A Phase 2 Study to Assess the Immunomodulatory Capacity of ...

Curcumin is a botanical with anti-tumor and immunomodulatory properties. We hypothesized that curcumin supplementation might influence ... ThisarticleispartoftheResearchTopic ImmunonutrientSupplementation Viewall 15 Articles Articles TheaMagrone UniversityofBariAldoMoro,Italy ZhijingTan MichiganMedicine,UniversityofMichigan,UnitedStates LeiShi GeorgiaStateUniversity,UnitedStates Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction MaterialsandMethods ResultsandDiscussion Discussion EthicsStatement AuthorContributions Funding ConflictofInterestStatement Acknowledgments References SuggestaResearchTopic> DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex totalviews ViewArticleImpact SuggestaResearchTopic> SHAREON OpenSupplementalData CLINICALTRIALarticle Front.Nutr.,11January2019 |https://doi.org/10.3389/fnut.2018.00138 APhase2StudytoAssesstheImmunomodulatoryCapacityofaLecithin-basedDeliverySystemofCurcumininEndometrialCancer SandraTuyaerts1,2*,KlaraRombauts3,TinaEveraert1,AnM.T.VanNuffel3andFrédéricAmant1,2,4 1LaboratoryofGynaecologicOncology,DepartmentofOncology,KULeuven,Leuven,Belgium 2LeuvenCancerInstitute,Leuven,Belgium 3AnticancerFund,Strombeek-Bever,Belgium 4CenterforGynecologicOncologyAmsterdam,AmsterdamUniversityMedicalCenter,AntonivanLeeuwenhoekHospital,NetherlandsCancerInstitute,Amsterdam,Netherlands Curcuminisabotanicalwithanti-tumorandimmunomodulatoryproperties.Wehypothesizedthatcurcuminsupplementationmightinfluenceinflammatorybiomarkerlevelsinendometrialcarcinoma(EC).Inthisopen-label,non-randomizedphase2study(NCT02017353),sevenECpatientsconsumed2g/dayCurcuminPhytosome(CP)orallyfor2weeks.Bloodwastakenatbaseline,days1,7,14,and21.Thefollowinganalytesweremeasured:curcuminoidsandmetabolites,56inflammatorybiomarkers,COX-2,frequenciesofmyeloid-derivedsuppressorcells,dendriticcellsandNKcells,expressionofMHCmoleculesonleukocytesandmonocytesandactivation/memorystatusofTcells.Patientscompletedqualityoflife(QoL)questionnairesatbaselineandendoftreatment.CurcuminmetabolitesweredetectableinplasmauponCPintake.CPdownregulatedMHCexpressionlevelsonleukocytes(P=0.0313),thefrequencyofmonocytes(P=0.0114)andICOSexpressionbyCD8+Tcells(P=0.0002).However,CPupregulatedCD69levelsonCD16−NKcells(P=0.0313).Nodifferenceswereobservedregardinginflammatorybiomarkers,frequenciesofotherimmunecelltypes,TcellactivationandCOX-2expression.Anon-significanttrendtoimprovedQoLwasobserved.Overall,CP-inducedimmunomodulatoryeffectsinECweremodestwithoutsignificantQoLchanges.Giventhesmallpopulationandtheobservedvariabilityininter-patientbiomarkerlevels,moreresearchisnecessarytoexplorewhetherbenefitsofCPcanbeobtainedinECbydifferentsupplementationregimens. ClinicalTrialRegistration:www.ClinicalTrials.gov,identifierNCT02017353;www.clinicaltrialsregister.eu,identifier2013-001737-40. Introduction