Flavone Effects on the Proteome and Transcriptome of Colonocytes in Vitro and in Vivo and Its Relevance for Cancer Prevention and Therapy
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TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Ernährungsphysiologie Flavone effects on the proteome and transcriptome of colonocytes in vitro and in vivo and its relevance for cancer prevention and therapy Isabel Winkelmann Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. D. Haller Prüfer der Dissertation: 1. Univ.-Prof. Dr. H. Daniel 2. Univ.-Prof. Dr. U. Wenzel (Justus-Liebig-Universität Giessen) 3. Prof. Dr. E.C.M. Mariman (Maastricht University, Niederlande) schriftliche Beurteilung Die Dissertation wurde am 24.08.2009 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 25.11.2009 angenommen. Die Forschung ist immer auf dem Wege, nie am Ziel. (Adolf Pichler) Table of contents 1. Introduction .......................................................................................................... 1 1.1. Cancer and carcinogenesis .................................................................................. 2 1.2. Colorectal Cancer ............................................................................................... 3 1.2.1. Hereditary forms of CRC ........................................................................................ 4 1.2.2. Sporadic forms of CRC ........................................................................................... 5 1.2.3. Genetical conditions for CRC development ............................................................ 6 1.3. Apoptosis ............................................................................................................ 8 1.4. Treatment of cancer ............................................................................................ 9 1.5. Flavonoids ........................................................................................................ 10 1.5.1. Structure of flavonoids .......................................................................................... 10 1.5.2. Sources of flavonoids ............................................................................................ 12 1.5.3. Flavonoid functions in plants ................................................................................ 13 1.5.4. Flavonoids as dietary components and their proposed impacts on health ............ 14 1.6. Proteomics in nutritional sciences and cancer research .................................... 16 2. Aims of this study ............................................................................................... 18 3. Materials and Methods ...................................................................................... 19 3.1. Cell culture........................................................................................................ 19 3.1.1. Cultivation of the cell line HT-29 ......................................................................... 19 3.1.2. Experimental Treatment and harvesting of HT-29 ................................................ 19 3.2. Animals ............................................................................................................. 20 3.2.1. Experimental procedures ....................................................................................... 20 3.2.2. Experimental arrangements ................................................................................... 20 3.2.3. ACF preparation and determination ...................................................................... 21 3.2.4. Immunohistochemistry .......................................................................................... 21 3.3. Sample preparation ........................................................................................... 22 3.3.1. Isolation of proteins from HT-29 cells .................................................................. 22 3.3.1.1. Preparation of whole cell lysates ......................................................... 23 3.3.1.2. Preparation of fractionated cell lysates................................................ 23 3.3.2. Isolation of proteins from colons of mice ............................................................. 24 3.3.3. Photometric determination of the protein concentration ....................................... 24 3.4. Two dimensional gel electrophoresis ............................................................... 24 3.4.1. Pouring of SDS-gels .............................................................................................. 24 3.4.2. First dimension ...................................................................................................... 25 3.4.3. Second dimension .................................................................................................. 26 III Table of contents 3.4.4. Analysis of the gels by the software Proteom Weaver .......................................... 27 3.5. Mass spectrometry ............................................................................................ 27 3.5.1. Sample preparation and processing ....................................................................... 27 3.5.1.1. In-gel digestion by hand for in vitro experiments ............................... 27 3.5.1.2. Sample spotting on the target by Gobom method ............................... 27 3.5.1.3. Sample spotting for post-source decay analysis .................................. 28 3.5.1.4. In-gel digestion and spotting by robotic systems ................................ 28 3.5.2. MALDI-TOF MS .................................................................................................. 28 3.5.3. Computer-aided protein identification .................................................................. 28 3.6. Microarrays ....................................................................................................... 30 3.6.1. RNA-isolation out of the tissue ............................................................................. 30 3.6.2. RNA quality-check and quantification .................................................................. 30 3.6.2.1. DNA-Arrays ........................................................................................ 30 3.6.2.2. Processing of total RNA, followed by hybridization and scanning .... 30 3.6.2.3. Data interpretation by the Genomatix software ................................... 31 3.7. Calculations and statistics ................................................................................. 31 4. Results ................................................................................................................. 33 4.1. Establishment of 2-DE methods ....................................................................... 33 4.1.1. Staining methods ................................................................................................... 33 4.1.2. Fractionation of the whole cell lysate .................................................................... 34 4.2. Establishment of automated processes in mass spectrometry .......................... 35 4.2.1. Protein spot handling with robotic systems ........................................................... 35 4.2.2. Acquirement of mass spectra in automatic mode .................................................. 35 4.3. 2-DE analysis of whole cell lysates from flavone- or camptothecin-treated HT-29 cells ....................................................................................................... 35 4.3.1. Effect of flavone exposure on the proteome of HT-29 cells ................................. 36 4.3.2. Effect of camptothecin exposure on the proteome of HT-29 cells ........................ 42 4.4. 2-DE analysis of fractionated cell lysates from flavone-treated HT-29 cells ... 51 4.4.1. Fraction I – cytosolic proteins ............................................................................... 51 4.4.2. Fraction II – membrane/organelle proteins ........................................................... 57 4.4.3. Fraction III – nuclear proteins ............................................................................... 60 4.4.4. Fraction IV – cytoskeletal proteins ....................................................................... 62 4.5. Histological and histochemical analysis of colonic tissue derived from flavone- treated C57BL/6J mice ..................................................................................... 65 IV Table of contents 4.5.1. Frequency and multiplicity of aberrant crypts in the colonic tissue ...................... 65 4.5.2. Modification of apoptosis and proliferation rates in the colonocytes of mice in vivo ……… ........................................................................................................ 68 4.6. 2-DE analysis of colonic tissue derived from flavone-treated C57BL/6J mice 70 4.6.1. Proteome analysis of tissues from the blocking group .......................................... 70 4.6.2. Proteome analysis of tissues from the suppressing group ..................................... 74 4.6.3. Proteome analysis of tissues from the therapy group ............................................ 79 4.7. Transcriptome analysis of colonic tissue derived from flavone-treated C57BL/6J mice from the therapy group ........................................................... 84 5.