YEB Recent Publications in this Series ESSI PÄIVÄRINTA 15/2016 Johanna Rytioja Enzymatic Plant Cell Wall Degradation by the White Rot Fungus Dichomitus squalens 16/2016 Elli Koskela Genetic and Environmental Control of Flowering in Wild and Cultivated Strawberries 17/2016 Riikka Kylväjä Staphylococcus aureus and Lactobacillus crispatus: Adhesive Characteristics of Two Gram- DISSERTATIONES SCHOLA DOCTORALIS SCIENTIAE CIRCUMIECTALIS Cloudberry and Its Components as Chemopreventive Constituents in Cloudberry and Its Components as Chemopreventive , Positive Bacterial Species ALIMENTARIAE, BIOLOGICAE. UNIVERSITATIS HELSINKIENSIS 6/2017 18/2016 Hanna Aarnos Photochemical Transformation of Dissolved Organic Matter in Aquatic Environment – From a Boreal Lake and Baltic Sea to Global Coastal Ocean 19/2016 Riikka Puntila Trophic Interactions and Impacts of Non-Indigenous Species in the Baltic Sea Coastal Ecosystems ESSI PÄIVÄRINTA 20/2016 Jaana Kuuskeri Genomics and Systematics of the White-Rot Fungus Phlebia radiata: Special Emphasis on Wood-Promoted Transcriptome and Proteome Cloudberry (Rubus chamaemorus) and Its Components 21/2016 Qiao Shi as Chemopreventive Constituents in ApcMin Mice and Synthesis and Structural Characterization of Glucooligosaccharides and Dextran from Weissella confusa Dextransucrases Human Colon Adenocarcinoma Cells 22/2016 Tapani Jokiniemi Energy Efficiency in Grain Preservation 23/2016 Outi Nyholm Virulence Variety and Hybrid Strains of Diarrheagenic Escherichia coli in Finland and Burkina Faso 24/2016 Anu Riikonen Some Ecosystem Service Aspects of Young Street Tree Plantings 25/2016 Jing Cheng The Development of Intestinal Microbiota in Childhood and Host-Microbe Interactions in Pediatric Celiac Diseases 26/2016 Tatiana Demina Molecular Characterization of New Archaeal Viruses from High Salinity Environments 27/2016 Julija Svirskaitė Apc Life in Extreme Environments: Physiological Changes in Host Cells during the Infection of Min Halophilic archaeal Viruses Mice and Human Colon Adenocarcinoma Cells 1/2017 Pauliina Kokko Towards more Profitable and Sustainable Milk and Beef Production System 2/2017 Juha Kontturi Type III Polyketide Synthases from Gerbera hybrida 3/2017 Pauliina Kärpänoja Antimicrobial Resistance in the Major Respiratory Tract Pathogens - Methods and Epidemiology 4/2017 Clara Isabel Lizarazo Torres Diversifying Boreal — Nemoral Cropping Systems for Sustainable Protein Production DEPARTMENT OF FOOD AND ENVIRONMENTAL SCIENCES 5/2017 Festus Anasonye FACULTY OF AGRICULTURE AND FORESTRY Treatment of Organic Aromatic Contaminants in Soil with Fungi and DOCTORAL PROGRAMME IN FOOD CHAIN AND HEALTH Biochar UNIVERSITY OF HELSINKI 6/2017 Helsinki 2017 ISSN 2342-5423 ISBN 978-951-51-3116-4 Department of Food and Environmental Sciences University of Helsinki Cloudberry (Rubus chamaemorus) and its components as chemopreventive constituents in ApcMin mice and human colon adenocarcinoma cells Essi Päivärinta ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public examination in Walter Hall, EE Building, on 12th May 2017, at 12 o’clock Helsinki 2017 Supervisors: Professor Marja Mutanen Department of Food and Environmental Sciences University of Helsinki, Finland Adjunct Professor Anne-Maria Pajari Department of Food and Environmental Sciences University of Helsinki, Finland Adjunct Professor Mikael Niku Faculty of Veterinary Medicine University of Helsinki, Finland Reviewers: Adjunct Professor Anna-Marja Aura VTT Technical Research Centre of Finland Ltd, Finland Professor Jan Erik Paulsen Department of Food Safety and Infection Biology Norwegian University of Life Sciences, Norway Opponent: Professor Sari Mäkelä Institute of Biomedicine University of Turku, Finland Cover photograph: Jyrki Päivärinta ISBN 978-951-51-3116-4 (paperback) ISBN 978-951-51-3117-1 (PDF) Dissertationes Schola Doctoralis Scientiae Circumiectalis, Alimentariae, Biologicae ISSN 2342-5423 (print) ISSN 2342-5431 (online) Hansaprint Helsinki 2017 To my family 3 ABSTRACT A diet rich in fruits and vegetables has been suggested to decrease the risk of colorectal cancer and the protective effects of these foods could probably be at least partly mediated by their polyphenolic compounds. The number of different polyphenols is huge and their effects on cells and tissues may differ. The promising results on chemopreventive effects of ellagitannin-rich pomegranate suggest that the other dietary sources of ellagitannins may also be effective in cancer prevention. The overall aim of the research reported in this thesis was to study the effects of cloudberry (Rubus chamaemorus), which is rich in ellagitannins, on all phases of intestinal tumour development. Specifically, the effects of