmed
III. Haza AI, Glinghammar B, Grandien A, Rafter J (2000). "Effect of colonic luminal components on induction of apoptosis in human colonic cell lines. " Nutr Cancer 36(1): 79-89
Pubmed
IV. Glinghammar B, Inoue H, Rafter J (2001). "Colonocytes surviving bile acid induced apoptosis accumulate in G0/G1 and show a dose-dependent increase in COX-2 promoter activity and the transcription factors NF-kb and AP-1." (Submitted)
V. Venturi M, Hambly RJ, Glinghammar B, Rafter JJ, Rowland IR (1997). "Genotoxic activity in human faecal water and the role of bile acids: a study using the alkaline comet assay. " Carcinogenesis 18(12): 2353-9
Pubmed
VI. Glinghammar B, Venturi M, Rowland IR, Rafter JJ (1997). "Shift from a dairy product-rich to a dairy product-free diet: influence on cytotoxicity and genotoxicity of fecal water--potential risk factors for colon cancer. " Am J Clin Nutr 66(5): 1277-82
Pubmed
I. Glinghammar B, Holmberg K, Rafter J (1999). "Effects of colonic lumenal components on AP-1-dependent gene transcription in cultured human colon carcinoma cells. " Carcinogenesis 20(6): 969-76
Pubmed
II. Glinghammar B, Rafter J (2001). "Colonic luminal contents induce cyclooxygenase 2 transcription in human colon carcinoma cells. " Gastroenterology 120(2): 401-10
Pubmed
III. Haza AI, Glinghammar B, Grandien A, Rafter J (2000). "Effect of colonic luminal components on induction of apoptosis in human colonic cell lines. " Nutr Cancer 36(1): 79-89
Pubmed
IV. Glinghammar B, Inoue H, Rafter J (2001). "Colonocytes surviving bile acid induced apoptosis accumulate in G0/G1 and show a dose-dependent increase in COX-2 promoter activity and the transcription factors NF-kb and AP-1." (Submitted)
V. Venturi M, Hambly RJ, Glinghammar B, Rafter JJ, Rowland IR (1997). "Genotoxic activity in human faecal water and the role of bile acids: a study using the alkaline comet assay. " Carcinogenesis 18(12): 2353-9
Pubmed
VI. Glinghammar B, Venturi M, Rowland IR, Rafter JJ (1997). "Shift from a dairy product-rich to a dairy product-free diet: influence on cytotoxicity and genotoxicity of fecal water--potential risk factors for colon cancer. " Am J Clin Nutr 66(5): 1277-82
Pubmed
Issue date: 2001-05-11
Publication year: 2001
ISBN: 91-628-4816-X
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