Selecting Dietary-derived Bioactives which Protect Cartilage from Destruction in Osteoarthritis Thesis by: Jonathan Alex Green Thesis submitted for the degree of Doctor of Philosophy University of East Anglia School of Biological Sciences September 2015 © 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 there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. II Abstract Osteoarthritis (OA) is a disease that affects the whole joint, with progressive articular cartilage loss. Healthy cartilage must maintain a balance between extracellular matrix synthesis and degradation. Metalloproteinases (Matrix metalloproteinases, MMPs and a disintegrin and metalloproteinase domain with thrombospondin motifs, ADAMTSs) play an important role in cartilage degradation. The collagenases (MMP-1 and MMP-13) and aggrecanases (ADAMTS-4 and ADAMTS-5) are implicated in the development of OA because of their ability to degrade type 2 collagen and aggrecan. This project aims to identify compounds from the diet that could offer protection or slow the progression of OA via collagenase and/or aggrecanase inhibition. Ninety-six dietary derived compounds selected from a Natural Product Library were screened at 10µM for IL1-induced and basal MMP13 inhibition in SW1353 and C28/I2 cells. Several compounds inhibited MMP13 at 10µM in both cell lines. Twenty compounds were selected for study in primary human articular chondrocytes. Of these compounds, apigenin and isoliquiritigenin showed the greatest inhibition of MMP13 while also inhibiting MMP1, ADAMTS4 and ADAMTS5. The effect of apigenin or isoliquiritigenin pretreatment on three pathways implicated in OA, the NFκB, TGFβ and Wnt pathways was analysed. Apigenin and isoliquiritigenin showed differences in the patterns of inhibition in the NFκB and TGFβ pathways. The Human Phospho-Kinase Array Kit was used to perform an unbiased dissection of the kinase pathways affected by apigenin and isoliquiritigenin. Apigenin and isoliquiritigenin had similar effects on many phosphorylation events, however there were kinase phosphorylation events that were specific to either apigenin or isoliquiritigenin treatment. Taken together these data suggest that the chondro-protective gene expression changes seen with apigenin and isoliquiritigenin treatment involves a complex network of signalling pathways that require further characterisation. If these compounds are able to get to the site of OA damage they may prevent progression of OA offering an alternative treatment. III List of Contents Abstract ........................................................................................................................ II List of Contents ............................................................................................................ III List of Tables ............................................................................................................... VII List of Figures ............................................................................................................ VIII Acknowledgements ..................................................................................................... XI Abbreviations ............................................................................................................. XII Chapter 1: Introduction ................................................................................................ 1 1.1 Synovial Joint structure ................................................................................................ 1 1.1.1 Articular cartilage and structure ........................................................................... 2 1.1.2 The extracellular matrix structure and function ................................................... 3 1.1.3 The structure and function of type 2 collagen ...................................................... 5 1.1.4 The structure and function of aggrecan ............................................................... 9 1.1.5 Chondrocytes and their function in cartilage ..................................................... 11 1.2 Osteoarthritis ............................................................................................................. 13 1.2.1 Epidemiology ....................................................................................................... 13 1.2.2 Pathology ............................................................................................................ 14 1.2.3 Aetiology ............................................................................................................. 15 1.3 The Metalloproteinases ............................................................................................. 20 1.3.1 ADAMs and ADAMTSs ......................................................................................... 21 1.3.2 Matrix Metalloproteinases ................................................................................. 25 1.3.3 The regulation of Matrix Metalloproteinases ..................................................... 29 1.3.4 Tissue inhibitors of metalloproteinases .............................................................. 