isothiocyanates Rose Davidson Biomedical Research Centre University of East Anglia School Of Biological Sciences Ian Clark Kirsty Culley Rose Davidson Orla Jupp Sarah Gardner Jonathan Green ID: 1043248
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1. Investigating the chondroprotective properties of isothiocyanatesRose DavidsonBiomedical Research CentreUniversity of East Anglia
2. School Of Biological SciencesIan Clark Kirsty CulleyRose DavidsonOrla JuppSarah GardnerJonathan Green Kimberley Hirst-Jones Tracey SwinglerYvette WormstoneNatalie CroweLinh Le School Of MedicineYongping BaoAedin Cassidy Alex MacgregorGeoffrey GuileColin KayRachel de FerrarsSchool Of PharmacyRosemary NortonMaria O’ConnellNIHR Flexibility and Sustainability FundingInstitute of Orthopaedics, NNUHSimon DonellSue ButtersAngela BulloughTracey PotterLaura WattsKings College, LondonFrances WilliamsTim SpectorKennedy Institute of Rheumatology, OxfordTonia VincentOlga BorucClare DriscollHG Blake LtdInstitute for Food ResearchRichard MithenCharlotte ArmahMaria TrakaNIHR Flexibility and Sustainability FundingHG Blake Ltd
3. Osteoarthritis – the problemDevelopment of functional foods or food products, beneficial in the treatment or prevention of OA.8.5 million people have OA in UK, 71% of which are in constant pain.By 2030 >17 million projected to have OA (50% population >50yrs).Wang, McPherson et al (2011). OANation 2012 Report: 49% of OA patients visiting GP:‘would like more information about other areas of life that might affect their OA such as diet and exercise.’
4. How can we stop cartilage degradation?Dietary factors
5. Biochemical changesdecrease in aggrecandamage to the type II collagen networkMorphological changesfibrillations/pittingsoftening and loss of cartilage thicknessChanges in gene expression OSTEOARTHRITICNORMALChanges in cartilage during OA
6. Metalloproteinases and their inhibitors Matrix metalloproteinases23 human enzymes(Collagenases MMP-1, -8, -13, -2, -14?)ADAMTSs19 human enzymes(Aggrecanases ADAMTS-1, -4, -5, -8, -9, -15?)TIMPs4 human inhibitors
7. How can we stop cartilage degradation?Dietary factorsWow! What a drug… and no side effects so far…
8. Dietary-derived factors and osteoarthritisBest studied: chondroitins and glucosamineSystematic analyses: small effect for pharmaceutical grade chondroitin sulfate.
9. Dietary-derived factors and osteoarthritis - ModelsVitamins A, B(1, 2, 3, 6), C, D, E, Kn3 vs n6 PUFAFlavonols (quercetin, kaempferol, myricetin)Flavones (apigenin, luteolin, tangeritin, nobiletin)Flavan-3-ols (EGCG, proanthocyanadins)AnthocyaninsIsoflavones (soy, genistein)Flavanones (hesperetin, naringen)Carotenoids (lutein, zeaxanthin, beta-cryptoxanthin)Plant sterols (stigmasterol, campesterol)Glucosinolates (glucoraphanin)ResveratrolCurcuminAvocado-soybean unsaponifiablesGinger(extract of edible bird’s nest)Cell dataExplant workAnimal model dataDose?Method of delivery?Thoroughness?
10. Intervention trials - osteoarthritisVitamin CPeregoy et al 2011 Public Health Nutr 14:709-15Vitamin ECanter et al 2007 Rheumatology 46:1223-33Vitamin DMcAlindon et al 2013 JAMA 309:155-62Tart cherry juiceSchumacher et al 2013 Osteoarthritis Cart 21:1035-41SoyArjmandi et al 2004 Phytomedicine 11:567-75Curcumin (phosphatidyl choline complex)Belcaro G et al 2010 Panminerva Med 52:55-62Avocado-soybean unsaponifiablesMaheu et al 2014 Ann Rheum Dis 73:376-84GingerLeach et al 2008 Int J Evid Based Healthc 6:311-20Small scaleInterpretation
11. Observational StudySt Thomas’ Twins UK registryMatched co-twin case control design compares discordant exposure- disease status Radiographic OA determined at the hand hip and kneeLumbar and cervical degeneration assessed by MRI
12. Peripheral Joints
13. Fruit and vegetable pattern at the hipAllium vegetablesCruciferous vegetablesGreen leafy vegetablesYellow vegetablesOther vegetablesCitrus fruitNon-citrus fruitChips and roast potatoesOdds ratio
14. Laboratory studiesOrganosulfur compounds derived from garlic oil
15. Diallyl disulphide regulates the expression of key MMP genes
16. SummaryMatched analysis indicates that these findings are less likely to be confounded by other lifestyle factorsHowever, the effects are small effects and (despite the dietary pattern approach) not robust to multiple testing
17. Fruit and vegetable pattern at the hipAllium vegetablesCruciferous vegetablesGreen leafy vegetablesYellow vegetablesOther vegetablesCitrus fruitNon-citrus fruitChips and roast potatoesOdds ratio
18.