cloudberry on the first two phases of carcinogenesis, initiation and promotion, were studied using the ApcMin mouse model. In addition, the effects of cloudberry extract on the indicators of the last phase of cancer development, progression and metastasis, and particularly cancer cell migration that is essential for this phase, were studied using scattering and wound healing assays in hepatocyte growth factor-induced HT29 and HCA7 human colon adenocarcinoma cell lines. A whole-cloudberry diet containing 10% (w/w) freeze-dried cloudberries significantly decreased the number and size of intestinal adenomas in ApcMin mice. In contrast, cloudberry seeds, cloudberry pulp, or pure ellagic acid, when incorporated into the diets in concentrations that corresponded to the whole- cloudberry diet, had no apparent effect. The effects of cloudberry on the development of adenomas were also compared with the effects of bilberry (Vaccinium myrtillus), which is rich in anthocyanins. Both berries decreased the number of adenomas, but their effects of adenoma size were different: thus, the adenoma size was decreased by cloudberry and increased by bilberry in comparison to the control diet. The opposite effects of the berries on tumour growth were associated with the changes in the gut microbiota, intestinal immunity, and the expression of energy metabolism-related genes. The activation of Met, which is also known as hepatocyte growth factor receptor induces cell migration and is considered to play an important role in tumour metastasis. Cloudberry extract inhibited cell migration by inhibiting the activation of Met in hepatocyte growth factor-induced human HT29 and HCA7 colon adenocarcinoma cells, and thus Met signalling and consequent activation of the phosphatidylinositol 3-kinase/AKT pathway. The activation of Met signalling in the tumours of cloudberry-fed ApcMin mice was also inhibited. 4 The results of the research presented in this thesis suggest that consumption of cloudberry or cloudberry preparations may reduce the risk of colon cancer, slow down the growth of colon adenomas, and have therapeutic value in reducing cancer progression and metastasis. Long-term studies in human subjects are needed to confirm these results. 5 ACKNOWLEDGEMENTS This study was carried out at the Department of Food and Environmental Sciences, Division of Nutrition, University of Helsinki. The work was financially supported by the Ministry of Agriculture and Forestry, the Finnish Cultural Foundation, the Yrjö Jahnsson Foundation, the Finnish Food Research Foundation, the Ida Montin Foundation, the Faculty of Agriculture and Forestry and the Association of Academic Agronomists. I owe my warmest thanks to my supervisor, Professor Marja Mutanen, for her enthusiastic and inventive guidance throughout this work. There were many times I lost my confidence in my ability to finish this work but you always helped me to continue. Thank you, Maisa, for your patience, understanding and optimism during this long process. My deepest thanks also belong to my other supervisors, Adjunct Professors Anne-Maria Pajari and Mikael Niku. Thank you, Anne-Maria, for the idea of focusing this work on cloudberry, for all those long sessions with studies II, IV and the manuscript of this thesis, and for always trusting in me and being one of my closest friends. And Mikael, thank you for your tireless and careful work while commenting on study II and the manuscript of this thesis. Your comments as a “non-nutritionist” were always so valuable! And, of course, thank you for guiding me through the jungle of immunology and immunohistochemical stainings! I sincerely thank Adjunct Professor Anna-Marja Aura and Professor Jan Erik Paulsen for reviewing my thesis and giving their valuable comments and suggestions to improve the manuscript. I am grateful to Dr. Alisdair Mclean for reviewing the English language of the manuscript, and also for raising important questions about the content of the thesis. I am grateful to all my co-authors for their contributions to the articles. Special thanks go to Dr. Markus Storvik for the pathway analyses and to Dr. Johanna Maukonen for the microbiological analyses of study II, to PhD student Tuuli Koivumäki for analysing the composition of cloudberry extract, to Dr. Lassi Paavolainen for analysing the time-lapse photograph data, and also to PhD student Elina Vaara for her excellent statistical analyses of study IV. My warmest thanks to laboratory technician Anu Heiman-Lindh for everything: you have really been so much more than my “right-hand-man” in the lab! I also want to thank all my present and former colleagues at the Division of Nutrition. 6 My special thanks go to Virpi Fredriksson for teaching me how to