33 1.3.5 Matrix metalloproteinases in osteoarthritis ....................................................... 35 1.4 Current Treatments .................................................................................................... 37 1.5 Diet derived bioactives as a potential therapy .......................................................... 40 1.5.1 Flavonoids ........................................................................................................... 41 1.5.2 Flavan-3-ols ......................................................................................................... 42 1.5.3 Anthocyanins ....................................................................................................... 42 1.5.4 Flavonols ............................................................................................................. 42 1.5.4 Flavones .............................................................................................................. 43 1.5.5 Isoflavones .......................................................................................................... 43 1.5.6 Flavanones ............................................................................................................... 44 1.5.7 Carotenoids ............................................................................................................. 45 IV 1.5.8 Plant sterols ............................................................................................................. 46 1.5.9 Glucosinolates ......................................................................................................... 46 1.6 Conclusion and Aims................................................................................................... 48 Chapter 2: Materials and Method ............................................................................... 49 2.1 Materials .................................................................................................................... 49 2.1.1 Cell lines .................................................................................................................. 49 2.1.1.1 SW1353 ............................................................................................................ 49 2.1.1.2 C28/I2 ............................................................................................................... 49 2.1.1.3 Primary Articular Chondrocytes ....................................................................... 49 2.1.2 Diet derived bioactives............................................................................................ 50 2.1.2.1 Compound screens........................................................................................... 50 2.1.2.2 Selected compounds ........................................................................................ 50 2.1.3 Cytokines ................................................................................................................. 50 2.1.4 Immunoblotting ...................................................................................................... 50 2.2 Methods ..................................................................................................................... 51 2.2.1 Cell culture .............................................................................................................. 51 2.2.1.1 Cell treatments ................................................................................................. 51 2.2.1.2 Cytotoxicity assay cell necrosis ........................................................................ 52 2.2.1.3 Cytotoxicity assay cell apoptosis ...................................................................... 52 2.2.2 cDNA synthesis – Cells-to-cDNA II with MMLV RNA ............................................... 53 2.2.3 Quantitiative Real Time PCR (qRT-PCR) .................................................................. 53 2.2.3.1 Standard Probe-based Real-Time qRT-PCR ...................................................... 53 2.2.3.2 Universal Probe-based Real-Time qRT-PCR ..................................................... 55 2.2.4 Sub-cloning .............................................................................................................. 56 2.2.4.1 Preparation of competent DH5α cells Plasmid extraction (Qiagen mini preps) ...................................................................................................................................... 56 2.2.4.2 Plasmid extraction (Qiagen mini preps) ........................................................... 56 2.2.4.3 SW1353 transfection ........................................................................................ 57 2.2.5 Western blotting ..................................................................................................... 57 2.2.5.1 Bicinchoninic acid (BCA) protein quantification assay ..................................... 58 2.2.5.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ....... 58 2.2.5.3 Semi-dry blot transfer ...................................................................................... 