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Transient, deoxycholate induced, hypercalcemia in human colon cancer cells
April 1 9 9 5
• TRANSIENT, DEOXYCHOLATE INDUCED, HYPERCALCEMIA IN HUMAN COLON CANCER CELLS. PK Bamber,qer, MD Duncan, M Molloy, S Batzri, WJ Goldberg, JW Harmon. Departments of Surgery, VA Medical Center and Walter Reed Army Medical Center, Washington D.C. Increased fecal concentration of Deoxycholic acid (DC) is associated with colon cancer by epidemiologic and experimental studies. DC may act via cytosolic calcium in e~(~rting its carcinogenic effect on colonic mucosal cells. Aim: This study investigates the effect of DC exposure on cytosolic ionized calcium concentration ([Ca]=) in a hfJman colon cancer cell line. Methods: Cultured Caco-2 cells were loaded with FURA-2-AM and imaged with a fluorescent microscope. The ratio of the intensity of emission after excitation at wavelengths of 340 nm and 380 nm (340•380 ratio) is a function of [Cain. The ratio is assigned a color by the imaging system and changes in color reflect the change in [Ca]~. Results: In the presence of 1 mM extracellular calcium the 340•380 ratio increased from a 12 baseline of 0.52+0.02 to 1.02"+0.07 within 5 minutes 1.0 following the addition of 1.25 mM DC to the media (*P<.001 0.9 1 0,8 _o0.7 eaired t-test). [Ca]i calculated with the Grynkiewicz equation ~ 0.5rose from a baseline of 125 nM to a peak of 459 nM. The 0.3 intracellular calcium levels then 02~ • l~t~a returned to baseline, In calcium free media a rise from e 0.0 baseline of 0.41+0.005 (24 nM) 0 2 3 4 5 6 7 8 9 10 to a peak of 0.55*+0.02 (140 riM) was recorded (*P<.001), a return to baseline was also observed (Figure). Conclusions: These results indicate that DC in physiologic concentrations induces a transient hypercalcemia in Caco-2 cells. A significant response persists despite chelation of extracellular calcium. DC associated transient hypercalcemia may be critical to the carcinogenic effect of bile acids in the colon.
• ELIAGIC ACID INDUCES TRANSCRIFrION OF MICROSOMAL EPOXIDE HYDROLASETHROUGHA PROMOTER WHICH IS NOT AN ANTIOXIDANT RESPONSIVE ELEMENT. D.H. Barch, LM. Rundhaugen. Dept of Med Lakeside Veterans Affairs Medical Center, Northwestern University Lurie Cancer Center & Northwestern Univ Medical School, Chicago, IL 60611 The dietary anticarcinogen ellagic acid has been shown to induce transcription of the Phase II detoxification enzymes glutathione S-transferase Ya and NAD(P)H:quinone reductase through activation of the Antioxidant Responsive Elements in these genes. Compounds which induce glutathione S-transferase Ya and NAD(P)H:quinone reductase have also been shown to induce microsomal epoxide hydrolase (mEH) and we proposed that ellagic acid would also induce the transcription of mEH through a related Antio×idant Responsive Element. Antioxidant Responsive Elements contain two modified AP-1 sites and we have identified a similar structure between -171 bp and -271 bp in the 5' regulatory region of the mEH gene. To evaluate this hypothesis we examined the ability of ellagic acid to activate transcription of mEH in a transient transfection system. Three plasmid constructs, containing 891, 271 and 171 bp 5' from the transcription start site of the rat mEH gene, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, were transfected into Hep-G2 hepatoma cells. The transfected cells were then exposed to ellagic acid, tert-butylhydroquinone (a known Antioxidant Responsive Element inducer) or control media. Quantitation of CAT activity demonstrated that ellagic acid significantly (p<0.001) induced transcription of the mEH-891, mEH-271 and mEH-171 constructs demonstrating that an ellagic acid responsive element is present in the mEH gene. Ellagic acid induction of the mEH-171 construct indicates that the ellagic acid responsive element is not between -171 and -271 bp in the mEH gene. t-Butylhydroquinone did not induce any of the mEH constructs indicating that the eUagic acid responsive element in the mEH gene is not an Antioxidant Responsive Element. (Supported by the VA Research Service, the American Institute for Cancer Research and NIH-CA60553).