CurcuminisapolyphenolderivedfromtheplantCurcumalonga(commonnameTurmeric).ItisusedintraditionalAyurvedicmedicine.Besidescurcumin,turmericalsocontainsdemethoxycurcuminandbisdemethoxycurcumin,togetherformingthecurcuminoids(1).Severalpreclinicalstudiesdocumentedtheanticancereffectsofcurcumin,bymodulatingmoleculesimplicatedincancer,suchasNF-κB,COX-2,lipooxygenase,andproteinkinaseC(2,3).Furthermore,curcuminhasalsobeenshowntopotentiatetheanticancereffectsofconventionalanticancertherapiessuchaschemotherapyorradiationbysensitizingcancercellstotheircytocidaleffects(4,5). Besidesitsdirecteffectsoncancercells,emergingdatapointtowardanti-inflammatoryandimmune-modulatoryeffectsofcurcuminthatcouldplayaroleinitsanti-tumoreffects(6).Curcuminhasbeenshowntoinhibittheaccumulationofmyeloid-derivedsuppressorcells(MDSC)andtheirinteractionwithcancercellsandinducesthedifferentiation/maturationofMDSC(7).CurcuminreducedintratumoralIL-6productionandmetastasisformationinabreastcancermodeland,whencombinedwithcryoablation,inducedrobustanti-tumorTcellimmunityandreducedtumorgrowth(8).InRAW264.7murinemacrophages,acurcuminformulationsignificantlydecreasedtheLPS-inducedpro-inflammatorymediatorsNO,PGE2,andIL-6byinhibitingactivationofNF-κB(9).InIFNγ-stimulatedmurinebonemarrow-deriveddendriticcells(DCs),curcuminhasshowntoinhibittheexpressionandfunctionalityofindoleamine-2,3-dioxygenase,amajorimmunosuppressiveenzymeintumorimmunology(10).ThroughinhibitionofCOX-2,curcuminalsoreducedPGE2production,whichexertspotentimmunosuppressiveeffectsinthetumormicroenvironment(1).Recently,curcuminhasshowntoinhibitinflammation-mediatedPD-L1expression,animmunecheckpointenablingtumorstoevadetheimmuneresponse(11).Incontrast,severalfeaturesthatcouldbedetrimentaltoanti-tumorimmunityhavealsobeenattributedtocurcumin,suchasinductionofIL-10(12),inhibitionofTcellresponses(13),inhibitionofdendriticcellmaturation(13),andinductionofregulatoryTcells(14). Amajorobstaclehamperingtheimplementationofcurcuminintheclinicisitspoorbioavailability.Mostpreclinicalstudieshaveinvestigatedtheeffectsofcurcuminatdosagesimpossibletoobtainafteroralintakeofcurcumin.Variousapproacheshavebeendevelopedtoimprovethebioavailabilityofcurcumin.Afirstapproachistheuseoftheadjuvantpiperine,whichincreasescurcuminbioavailabilitybyinhibitingtheenzymesUDP-glucuronyltransferase(UGT)andsulfotransfereases(SULT)thatareresponsiblefortransformationofcurcuminintocurcuminglucuronideandcurcuminsulfate(15–17).Second,variousformulationsofcurcumindeliverysystemshavebeendevelopedtoimprovebioavailability.Thiscomprisestheuseofnanoparticles/nanoemulsions,complexeswithphospholipids,formulationwithsolubledietaryfibers,micronization,micellization,andotheragents(17–22).Finally,curcuminderivativesandanalogshavebeensynthesizedtoimprovethebiologicalactivityofcurcumin.However,althoughmanycurcuminanalogshaveshownimprovedbiologicalactivityovercurcumin,specificevaluationsofstructuralanalogsand/orderivativesofcurcumintoimprovedtissueandplasmadistributionarelacking(17).Althoughalltheseformulationsclaimtoimprovecurcuminbioavailability,plasmalevelsremainquitelow,duetorapidmetabolismandpossiblyuptakeintotissues.Moreover,extensivevariabilityinthestudiesmakesitdifficulttodirectlycompareandconcludewhichformulationisbetterthantheother.CurcuminPhytosome(CP)isapatentedformulationofturmericextractwithsoylecithin.Thesetwocomponentsformanon-covalentadductina1:2ratio,andtwopartsofmicrocrystallinecelluloseareaddedtoimproveformulation,withanoverallcurcuminoidcontentof20%(15).Thisformulationimprovestheplasmalevelsofcurcuminanditsmetabolites(23)andisdocumentedwithpreclinicalandclinicalpharmacokineticstudies(23,24),supportedbyGLPpreclinicalsafetystudies(personalcommunicationwithIndenaS.p.A.,Investigator'sBrochure)andhasbeenusedinanumberofclinicalstudies(25–28).Anotherdrawbackofcurcuminisitspotentialtointerferewithseveralassays(pan-assayinterferencecompoundsorPAINS),whichmightresultinoverestimationofitsbiologicalactivities(29). Curcumin-containingdietarysupplementshavebeenusedinvariousclinicaltrialsincancerorotherdiseaseswithoutmajorsideeffectsandaregenerallyregardedassafe(GRAS)bytheUSFoodandDrugAdministration(FDA).Inthisphase2study,weevaluatedtheeffectsofadailyintakeof2gCPbyECpatientsduringa2-week,oncologicaltreatment-freeinterval.TheobjectivesofthestudyincludedevaluationoftheimmunomodulatoryeffectsofCP,bioavailabilityandimpactofthetreatmentonpatient'squalityoflife. MaterialsandMethods PatientRecruitmentandTreatment ThetrialwasapprovedbythelocalethicscommitteeoftheUniversityHospitalLeuven(S55201)andbytheFederalAgencyforMedicinesandHealthProducts(FAMHP;EudraCT:2013-001737-40).PatientswithhistologicallyconfirmedECandnolife-threateningmetastaseswererecruitedbythedepartmentofgynecologicaloncologyofUZLeuven.Exclusioncriteriawere:otheractivemalignancy,documentedautoimmunediseaseorimmunedeficiency,ongoingimmunosuppressivetherapyandcurrentenrollmentinotherclinicaltrials.AllpatientswereaskedtocompleteaquestionnaireconcerningtheQoLbeforeandafterCPsupplementation.Eachpatienthadtodocumenttheirdailyconsumptionofcertainfoodsorfoodsupplementsspecifiedinadietarylist.Writteninformedconsentwasobtainedfromeachpatientbeforeenrollment. CurcuminPhytosome(Meriva®,CP)wasprovidedbyIndenaSpAandmanufacturedintocapsules(Curcuphyt®)bynutrisannv.Thecapsulescontained500mgofCP,correspondingto100mgofcurcuminoids.Patientsweresupplementedfor2weekswith2gCPperdayinatimeperiodduringwhichtheydidnotreceiveanyoncologicaltreatment. BloodandUrineCollection Bloodsampleswerecollectedatbaseline,onthefirstdayofcurcuminintake,thenonceweeklyduringthesupplementationperiod,andfinally1weekaftertheendofsupplementation.Oneachdayofbloodcollection,patientswererequestedtotaketheirnoonintakeofcurcumininthehospitalandbloodsampleswerecollectedatdifferenttimepointsfollowingcurcuminintake(15-30-60-120min).BloodwascollectedinoneEDTAandoneheparintubeforthemeasurementofhemoglobin,redbloodcells,whitebloodcells,thrombocytes,CA125andCRPatthecentrallaboratory.Inaddition,bloodwascollectedin1supplementaryEDTAtubeand4supplementaryheparintubesandtransferredtothelaboratoryofgynecologicaloncologyforseparationofplasmaandcellularfraction.IndomethacinwasaddedtotheEDTAtubebeforecentrifugation.Plasmawasaliquotedandstoredat−80°C.Onthefirstdayofcurcuminintake,patientswererequestedtoperforma24-hurinecollection.Thecollectedurinewascentrifuged,aliquoted,andstoredat−80°C. MeasurementofPlasmaCurcuminLevels Forextraction,500μLofacetone/0.25Mformicacid(9:1,v/v)wasaddedto250μLofplasmasample.Aftervortexing,themixturewaskeptat−20°Cfor30minandsubsequentlycentrifugedat16,100×gfor20minat4°C.ThesupernatantwasevaporatedtodrynessovernightusingaSavantDNAspeedvacDNA120centrifugalevaporator.Thedriedresiduewasresuspendedin50μLof0.1%aceticacid/acetonitrile(0.1%aceticacid)(40:60,v/v),centrifugedat16,100×gfor3minandthevolumeinjectedontothecolumnwas20μLinduplicate.CurcuminoidswereseparatedandquantifiedusingaWatersAlliance2695separationsmodulewitha100μLinjectionloopandWaters2487UVdetector,withaHyPurityC18(2.1×150mm,3μm)columnconnectedtoaHyPurityC18(2.1×10mm,3μm)guardcartridgeplusaKrudKatcher(5μm)disposablepre-columnfilter.Thesampleswereanalyzedinnegativeelectrosprayionization(ESI)mode.ThedatawasacquiredusingMassLynxsoftwarev4.0.Asingleinjectionforeachsamplewasperformed.Thecalibrationlineswereconstructedusingpurestandardsforcurcumin,curcuminglucuronideandcurcuminsulfatebyinjectionofa10μLaliquotforeachstandardontotheliquidchromatography/electrosprayionizationmassspectrometry(LC-ESI-MS/MS).Fordesmethoxycurcumin,astandardwasnotavailableandlevelswereestimatedusingthecurcumincalibrationline. QualityofLifeAssessment