59 2.2.6 Human Phospho-Kinase Antibody Array ................................................................. 59 2.2.7 Statistical analysis ................................................................................................... 60 Chapter 3: Dietary Derived Bioactive Screen ............................................................... 61 3.1 Introduction .......................................................................................................... 61 V 3.2 Results .................................................................................................................. 63 3.2.1 Designing the custom dietary derived library ......................................................... 63 3.2.2 Cell cytotoxicity screen ........................................................................................... 64 3.2.2.1 Dietary derived compounds effects on cell necrosis - Lactate dehydrogenase assay ............................................................................................................................. 64 3.2.3 Bioactive compound screen in SW1353 cells ......................................................... 65 3.2.3.1 SW1353 IL1 induced MMP13 expression screen ............................................. 65 3.2.3.2 SW1353 Basal MMP13 expression screen ....................................................... 66 3.2.4 Bioactive compound screen in C28/I2 cells ............................................................ 67 3.2.4.1 C28/I2 IL1 induced MMP13 expression screen................................................ 67 3.2.4.2 C28/I2 Basal MMP13 expression screen .......................................................... 68 3.2.4 Selecting compounds of interest ............................................................................ 69 3.3 Discussion ............................................................................................................. 71 3.3.1 Conclusion............................................................................................................... 77 3.3.2 Strengths of Chapter 3 ............................................................................................ 77 3.3.3 Weaknesses of Chapter 3 ........................................................................................ 77 Chapter 4: Short-listing dietary-derived bioactives in human primary knee osteoarthritis chondrocytes .............................................................................................................. 78 4.1 Introduction .......................................................................................................... 78 4.2 Results .................................................................................................................. 81 4.2.1 The effect of the twenty shortlisted compounds on genes of interest in KOA chondrocyte cells ............................................................................................................. 81 4.2.1.1 Effect on IL1 induced MMP13 in KOA chondrocytes ....................................... 81 4.2.1.2 Effect on basal MMP13 in KOA chondrocytes ................................................. 83 4.2.1.3 Effect on IL1 induced MMP1 in KOA chondrocytes ......................................... 84 4.2.1.4 Effect on basal MMP1 in KOA chondrocytes ................................................... 86 4.2.1.5 Effect on IL1 induced ADAMTS4 in KOA chondrocytes .................................... 87 4.2.1.6 Effect on basal ADAMTS4 in KOA chondrocytes .............................................. 88 4.2.1.7 Effect on basal ADAMTS5 in KOA chondrocytes .............................................. 89 4.2.2 Selecting compounds for further study .................................................................. 90 4.2.3 IL1 stimulated MMP13 Time course ....................................................................... 91 4.2.3.1 Apigenin Time course ....................................................................................... 91 4.2.3.2 Isoliquiritigenin Time course ............................................................................ 92 4.2.4 Dose response curves - Apigenin ............................................................................ 93 4.2.4.1 Apigenin dose response curve - effect on IL1 induced MMP13 in KOA chondrocytes ................................................................................................................ 93 4.2.4.2 Apigenin dose response curve - effect on IL1 induced MMP1 in KOA chondrocytes ................................................................................................................ 94 VI 4.2.4.3 Apigenin dose response curve - effect on IL1 induced ADAMTS4 in KOA chondrocytes ................................................................................................................ 95 4.2.4.4 Apigenin dose response curve - effect on Basal ADAMTS5 in KOA chondrocytes ...................................................................................................................................... 