Nutr Metab Cardiovasc Dis (2001) 11: Suppl. to No. 4
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Functional foods against colorectal cancer. An example project integrating functional genomics, nutrition and health R. Stierum1, R. Burgemeister2, A. van Helvoort3, A. Peijnenburg4, K. Schiitze2, M.. Seidelin5,0. Vang5, and B. van Ommen1 'TNO Nutrition and Food Research, Zeist, Netherlands, 2P.A.L.M. Microlaser Technologies AG. Bernried. Germany. NUMICO Research. Wageningen, Netherlands, 4 RIKILT, Wageningen. Netherlands, and 'Department of Life Sciences and Chemistry, Roskilde University. Roskilde. Denmark. 3
Summary Functional Food Against Colorectal Cancer is one of the first European Union funded Research Projects at the crossroad of functional genomics [comprising transcriptomics, the measurement of the expression of all messengers RNA (mRNAs) and proteomics, the measurement of expression/state of all proteins], nutrition and human health. The goal of Functional Foods Against Colorectal Cancer is to develop a colon epithelial cell line-based screening assay for nutrients with presumed anti-colorectal carcinogenic properties. Genes in ol ed in colon carcinogenesis are identified at the RNA and protein le el, using a ariety of methods (subtracti e hybridisation, DNA microarray, proteomics) in combination with models for colorectal cancer de elopment (human biopsies, rat model for colorectal carcinogenesis, colorectal cancer epithelial cell lines). Secondly, colorectal cancer epithelial cell lines are selected, in terms of their capacity to undergo gene/protein expression changes representing different phases in the colorectal carcinogenesis. Thirdly, these cell lines are used to determine the effects of nutrients with presumed anticarcinogenic properties (eg res eratrol, fla onoids) on functional genomics-deri ed endpoints. Once alidated against the effects of these nutrients in in vivo animal models and classical biomarkers for colorectal carcinogenesis, these
Key words:...... Correspondence to: Ben van Ommen. TNO Nutrition and Food Research. P.O. Box 360,3700 AJ Zeist. Netherlands. E-mail:
[email protected].
cell line models combined with functional genomics represent useful tools to study colorectal carcinogenesis and screen for nutrients with anti-carcinogenic properties. Nutr Metab Cardiovasc Dis (2001) 11: Suppl. to No. 4 ®2001,Medikal Press
Functional genomics in nutrition (nutrigenomics) and health research The mechanisms by which dietary components potentially influence disease are partially unknown because of lack of appropriate research tools to elucidate the complex mechanisms involved. Single nutrients may have multiple unknown possible biochemical targets and physiological actions, which cannot be studied with the 'single-gene/protein' approach using conventional molecular biology. Instead, the complexity of the effects of diet on health and disease warrants the use of novel research technologies. Novel post-genomic technologies which allow the simultaneous determination of the expression of multiple genes at the mRNA (transcriptomics) and protein (proteomics) level are being developed. The human genome consists of approximately 30,000 genes. Physiological processes are not merely regulated at the genome sequence itself, but are eg influenced by alternative mRNA splicing variants or protein translation products. Functional genomics can be defined as the research discipline that entails the explanation of complete biological processes, based on the information obtained at the
R. Stierum, et al
genome (DNA), transcriptome (RNA), proteome (protein) and metabolome (metabolism) level. The combination of functional genomics disciplines genomics, transcriptomics, proteomics, metabolomics with nutritional sciences shapes the new research discipline of nutrigenomics. Functional Food Against Colorectal Cancer (FFACC) is one of the first European Union-funded Research Projects on the integration of nutrigenomics with human health and disease. To further illustrate the concept of nutrigenomics, the present paper summarizes the activities within FFACC. The aim of FFACC is to develop a validated cell-based screening assay for the selection of (functional) food ingredients with a specific preventive or inhibitory effect on the development of colorectal cancer. The assay will be based upon evaluation of the expression of a large portfolio of genes and proteins involved in colorectal carcinogenesis and their presumed preventive or protective mechanisms. For this purpose, colonic epithelial cell lines will be selected based on transcriptome and proteome similarities with certain stages from colorectal tumors from rat and man. The usefulness of these cell lines to screen for anti-carcinogenic compounds is further evaluated by comparing the direction of the changes induced by these compounds in the transcriptome/proteome with the changes during stages of colon cancer development in humans in i o and chemically-induced tumors in the rat. Transcriptome and proteome studies within FFACC, performed to select appropriate colon cancer cell lines, are discussed below. Subtractive hybridisation is used to reveal genes involved in colon carcinogenesis and anti-carcinogenesis. The importance of the selection of appropriate cell lines representative for colorectal cancer stages and for screening of bioactive compounds that may prevent colorectal carcinogenesis is mentioned. The promising tool of laser microdissection (laser pressure catapulting) of colorectal tissue samples to reveal at the cellular level genes involved in the carcinogenic/anti-carcinogenic process is discussed.
human and rat cell lines, representative for specific stages in the development of colorectal cancer. Eventually, these will be used for the screening of food compounds for functionality. Human and rat-specific DNA microarrays are tailored for this particular application. The arrays contain genes which are known or suspected from literature and databases on the internet to be related to colorectal cancer and are obtained from commercial cDNA collections. In addition, the arrays contain clones from cDNA libraries which are normalised and enriched for colorectal cancerrelated genes by a subtractive cloning approach as described later. The microarrays are used for gene expression profiling of rat and human colon carcinoma cell lines exposed or not exposed to food compounds with putative anticarcinogenic activity.