Gastrointestinal Oncology
A447
ISOLATION O F DIFFERENTIALLY EXPRESSED MESSENGER RNAS F R O M PERI-AMPULLARY CANCER ASSOCIATED W I T H FAMILIAL ADENOMATOUS POLYPOSIS (FAP) B. Ba_oat, L. Xia, T. Bcrk, Y.H. Nong, S. Gallingcr. Depts. of Pathology & Surgery, Mount Sinai Hospital, University of Toronto, Ontario, CANADA FAP patients manifest peri-ampullary adenomas and cancer at a significantly higher frequency than the general population. Molecular genetic alterations underlying the formation of these pre-malignant and malignant lesions are incompletely understood. We have used a recently described method; DDRT-PCR (differential display reverse transcribed PCR) ~ to isolate differentially expressed transcripts that may be intportant in peri-ampullary tumorigenesis. RNA isolated from matched normal and tumor peri-ampullary samples from two FAP patients was screened by DDRT-PCR in three replicate experiments. A total of fifteen unique and reproducible complementary DNA (eDNA) fragments ranging in size from 300 - 650 bp were isolated. Eight cDNAs were subcloned and further characterized by sequence analysis. Of these, three eDNA sequences showed no significant homology to sequences in current databases. One eDNA (355 bp) was 88% homologous over 334 bp to human Guanosine binding protein (GBP). Northern hybridization analysis of a eDNA clone, AR3a, indicated differences in the expression levels, in RNA extracted from various cell lines as follows; normal lymphoblast, colon carcinoma and melanoma (high) and lung and breast carcinoma (low). Identification and characterization of differentially expressed genes may yield novel molecular markers useful in understanding tumorigenesis in the peri-ampullary region of FAP patients. tLiang P. and Pardee A, B. (1992) Science vol. 257, pp 967-970
• DIETARY FISH OIL DECREASES FECAL CONTENT OF THE PUTATIVE COLON-CARCINOGEN 4-CHOLESTEN-3-ONE IN HEALTHY VOLUNTEERS. H.-P. Bartram, A. Gostner, E. Kelber, A. Weimer, G. Dusel, W. Scheppach, H. Kasper. Dept. of Medicine, University of Wurzburg, Germany. There is strong evidence from various epidemiological and experimental studies that high consumption of fish oil (FO) may protect against colon cancer. In this study the effects of FO on fecal excretion of neutral sterols was investigated, knowing that certain neutral sterols (e.g. 4-cholesten-3-one) can act as initiators of colon carcinogenesis in animal models (Mutat.Res. 141:35, 1984). METHODS: 12 healthy volunteers (20-31 yrs.) received either 20 capsules of FO (= 4.4 g (o-3 fatty acids) or corn oil (CO) per day in addition to a controlled basal diet (50% of energy derived from fat, 15% protein, 35% carbohydrates) for two 4-week periods (double-blind, cross-over). At the end of each test period stool was collected for 5 days and neutral sterols were determined in the dried feqat samples by gas liquid chromatography using a Hewlett Packard 5890 GC equipped with a megabore DB1 capillary column (15 m, 113 530 }am). Data on fecal concentrations (mg/g dry stool) and daily excretion (mg/day) are given as mean + SEM, differences between the two test periods were calculated by Wilcoxon's signed rank test. RESULTS: Significant differences in fecal concentrations between the FO and CO period were found for coprostanol (7.41 ± 0.75 vs. 4.91 ± 0.6, p = 0.01), campesterol (3.64 ± 0.47 vs. 5.03 ± 0.51, p-~0.005), and 13sitosterol (0.91 ± 0.14 vs. 1.39 ± 0.28, p=0.02). A trend to lower 4-cholesten-3-one concentrations was found under FO compared to CO (0.79 + 0.07 vs. 1.06 ± 0,1 l, p=0.05) and daily excretion of this putative carcinogen was significantly lower during FO compared to COconsumption (22.62 ± 1.57 vs. 33.49 ~- 4.73 mg/day, p-N).04). CONCLUSION: Fish oil supplementation leads to considerable changes in the fecal content and daily excretion of several neutral sterols. Aside from other biochemical mechanisms, the decreased fecal excretion of 4-cholesten-3-one during FO-consumption may give an explanation for the protective effect of fish oil diets in colon carcinogenesis. (This study was funded by a BMFT grant 07ERG09)
• TRANSIENT, DEOXYCHOLATE INDUCED, HYPERCALCEMIA IN HUMAN COLON CANCER CELLS. PK Bamber,qer, MD Duncan, M Molloy, S Batzri, WJ Goldberg, JW Harmon. Departments of Surgery, VA Medical Center and Walter Reed Army Medical Center, Washington D.C. Increased fecal concentration of Deoxycholic acid (DC) is associated with colon cancer by epidemiologic and experimental studies. DC may act via cytosolic calcium in e~(~rting its carcinogenic effect on colonic mucosal cells. Aim: This study investigates the effect of DC exposure on cytosolic ionized calcium concentration ([Ca]=) in a hfJman colon cancer cell line. Methods: Cultured Caco-2 cells were loaded with FURA-2-AM and imaged with a fluorescent microscope. The ratio of the intensity of emission after excitation at wavelengths of 340 nm and 380 nm (340•380 ratio) is a function of [Cain. The ratio is assigned a color by the imaging system and changes in color reflect the change in [Ca]~. Results: In the presence of 1 mM extracellular calcium the 340•380 ratio increased from a 12 baseline of 0.52+0.02 to 1.02"+0.07 within 5 minutes 1.0 following the addition of 1.25 mM DC to the media (*P<.001 0.9 1 0,8 _o0.7 eaired t-test). [Ca]i calculated with the Grynkiewicz equation ~ 0.5rose from a baseline of 125 nM to a peak of 459 nM. The 0.3 intracellular calcium levels then 02~ • l~t~a returned to baseline, In calcium free media a rise from e 0.0 baseline of 0.41+0.005 (24 nM) 0 2 3 4 5 6 7 8 9 10 to a peak of 0.55*+0.02 (140 riM) was recorded (*P<.001), a return to baseline was also observed (Figure). Conclusions: These results indicate that DC in physiologic concentrations induces a transient hypercalcemia in Caco-2 cells. A significant response persists despite chelation of extracellular calcium. DC associated transient hypercalcemia may be critical to the carcinogenic effect of bile acids in the colon.
• ELIAGIC ACID INDUCES TRANSCRIFrION OF MICROSOMAL EPOXIDE HYDROLASETHROUGHA PROMOTER WHICH IS NOT AN ANTIOXIDANT RESPONSIVE ELEMENT. D.H. Barch, LM. Rundhaugen. Dept of Med Lakeside Veterans Affairs Medical Center, Northwestern University Lurie Cancer Center & Northwestern Univ Medical School, Chicago, IL 60611 The dietary anticarcinogen ellagic acid has been shown to induce transcription of the Phase II detoxification enzymes glutathione S-transferase Ya and NAD(P)H:quinone reductase through activation of the Antioxidant Responsive Elements in these genes. Compounds which induce glutathione S-transferase Ya and NAD(P)H:quinone reductase have also been shown to induce microsomal epoxide hydrolase (mEH) and we proposed that ellagic acid would also induce the transcription of mEH through a related Antio×idant Responsive Element. Antioxidant Responsive Elements contain two modified AP-1 sites and we have identified a similar structure between -171 bp and -271 bp in the 5' regulatory region of the mEH gene. To evaluate this hypothesis we examined the ability of ellagic acid to activate transcription of mEH in a transient transfection system. Three plasmid constructs, containing 891, 271 and 171 bp 5' from the transcription start site of the rat mEH gene, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, were transfected into Hep-G2 hepatoma cells. The transfected cells were then exposed to ellagic acid, tert-butylhydroquinone (a known Antioxidant Responsive Element inducer) or control media. Quantitation of CAT activity demonstrated that ellagic acid significantly (p<0.001) induced transcription of the mEH-891, mEH-271 and mEH-171 constructs demonstrating that an ellagic acid responsive element is present in the mEH gene. Ellagic acid induction of the mEH-171 construct indicates that the ellagic acid responsive element is not between -171 and -271 bp in the mEH gene. t-Butylhydroquinone did not induce any of the mEH constructs indicating that the eUagic acid responsive element in the mEH gene is not an Antioxidant Responsive Element. (Supported by the VA Research Service, the American Institute for Cancer Research and NIH-CA60553).