96 4.2.4.5 Apigenin dose response curve - effect on Basal HO-1 in KOA chondrocytes .. 97 4.2.4.6 Apigenin dose response curve - effect on Basal AXIN2 in KOA chondrocytes 98 4.2.5 Dose depended response curves - Isoliquiritigenin ................................................ 99 4.2.5.1 Isoliquiritigenin dose response curve - effect on IL1 induced MMP13 in KOA chondrocytes ................................................................................................................ 99 4.2.5.2 Isoliquiritigenin dose response curve - effect on IL1 induced MMP1 in KOA chondrocytes ..............................................................................................................100 4.2.5.3 Isoliquiritigenin dose response curve - effect on IL1 induced ADAMTS4 in KOA chondrocytes ..............................................................................................................101 4.2.5.4 Isoliquiritigenin dose response curve - effect on Basal ADAMTS5 in KOA chondrocytes ..............................................................................................................102 4.2.5.5 Isoliquiritigenin dose response curve - effect on Basal HO-1 in KOA chondrocytes ..............................................................................................................103 4.2.5.6 Isoliquiritigenin dose response curve - effect on Basal AXIN2 in KOA chondrocytes ..............................................................................................................104 4.2.6 Cell cytoxicity screens ...........................................................................................105 4.2.6.1 Apigenin effect on cell necrosis - Lactate dehydrogenase assay ...................105 4.2.6.2 Isoliquiritigenin effect on cell necrosis - Lactate dehydrogenase assay ........106 4.2.6.3 Apigenin effect on cell apoptosis - Caspase-Glo® 3/7 Assay .........................107 4.2.6.4 Isoliquiritigenin effect on cell apoptosis - Caspase-Glo® 3/7 Assay ..............108 4.3 Discussion ........................................................................................................... 109 4.3.1 Conclusion.............................................................................................................120 4.3.2 Strengths of Chapter 4 ..........................................................................................121 4.3.3 Weaknesses of Chapter 4 ......................................................................................121 Chapter 5: Mechanism of action of apigenin and isoliquiritigenin .............................. 122 5.1 Introduction ........................................................................................................ 122 5.2 Results ................................................................................................................ 125 5.2.1 NFκB, TGFβ and Wnt pathways in SW1353 cells ..................................................125 5.2.1.1 Cytokine stimulated luciferase assay Time course ........................................126 5.2.1.2 Effect of apigenin on the NFκB pathway in SW1353 cells .............................127 5.2.1.3 Effect of apigenin on the Smad2/3/4 pathway in SW1353 cells ...................129 5.2.1.4 Effect of apigenin on the Wnt pathway in SW1353 cells ...............................131 5.2.1.5 Effect of isoliquiritigenin on the NFκB pathway in SW1353 cells ..................133 5.2.1.6 Effect of isoliquiritigenin on the Smad2/3/4 pathway in SW1353 cells ........135 VII 5.2.1.7 Effect of isoliquiritigenin on the Wnt pathway in SW1353 cells ...................137 5.2.2 The effect of apigenin on MAPK kinase expression and activation ......................139 5.2.3 The effect of isoliquiritigenin on MAPK kinase expression and activation ...........141 5.2.4 Human Phospho-Kinase Array ..............................................................................143 5.3 Discussion ........................................................................................................... 149 5.3.1 Conclusion.............................................................................................................159 Chapter 6: General discussion ................................................................................... 164 6.1 Summary of findings ...............................................................................................164 6.1.1 Dietary Derived Bioactive Screen ......................................................................164 6.1.2 Short-listing dietary-derived bioactives in human primary knee osteoarthritis chondrocytes ..............................................................................................................166 6.1.3 Ascertaining mechanism of action ....................................................................168 6.2 Future directions .....................................................................................................172 6.3 Conclusions ..............................................................................................................174 Bibliography ............................................................................................................. 