Proteomics Proteomics aims at identifying the expression levels of all proteins in a cell. In addition, proteomics aims at defining the post-translational modifications, structural properties and interactions of all proteins. Currently, the most frequently used proteomics set up is based on two dimensional protein gel electrophoresis (2D) in combination with advanced image analysis and mass spectrometry. The basic principle is that all proteins of a cell are separated in two dimensions. Subsequently, proteins are visualised as a spot pattern, a proteome pattern (Fig 1).
FIGURE 1 Colon cell proteome.
Transcriptomics Methods to determine the transcriptome of a cell include differential display, serial analysis of gene expression and DNA microarray technology. DNA microarray technology has been applied in various studies in biology and medicine (1), but has been rarely used in nutritional research. For a comprehensive description of the microarray technology see van Hal et al (2). In the FFACC Project, the microarray technology is applied to select and validate
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Functional foods against colorectal cancer
Each spot in the pattern represents the amount of an expressed protein present in the cell. By comparison of proteomes obtained under different conditions (eg treatment with a nutrient --s control), the effect of a treatment on the expression of each protein in a cell can be assessed. Thousands of proteins are visualised at the same time. Computer software (eg Progenesis from Nonlinear Dynamics) helps to find protein expression differences for each individual spot. The next step is to identify the protein spots that change upon treatment (probably the most interesting proteins) in the gel, using mass spectrometry techniques. In FFACC, proteomics is used, together with transcriptomics, to select colon epithelial cell lines with respect to their resemblance to a certain cancer stage. To implement 2D-based proteomics within FFACC, proteome changes during differentiation of the colorectal cancer cell line Caco-2 have been studied. Interestingly, proteins that were found to change during in 'vitro differentiation were also reported to be associated with in i o colon carcinogenesis. Currently, 2D gels are prepared from various other colon epithelial cell lines described in "Use of cell lines in colon cancer bioactives screening". These images are compared to 2D images obtained from a large set of normal and human colon cancerous tissues to determine which of the colon cell lines is representative for a particular stage of colorectal cancer. Also, the first studies on nutrientinduced changes in the colon proteome are initiated using selected cell lines.
Colon anti-carcinogenesis: identification of differentially expressed genes using subtractive hybridisation The goal of this part of FFACC is to isolate human and rat genes that are differentially expressed during colon carcinogenesis and anti-carcinogenesis, using subtractive hybridisation. The isolated cDNAs are used to generate rat and human colon cancer chemopreventive specific DNA microarrays which are used to further characterize and validate appropriate colon cell lines for bioactive screening. The principle of subtractive hybridisation is that two different pools of poly A RNA's - pools A and B - are reverse trancribed to generate cDNAs. The cDNAs from pool A are then hybridised with an excess of cDNAs from pool B. After removal of the hybridised cDNAs, cDNAs specific to pool A remains. During the reverse subtraction, cDNAs specific to pool B will be isolated. In the present experiments, we used the PCR-select cDNA subtraction kit to
generate a plasmid vector library of differentially expressed genes. Cloned plasmid vector inserts were then arrayed on a glass slide and original cDNAs from the two pools of RNA hybridised to the arrays to provide a quick verification of differential expression of cloned cDNAs. Subsequently, inserts of positive clones were sequenced. In our studies, we are applying the subtractive hybridisation technique on rat samples. The pools of RNA used in the subtractive hybridisations came from in '> n o experiments, where the colon carcinogenic process was induced in male F344 rats by the colon carcinogen 2-amino-l-methyl-6phenylimidazo(4,5-b)-pyridine (PhIP). In the first study, rats were fed a PhIP diet for up to 5 weeks. In the second study, rats drank water containing trans-resveratrol - a very promising food-born cancer chemopreventive substance (3) - and were subsequently fed a PhIP diet for 1 week. Two subtractive hybridisations were performed using colon RNA from i) rats fed the control and PhIP diet for 5 weeks (resembling the promotion part of the carcinogenic process) and ii) rats given water bottles containing drinking water with or without trans-resveratrol and subsequently fed PhIP diet for 1 week (resembling the initiation part of the carcinogenic process). The rat cDNAs from the 1400 clone libraries generated from each subtraction were subjected to microarray analysis to verify their differential expression. From the first study more than 150 clones, which were detected as containing inserts from colon genes whose expression was more than 2-fold up- or down-regulated by the PhIP diet, have now been sequenced. The cDNAs obtained from the human and rat subtractive hybridisations will be used, together with cDNAs of genes whose products are already known to be involved in the colon carcinogenic process, to generate the final rat and human colon cancer chemopreventive specific DNA microarrays.
