Browsing by Subject "Colon carcinogenesis"

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Einfluss kurzkettiger Fettsäuren und mikrobieller Fermentationsprodukte neuartiger Oligosaccharide auf Cytotoxizität, Proliferation und Apoptose von humanen Coloncarcinom-Zelllinien
(2006) Roser, Silvia; Rechkemmer, Gerhard
Colon cancer is the second most common cancer in Germany. The role of dietary fibre in the prevention of colon cancer is still controversial: Promising results from in vitro and animal studies are contradictory to inconsistent results from epidemiological stu-dies. Functional carbohydrates as constituents of prebiotic food can modify the colonic microflora for the benefit of short chain fatty acid (SCFA)-producing microbial strains. The SCFA-concentrations should also be increased in the distal part of the colon where most colon carcinomas are developing. SCFA are considered to be preventive against colon cancer. For this study, three different new functional oligosaccharides (OS, made of Isomaltulose and resistant starch) were produced from the Südzucker company and fermented in vitro with human feces of healthy test subjects. The resulting fermentation supernatants (FS) were tested in a cell culture system, using colon carcinoma cell lines of various degrees of differentiation (HT29, HT29 Clone 19A, T84). Cytotoxicity, proliferation, the induction of apoptosis, influences on the cell cycle and electrophysiological parameters were measured. Spectral photometric and flow cytometric methods were performed, as well as measurements in vertical diffusion chambers (Ussing chambers). The parallel testing of SCFA-mixtures with the same SCFA-concentrations as in the FS was included, as well as the testing of a FS ?Control? which was produced without OS-fermentation. Several independent fermentations revealed reproducible results regarding the SCFA-concentrations of the FS. After OS-fermentation, the ratio of the three major SCFA in the FS, acetate, propionate, and butyrate, was similar to that observed in vivo. The FS and SCFA-mixtures tested had a cytotoxic effect on all cell lines at the con-centration of 50 %. A dose dependent decrease in cell proliferation could be found, as well as the induction of apoptosis at a concentration of 50 %. Parallel testing of the analogous SCFA-mixtures showed that cytotoxic and proliferation inhibiting effects of the FS could be primarily attributed to their SCFA-content. This could not be confirmed for apoptosis induction: the SCFA-mixtures were mostly able to induce a higher apoptosis rate than the FS. Similarly, the effects of FS and SCFA-mixtures on the cell cycle were different: The SCFA-mixtures showed more potent inhibition of DNA-synthesis than the analogous FS, which generally led to an arrest in the G2-phase of the cell cycle. Neither FS nor SCFA-mixtures had an impact on transepithelial resistance or short circuit current of differentiated cell monolayers in Ussing chambers. The difference in the fermentation patterns of the various FS and the SCFA-concentrations of the SCFA-mixtures was not great enough to achieve significantly different results in the test systems used. Also, the various differentiation grades of the cell lines showed inconsistent results after treatment with FS and their SCFA-mixtures, so that no correlation could be found between degree of differentiation and test compound action. This study shows that the in vitro fermentation of OS with human feces results in reproducible SCFA-patterns in the FS, similar to the in vivo situation. For the screening of FS and their SCFA-mixtures, respectively, a spectrum of methods was established for the incubation with colon carcinoma cell lines of various differentiation states and of all stages of growth (exponential, subconfluent, confluent, fully differentiated monolayer). Indeed, the effects measured after incubation with FS could only in part been ascribed to their SCFA content. Other FS components than SCFA that play a role, especially regarding to their apoptosis inhibiting and cell cycle influencing effects, remain to be identified. Also, this study allows no conclusions to be drawn, which of the fermented OS is more promising in it?s beneficial influence on colon cancer preventing factors, e.g. the induction of apoptosis, than the other. Future studies should investigate FS with greater differences in their SCFA-concentrations. The same OS which were used for the in vitro fermentation, should also be tested in animal studies and human intervention studies to elucidate their fermentation patterns in vivo.