175 Appendix .................................................................................................................. 208 List of Tables Table 1. Collagens of cartilage tissue Table 2. ADAMTS genes Table 3. MMP genes Table 4. Quantitative Real Time PCR primer probe sets Table 5. Twenty selected compounds Table 6. Selecting compounds for further study Table 7. The effect of isoliquiritigenin treatment on the Human Phospho Kinase Array. Table 8. The effect of apigenin treatment on the Human Phospho Kinase Array. VIII List of Figures Figure 1.1. Diagram showing the structure of the synovial joint Figure 1.2. The four distinct regions of matrix Figure 1.3. The assembly of aggregates and extracellular matrix components Figure 1.4. Structure of collagen Figure 1.5. Collagen fibril formation Figure 1.6. Schematic representation of aggrecan Figure 1.7. Common structure of ADAMTSs Figure 1.8. Common structure domain of an MMP Figure 1.9. MMP regulation Figure 1.10. MMP Promoters Figure 1.11. Structure of TIMP Figure 1.12. Structures of Flavonoid subgroups Figure 1.13. Structure of β-carotene Figure 1.14. Structure of Stigmasterol Figure 1.15. Structure of Sulforaphane Figure 3.1. Custom dietary derived compound library design Figure 3.2. Fold change in percentage cell necrosis relative to untreated control samples in SW1353 Figure 3.3. Fold change in MMP13 gene expression relative to IL1 treated control samples in SW1353 cells Figure 3.4. Fold change in MMP13 gene expression relative to untreated control samples in SW1353 cells Figure 3.5. Fold change in MMP13 gene expression relative to IL1 treated control samples in C28/I2 cells Figure 3.6. Fold change in MMP13 gene expression relative to untreated control samples in C28/I2 cells Figure 3.7. Four set Venn diagram Figure 4.1 Dietary derived compounds effect on IL1 stimulated MMP13 in KOA cells Figure 4.2. Dietary derived compounds effect on unstimulated MMP13 in KOA cells IX Figure 4.3 Dietary derived compounds effect on IL1 stimulated MMP1 in KOA cells Figure 4.4. Dietary derived compounds effect on unstimulated MMP1 in KOA cells Figure 4.5. Dietary derived compounds effect on IL1 stimulated ADAMTS4 in KOA cells Figure 4.6. Dietary derived compounds effect on unstimulated ADAMTS4 in KOA cells Figure 4.7. Dietary derived compounds effect on unstimulated ADAMTS5 in KOA cells Figure 4.8. Apigenin treated MMP13 inhibition Time course Figure 4.9. Isoliquiritigenin treated MMP13 inhibition Time course Figure 4.10. Apigenin IL1 induced MMP13 dose response Figure 4.11. Apigenin IL1 induced MMP1 dose response Figure 4.12. Apigenin IL1 induced ADAMTS4 dose response Figure 4.13. Apigenin basal ADAMTS5 dose response Figure 4.14. Apigenin basal HO-1 dose response Figure 4.15. Apigenin basal AXIN2 dose response Figure 4.16. Isoliquiritigenin IL1 induced MMP13 dose response Figure 4.17. Isoliquiritigenin IL1 induced MMP1 dose response Figure 4.18. Isoliquiritigenin IL1 induced ADAMTS4 dose response Figure 4.19. Isoliquiritigenin basal ADAMTS5 dose response Figure 4.20. Isoliquiritigenin basal HO-1 dose response Figure 4.21. Tukey Box and whisker plot showing fold change in AXIN2 gene expression relative to no treatment in KOA chondrocytes Figure 4.22. Apigenin has no effect on cell necrosis Figure 4.23. Isoliquiritigenin has no effect on cell necrosis Figure 4.24. Apigenin has no effect on cell apoptosis Figure 4.25. Isoliquiritigenin has no effect on cell apoptosis Figure 4.26. The chemical structure of ursolic acid Figure 4.27. The chemical structure of genistin Figure 4.28. The chemical structure of luteolin Figure 4.29.The chemical structure of emodin Figure 4.30. The chemical structure of aloe-emodin X Figure 4.31. The chemical structure of isoliquiritigenin Figure 4.32. The chemical structure of apigenin Figure 5.1. The NFκB, Smad2/3/4 and Wnt signalling reporters Figure 5.2. The regulation of the NFκB, TGFβ and Wnt pathways by IL1 Figure 5.3. The effect of apigenin on the NFκB pathway Figure 5.4. The effect of apigenin on the Smad2/3/4 pathway Figure 5.5. The effect of apigenin on the Wnt pathway Figure 5.6. The effect of isoliquiritigenin on the NFκB pathway Figure 5.7. The effect of isoliquiritigenin on the Smad2/3/4 pathway Figure 5.8. The effect of isoliquiritigenin on the Wnt pathway Figure 5.9. The effect of apigenin on MAPKs Figure 5.10. The effect of isoliquiritigenin on MAPKs Figure 5.11. Alteration of kinase signalling in KOA cells after isoliquiritigenin or apigenin treatment Figure 5.12a. Analysis of IL1 stimulated kinase signalling in KOA cells after isoliquiritigenin or apigenin treatment relative to IL1 fold change Figure 5.12b. Analysis of IL1 stimulated kinase signalling in KOA cells after isoliquiritigenin or apigenin treatment relative to unstimulated control fold change Figure 5.13. Summary of the effects of apigenin or isoliquiritigenin treatment on the Wnt pathway. Figure 5.14. Summary of the effects of apigenin or isoliquiritigenin treatment on the NFκB pathway. Figure 5.15. Summary of the effects of apigenin or isoliquiritigenin treatment on the AP-1 pathway.