Use of cell lines in colon cancer bioactives screening Many natural dietary phytonutrients found in fruits, vegetables, spices and tea have been shown to be protective against cancer in various animal models. In the light of the potential impact of these compounds on human health it is important to elucidate the mechanisms involved and to test new components that modulate these mechanisms. To study the mechanistically different steps involved in colon carcinogenes, various immortalized human epithelial cancer cell lines derived from the rectum and colon exist.
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FIGURE 2 Number of Medline references for the different colon cell lines. The name of the cell was combined with the term cell line, or with the term colon cancer, to minimize the number of irrelevant items. The names were spelled in different possible names (eg Caco-2, Caco2, and Caco 2). 3000 -
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Although numerous cell lines have been established, only limited cell lines are being used (Fig 2). The limited use is often due to availability, laboratory experience, growth characteristics, and lack of knowledge in the alternative models. In FFACC, we evaluate the characteristics of the different colon cancer cell lines, and the effect of selected model food components by a literature review of the available data and by the analysis of protein (proteomic) and RNA (transcriptomic) expression, as described above. Comparison of the cell line expression date with data obtained in human colon (tumor) biopsies enables us to update the information on the tumor stages and mechanisms affected in the colon cell line. For the characterization of the chemopreventive effect of model food components, the effects on protein and RNA expression obtained in relevant cell lines will be compared to results from animal studies and human clinical literature. With these model compounds we expect to affect different mechanisms involved in colon carcinogenesis and will identify marker genes/proteins that represent the mechanism involved in the therapeutic effect of these food components. Together, these results provide us with a panel of well-characterized
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colon cancer cell lines that should reflect the diversity of tumor phenotypes and provide adequate models for the study of tumor heterogeneity. The selected panel of colon cancer cell lines will be a useful tool to study the disease and to develop and test new therapeutic approaches in the appropriate cell line.
Microdissection of colon tissue. The "Laser Catapult" for high speed sample capture Microdissection of colonic tissues is of particular importance within FFACC to allow the comparison of basal transcriptomes/proteomes from cell lines -and changes induced by nutrients- with transcriptomes/proteomes occuring during colon carcinogenesis in humans and the rat. Colorectal tumours and normal colonic tissue obtained after surgical removal (human) or dissection (rat) may be 'contaminated' with non-epithelial cells. Microdissection can be performed to purify epithelial cells using laser pressure catapulting (LPCpat-) technology. This technology enables the dissection transfer of the selected cells from a tissue without any mechanical contact, but solely by the force of focused laser light (4). The PALM® MicroBeam provides a unique manipulating tool. Within a narrow laser focal spot the focused light generates forces that allow microsurgery or microdissection of microscopically small objects without any mechanical contact. The system precision combined with an especially designed RoboSoftware allows the preparation of samples as small as filaments or chromosomes but also the capture of large tissue areas, eg colonic tissues. Thus, the capture of thousands of cells within a very short time (minutes only) is possible, which enables high throughput specimen generation, which can be used in conjunction with DNA microarray and proteome technologies.
tumours with PhIP (a heterocyclic amine) and also after combination of PhIP with resveratrol, a polyphenol with possible anti-carcinogenic activity. Currently, 150 genes have already been found to be more than 2-fold up or down regulated in this model, which therefore may be involved in colorectal carcinogenesis. - DNA microarray and proteomics based expression analysis of human colorectal epithetlial cell lines and tumour samples and their corresponding "normal" counterparts. At present, the expression of 18 proteins has been found to change upon in itro differentiation of the colorectal cancer cell line Caco-2, using proteomics. Some of these proteins were reported to be associated with colorectal carcinogenesis in humans. - "Digital" differential display and SAGEnet database extraction on internet based gene expression data of colorectal carcinogenesis, thus exploiting already published data. In the near future, changes in gene and protein expression resulting from exposure to presumed bioactive food constituents are analysed in human cell lines representing different stages in colon carcinogenesis, and related to changes observed in different stages of the colorectal molecular pathogenesis. It is envisaged that this panel of cell lines combined with functional genomics technologies allows for medium throughput screening of potentially anti-carcinogenic food constituents.
Acknowledgements Financial Support for by the European Union within the 5th framework programme is greatly acknowledged (QLRT-1999-00706).
References Conclusions
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Functional Foods Against Colorectal Cancer demonstrates promising developments at the cross-road of functional genomics, nutrition and human health and disease. The activities employed in FFACC contribute to the development of a colon-epithelial cell-line screening assay for nutrients with presumed anti-colorectal carcinogenic properties. Through a variety of functional genomics methods, genes involved in colorectal cancer development are being identified at the RNA and protein level: - Subtractive hybridisation in rat after induction of colon
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