Gastrointestinal Oncology
A447
ISOLATION O F DIFFERENTIALLY EXPRESSED MESSENGER RNAS F R O M PERI-AMPULLARY CANCER ASSOCIATED W I T H FAMILIAL ADENOMATOUS POLYPOSIS (FAP) B. Ba_oat, L. Xia, T. Bcrk, Y.H. Nong, S. Gallingcr. Depts. of Pathology & Surgery, Mount Sinai Hospital, University of Toronto, Ontario, CANADA FAP patients manifest peri-ampullary adenomas and cancer at a significantly higher frequency than the general population. Molecular genetic alterations underlying the formation of these pre-malignant and malignant lesions are incompletely understood. We have used a recently described method; DDRT-PCR (differential display reverse transcribed PCR) ~ to isolate differentially expressed transcripts that may be intportant in peri-ampullary tumorigenesis. RNA isolated from matched normal and tumor peri-ampullary samples from two FAP patients was screened by DDRT-PCR in three replicate experiments. A total of fifteen unique and reproducible complementary DNA (eDNA) fragments ranging in size from 300 - 650 bp were isolated. Eight cDNAs were subcloned and further characterized by sequence analysis. Of these, three eDNA sequences showed no significant homology to sequences in current databases. One eDNA (355 bp) was 88% homologous over 334 bp to human Guanosine binding protein (GBP). Northern hybridization analysis of a eDNA clone, AR3a, indicated differences in the expression levels, in RNA extracted from various cell lines as follows; normal lymphoblast, colon carcinoma and melanoma (high) and lung and breast carcinoma (low). Identification and characterization of differentially expressed genes may yield novel molecular markers useful in understanding tumorigenesis in the peri-ampullary region of FAP patients. tLiang P. and Pardee A, B. (1992) Science vol. 257, pp 967-970
• DIETARY FISH OIL DECREASES FECAL CONTENT OF THE PUTATIVE COLON-CARCINOGEN 4-CHOLESTEN-3-ONE IN HEALTHY VOLUNTEERS. H.-P. Bartram, A. Gostner, E. Kelber, A. Weimer, G. Dusel, W. Scheppach, H. Kasper. Dept. of Medicine, University of Wurzburg, Germany. There is strong evidence from various epidemiological and experimental studies that high consumption of fish oil (FO) may protect against colon cancer. In this study the effects of FO on fecal excretion of neutral sterols was investigated, knowing that certain neutral sterols (e.g. 4-cholesten-3-one) can act as initiators of colon carcinogenesis in animal models (Mutat.Res. 141:35, 1984). METHODS: 12 healthy volunteers (20-31 yrs.) received either 20 capsules of FO (= 4.4 g (o-3 fatty acids) or corn oil (CO) per day in addition to a controlled basal diet (50% of energy derived from fat, 15% protein, 35% carbohydrates) for two 4-week periods (double-blind, cross-over). At the end of each test period stool was collected for 5 days and neutral sterols were determined in the dried feqat samples by gas liquid chromatography using a Hewlett Packard 5890 GC equipped with a megabore DB1 capillary column (15 m, 113 530 }am). Data on fecal concentrations (mg/g dry stool) and daily excretion (mg/day) are given as mean + SEM, differences between the two test periods were calculated by Wilcoxon's signed rank test. RESULTS: Significant differences in fecal concentrations between the FO and CO period were found for coprostanol (7.41 ± 0.75 vs. 4.91 ± 0.6, p = 0.01), campesterol (3.64 ± 0.47 vs. 5.03 ± 0.51, p-~0.005), and 13sitosterol (0.91 ± 0.14 vs. 1.39 ± 0.28, p=0.02). A trend to lower 4-cholesten-3-one concentrations was found under FO compared to CO (0.79 + 0.07 vs. 1.06 ± 0,1 l, p=0.05) and daily excretion of this putative carcinogen was significantly lower during FO compared to COconsumption (22.62 ± 1.57 vs. 33.49 ~- 4.73 mg/day, p-N).04). CONCLUSION: Fish oil supplementation leads to considerable changes in the fecal content and daily excretion of several neutral sterols. Aside from other biochemical mechanisms, the decreased fecal excretion of 4-cholesten-3-one during FO-consumption may give an explanation for the protective effect of fish oil diets in colon carcinogenesis. (This study was funded by a BMFT grant 07ERG09)