19. Sulforaphane1-isothiocyanato-(4R)-(methylsulfinyl) butaneglucoraphaninmyrosinaseglucoraphaninmyrosinaseSFNPlant injuryPathogen attackChewingFood prep
20. In man, 68g broccoli sprouts was sufficient to show HDACi in peripheral blood mononuclear cells (Myzack et al., 2007)
21. Histone acetylationSW1353 cell lineControlTSAIL-1/OSMIL-1/OSM/TSA51015305101530SFN (M)IL-1/OSMAcH3Total H3AcLys No differences in: Histone acetylation Cytotoxicity Apoptosis
22. 46kDa MMP-1 43kDa MMP-13 SFN (µM) 0 5 10 15 30 IL-1/OSM + + + + +Chondroprotection in vitrosulforaphane
23. Mechanism?
24. Deducing the pathways involved – Nrf2SFN inhibits MMP expression independently of Nrf2(cf HO-1 control)
25. Deducing the pathways involved – MAP kinases
26. BBBBBLuciferaseIB promoterReporterDeducing the pathways involved – NFκB
27. IL-6IL-8iNOSSOD2Deducing the pathways involved – NFκB
28. p65 phosphorylation and translocation
29. phos-p65IBGAPDHIL-1/OSM time (mins)0515306005153060+ SFNIB degradation and p65 phosphorylation
30. GAPDHp65CNControlCNIL-1CNSFNCNSIp65 translocationC – cytoplasmicN - nuclear
31. ControlSFN 30minIL1 45minSFN 30min + IL1 45minp65mergeDAPIp65 translocationDr Orla Jupp
32. NF-B DNA binding (EMSA)
33. Direct inhibition of NFκB bindingDr Orla Jupp
34. In vivo efficacy of sulforaphane DMM (destabilisation of the medial meniscus) model of murine osteoarthritis collaboration with Tonia Vincent, Kennedy Institute for RheumatologyArthritis Research UK Project: Dr Orla JuppGlasson SS et al Osteoarthritis Cartilage. 2007 15(9):1061-9.
35. Control ChowLow SFN chow0.18mg/g chow(1µmol/g)x8 DMMx4 Sham Histology to score osteoarthritis in right hind knee joint2. Collection of tissue (knee & serum) for LC-MS/MS12 mice in each groupTonia Vincent, Kennedy Institute for RheumatologyIn vivo efficacy of sulforaphane
36. In vivo efficacy of sulforaphane
37. In vivo efficacy of sulforaphaneGene expressionSFN inhibited genesInjury induced genes
38. SummarySFN (and DADS) is reported as an HDAC inhibitorIn chondrocytes, SFN can attenuate the expression of metalloproteinase genes in a dose-dependent mannerThis is not through the inhibition of histone deacetylases and not via Nrf2 but most likely via inhibition of NFκBSFN blocks cartilage destruction in vitro and in vivo
39. Where next?
40. “Having to eat broccoli twice a week is not child abuse”Proof-of-principle human study
41. 1 Week washout2 Week dietCollect bloodCollect knee joint tissuesAnalyse:Gene expressionITC tissue levelsIn vitro degradation ratesMetabolomics.Proof-of-principle human study
42. ITC content of broccoli intervention
43. Patients consented 35Patients completed 26Patients not completed 2Proof-of-principle human study
44. Other dietary-derived isothiocyanates
45. IsothiocyanateGlucosinolate(precursor)Food SourcesAllyl isothiocyanate(AITC)SinigrinBroccoli, Brussel sprouts, Cabbage, Horseradish, MustardBenzyl isothiocyanate(BITC)GlucotropaeolinCabbage, Garden cressPhenethyl isothiocyanate(PEITC)GluconasturtiinWatercressSulforaphaneGlucoraphaninBroccoli, Brussel Sprouts, CabbageIberinGlucoiberinBroccoliErucinGlucoerucinRocketErysolinGlucoerysolinBroccoli, RocketCruciferous vegetables contain a variety of glucosinolates, each of which forms a different isothiocyante when hydrolysed.Dr Orla Jupp
46. 0 1.25 2.5 5 10µMOther isothiocyanatesDr Orla JuppErysolinSFNIberinErucinBITCAITCPEITC
47. The three most efficacious ITC’s do not display synergistic activity
48. Synergism with other compound classes…Mr Jonathan Green
49. SummaryOther isothiocyanates can inhibit IL-1-induced MMP or ADAMTS expression with varying efficacyA screen of dietary-derived compounds in both SW1353 chondrosarcoma cells and C28I/2 transformed chondrocytes uncovers compounds which alter MMP expressionA number of compounds have been analysed in primary human articular chondrocytesMechanism of selected compounds will be dissected in detail
50. ConclusionEpidemiology shows a protective association between food groups and osteoarthritisDADS and SFN are reported as HDAC inhibitorsIn chondrocytes, SFN can attenuate the expression of metalloproteinase genes This is not through the inhibition of histone deacetylases and not via Nrf2 but most likely via inhibition of NFκBSFN blocks cartilage destruction in vitro and in vivoOther ITCs show efficacyProof-of-concept human trial underwayPotential new diet-derived bioactives have been identifed and are being characterised