Einfluss von Karotten- und Tomatensaft-Konsum auf Coloncarcinogenese-relevante Faecesmarker beim Menschen
(2006) Schnäbele, Kerstin; Briviba, Karlis
Colorectal cancer is one of the most common tumor diseases in the world. Most of the colorectal tumors are sporadic and develop somatically in epithelial cells. Nutritional factors can markedly affect tumor development. A high intake of fruits and vegetables is often associated with a reduced risk of colorectal cancer. Protective effects of fruits and vegetables are attributed to ingredients, such as fibers, vitamins, and secondary plant products (e.g. carotenoids), which have potential anticarcinogenic properties. The aim of this study was to investigate, by means of a human intervention trial with carrot and tomato juice consumption, whether a diet rich in carotenoids, especially high in beta-carotene and lycopene, can modify processes relevant to colon carcinogenesis in the gastrointestinal lumen. Therefore, several faecal markers had to be established and used in this study. In the randomized crossover trial, 22 healthy male subjects on a low-carotenoid diet consumed 330ml of carrot or tomato juice daily for a period of two weeks. The two juice intervention periods were preceded by two-week depletion phases. At the end of each study period the stool of twelve volunteers was collected over a 48-hour period. This stool was used to produce some preparations such as non-filtered and sterile-filtered faecal water, as well as faecal lipid extracts, in order to use them in cell culture systems. Spectral photometric and flow cytometric methods were used to determine the effects of the above-mentioned preparations on colon adenocarcinoma cells (HT-29), as well as to determine the activities of the bacterial enzymes beta- glucosidase and beta-glucuronidase in faecal water. HPLC methods were used to measure the concentrations of several bile acids in faecal water, as well as to determine the concentrations of carote-noids and malondialdehyde (MDA) in faecal samples. The concentrations of the major short chain fatty acids (SCFA) were measured via gas chromatography. Consumption of carrot juice led to a marked increase of beta-carotene and alpha-carotene in faeces and in non-filtered faecal water, as did lycopene after consumption of tomato juice. In the succeeding depletion phases, the contents of those carotenoids in faeces and faecal water returned to their initial values. Changes in faecal MDA concentrations by carrot and tomato juice interventions could not be observed. Faecal water showed high, dose-dependent cytotoxic effects on HT-29 cells. Those effects were, however, not markedly changed by carrot and tomato juice consumption. Neither bile acid concentrations nor the bile acid profile in faecal water changed after carrot and tomato juice consumption. Bacterial activities of beta-glucosidase and beta-glucuronidase also did not change. While tomato juice consumption did not significantly affect the pH value of faecal water, this value was, however, decreased by carrot juice consumption. Although faecal water concentrations of acetate and butyrate contributed to the decrease in faecal water pH values, SCFA were probably not responsible for the observed pH changes after carrot juice consumption. SCFA concentrations in faecal water and SCFA proportions did not change significantly. Neither bile and SCFA concentrations, nor the activities of tested bacterial enzymes, had any influence on the cytotoxic effects of sterile-filtered faecal water. These cytotoxic effects, however, decreased with increasing proportions of the primary bile acids cholic and chenodesoxycholic acid, independent of the study phases. As determined by multiple regression analysis, the most probable leading factors for the growth inhibitory effects of faecal water are the faecal MDA content and bacterial beta-glucosidase activity. Further studies should investigate whether the parameters mentioned directly influence cytotoxic and antiproliferative effects of faecal water or if those parameters are indirect markers for the activity of individual microflora. Carrot and tomato juice consumption strongly increased the cytotoxic effects of faecal lipid extracts in HT-29 cells, likely caused by the induction of apoptosis. Which mechanisms account for these effects and the consequences of these effects in the in vivo situation should be investigated in further studies. This work shows that two-week interventions with carotenoid-rich juices lead only to minor changes in luminal processes relevant to colon carcinogenesis in young healthy volunteers on an energy- and macronutrient-balanced diet. Lacking effects on 1) the toxic and antiproliferative properties of faecal water, 2) lipid peroxidation in faeces, 3) the bile and SCFA concentrations in faecal water, and 4) bacterial enzyme activities indicate that related physiological effects can not be influenced by a diet rich in carotenoids under the just described conditions. Other anticarcinogenic mechanisms seem to be of greater importance.