Exploring Plant Stilbenes For Healthy Skin A thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy By Erica Hawkins John Innes Centre Norwich September 2018 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived therefrom must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution Abstract Accumulating evidence supports pharmacological roles for the plant stilbene, resveratrol in aging and disease contexts. Pterostilbene, a derivative of resveratrol, has been shown to have higher bioavailability and bio-efficacy than resveratrol, but is not abundant in natural sources. Previous work demonstrated that expression of the stilbene synthase gene, isolated from grapevine Vitis vinifera, under the control of a plant-wide promoter, led to a variety of growth and fertility problems in the transformed plants. To circumvent this problem two transgenic tomato lines were developed in this thesis to produce resveratrol and pterostilbene specifically in the fruit of tomato. An ex vivo full thickness human skin explant model was used to investigate the biological effects of aqueous tomato juices on normal human skin, and in an inflammatory disease-like context by treatment with inflammatory cytokines. Microarray analysis and qRT-PCR validation identified the differential regulation of several genes in human skin with tomato juice treatment, including a significant downregulation in expression of the aging-associated matrix metalloproteinase, MMP-12, observed with both wild type and resveratrol-enriched tomato extracts. MMP-12 protein could be detected in human skin explant media but regulation at this level proved elusive. Analysis of elastin fibres, a crucial structural component of skin, revealed subtle effects of tomato extracts. The effect of tomato extracts on reepithelialisation aspects of wound-healing was assessed using a full thickness skin explant model, in addition to scratch-wounds of keratinocyte and fibroblast skin cell lines. Initial studies of resveratrol- and pterostilbene-enriched tomato extracts indicated a greater biological activity associated with pterostilbene, and a potential inhibitory effect on migration of cells. Overall novel tomato lines have been generated and their fruit showed effects which could be relevant in the prevention of skin aging and in overcoming the consequences of inflammation. i Acknowledgements There are many people that I would like to thank for their support over the last four years. Firstly, I would like to thank my supervisors Prof. Cathie Martin, and Dr Jelena Gavrilovic for giving me the opportunity to join their labs and providing me with scientific and emotional support throughout my PhD. I am extremely grateful to all of those in both the Martin and Gavrilovic labs, past and present, who helped to show me numerous techniques, and who shared their knowledge with me. I am fortunate to have had a lab filled with so many friends, and with whom I can share countless memories. A special thanks to Dr Eugenio Butelli and Dr Damon Bevan who taught me so much throughout my project, and kept the project going as I moved between the JIC and UEA! Dr Daniel Knevitt, thank you for being my office pal for two years, and keeping me entertained, and Dr Lionel Hill, thank you for helping me solve all the conundrums I came to you with, and for your friendship. None of this would have been possible without the support of my family. Mum, Dad and Kirsten, you put up with me through all my highs and lows, and I do not know what I would have done without you. To all my friends here in Norwich, thank you for making the last four years so memorable, I am lucky to have shared them with you. Finally, Athina and Fabi, all my love and gratitude go to you both, for your unending friendship and ability to always brighten my day. ii List of Abbreviations 3T3 "3-day transfer, inoculum 3×105 cells." Mouse fibroblast cell line 4CL 4-courmarate coenzyme A A. tumefaciens Agrobacterium tumefaciens ACN Acetonitrile AKT Protein kinase B ANS Anthocyanin Synthase BCA Bicinchoninic Acid C4H Cinnamate-4 hydroxylase CaMV Cauliflower mosaic virus cDNA Complementary DNA CHI Chalcone isomerase CHS Chalcone synthase COL17A1 Collagen, type 17, alpha 1 COL7A1 Collagen, type 7, alpha 1 COX Cyclooxygenase CT Cycle threshold DAB 3,3’ Diaminobenzidine DFR Dihydroflavonol-4-Reductase dH2O Distilled water DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide dNTPs Deoxyribonucleotide Triphosphate DW Dry Weight E. coli Escherichia coli EB Epidermolysis bullosa ECM Extracellular matrix EDN1 Endothelin 1 EDTA Ethylenediaminetetraacetic acid EGF Epidermal Growth Factor EGFR Epidermal Growth Factor Receptor EGTA Egtazic acid F3H flavanone-3-hydroxylase FCS Foetal Calf Serum FLS Flavonol Synthase FW Fresh weight iii GW Gateway HaCaT Human Keratinocyte cell line HPLC High Performance Liquid Chromatography HRP Horseradish Peroxidase IFS Isoflavone Hydroxylase IHC Immuno Histochemistry IL Interleukin IL-1A Interleukin 1 alpha iNOS Induced Nitric Oxide Synthase JIC John Innes Centre kb Kilobase pair kDa Kilo Dalton KO Knock Out LCMS IT-TOF Ion Trap and time of flight liquid chromatography/ mass spectrometer MAPK Mitogen-activated protein kinase MBW MYB-bHLH-WD40 complex MeOH Methanol MM Money Maker (WT tomato) MMP Matrix metalloproteinase mRNA Messenger RNA NF-KB Nuclear factor kappa-light-chain-enhancer of activated B cells) NHEK Normal Human Epidermal Keratinocytes OSM Oncostatin M PAL Phenylalanine PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PDA Photo Diode Detector PI3 Phosphoinositide 3-kinase PVDF Polyvinylidene difluoride qRT-PCR Quantitative Reverse Transcriptase Polymerase Chain Reaction RIPA Radioimmunoprecipitation assay buffer RNA Ribonucleic acid ROMT Resveratrol-o-methyltransferase SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis StSy Stilbene Synthase TCA Trichloroacteic acid iv UEA University of East Anglia UV Ultraviolet v/v Volume/volume w/v Weight/volume WB Western Blot WT Wild type v Table of Contents Abstract ....................................................................................................................................................... i Acknowledgements ..................................................................................................................................... ii List of Abbreviations .................................................................................................................................. iii Table of Contents ....................................................................................................................................... vi Table of Figures ........................................................................................................................................ xiii Table of Tables ........................................................................................................................................ xvii Chapter 1: Introduction ........................................................................................................................... 1 Plant secondary metabolites ............................................................................................................ 2 Stilbenes ............................................................................................................................................ 2 Resveratrol ................................................................................................................................... 4 Pterostilbene ................................................................................................................................ 4 Industrially synthesising stilbenes ..................................................................................................... 6 Plant Cell cultures ......................................................................................................................... 7 Engineering plants for increased stilbene production....................................................................... 7 Metabolically engineering a high-stilbene tomato ...................................................................... 8 Health benefits of polyphenols ....................................................................................................... 10 Pterostilbene and health ............................................................................................................ 11 Plant polyphenols and skin ............................................................................................................. 12 Introduction to Skin......................................................................................................................... 15 Skin structure and function ........................................................................................................ 15 Skin aging........................................................................................................................................ 17 Inflammation in skin ....................................................................................................................... 17 Matrix metalloproteinases ............................................................................................................