QoLscoreswereassessedusingtheEORTCQLQ-C30version3.0andEQ-5Dquestionnaires.Patientswereaskedtocompletethequestionnairesatbaselineandatthelastdayofcurcuminintake.QoLscoresarepresentedasmeans±standarddeviations. FortheEORTCQLQ-C30questionnaire,fivefunctionalscores(emotional,role,cognitive,physical,andsocial)werepooledandasummaryscorewascalculatedaccordingtoGiesingeretal(30)usingSPSSsoftware.Ahigherscoreindicatesabetterhealthforfunctioningandglobalhealthstatus,whereasforthesymptomscalesalowerscoreindicatesalowerlevelofsymptomburden. TheEQ-5Dquestionnaireconsistsof2parts—theEQ-5DdescriptivesystemandtheEQvisualanalogscale(EQVAS).TheEQ-5D-3Ldescriptivesystemcomprisesthefollowing5dimensions:mobility,self-care,usualactivities,pain/discomfort,andanxiety/depression.EQ-5Dhealthstates,definedbytheEQ-5Ddescriptivesystem,wereconvertedintoasinglesummaryindexaccordingtotheEQ-5Duserguide.TheEQVASrecordstherespondent'sself-ratedhealthonavertical,analogscalewheretheendpointsarelabeled“Bestimaginablehealthstate”and“Worstimaginablehealthstate.”ForboththeEQ-5DindexandEQVAS,ahigherscoreindicatesabetterhealthstatus. PeripheralBloodMononuclearCells(PBMC)Isolation Peripheralbloodfrom4heparintubeswasdiluted1:2inPBSandPBMCwereisolatedusingLymphoprep™(AXIS-SHIELD)densitygradientcentrifugationandcountedwithTürck'ssolution.PBMCwerecryopreservedin90%humanABserum(SeraLaboratoriesInternational)with10%DMSOat5–10×106cellspervialusingCoolCellfreezingcontainers(BioCision),andstoredinliquidnitrogenuntilfurtheruse. MeasurementofSolubleAnalytes PGE2wasmeasuredfromindomethacin-treatedEDTAplasmausingthecompetitiveBiotrak™enzymeimmunoassaysystem(GEHealthcare).Neopterinlevelsweremeasuredfromheparinplasmausinganenzyme-linkedcompetitiveimmunosorbentassay(NeopterinELISA;ImmunoBiologicalLaboratories).MeasurementofHMGB1wasperformedusingtheHMGB1ELISAkitfrom(IBL).LactatewasmeasuredinheparinplasmabyuseoftheL-Lactateassaykitcolorimetric(Abcam).Adeproteinizationstepwasperformedonthesamplespriortotheassaybyaddingtrichloroaceticacid.Forallassays,opticaldensitywasreadat450nmusingtheMultiscanFCreaderandScanItsoftware(ThermoScientific). Luminexassayswereperformedonheparinplasmasamples:customorderedkit(EMDMillipore,HCCBP1MAG-58K)toanalyzeCA15-3,CEA,Leptin,MIFandProlactinandProcartaplexImmunoassayKit(Affymetrix-eBioscience)toanalyzeBDNF,Eotaxin/CCL11,EGF,FGF-2,GM-CSF,GROα/CXCL1,HGF,NGFβ,LIF,IFNα,IFNγ,IL-1β,IL-1α,IL-1RA,IL-2,IL-4,IL-5,IL-6,IL-7,IL-8/CXCL8,IL-9,IL-10,IL-12p70,IL-13,IL-15,IL-17A,IL-18,IL-21,IL-22,IL-23,IL-27,IL-31,IP-10/CXCL10,MCP-1/CCL2,MIP-1α/CCL3,MIP-1β/CCL4,RANTES/CCL5,SDF-1α/CXCL12,TNFα,TNFβ/LTA,PDGF-BB,PlGF,SCF,VEGF-A,VEGF-D.PlateswerereadonaLuminex200system(Bio-RadLaboratories).DatawereanalyzedusingBio-PlexManagersoftware(Bio-RadLaboratories). FlowCytometricAnalyses CryopreservedPBMCfromeachtimepointofeverypatientwerethawedandcountedusingtrypanblue(Sigma-Aldrich). ForanalysisofCOX-2expression,1.5×106cellswereplatedper24-well(2wellsforeachtimepoint)ofalow-adherence24-wellplate.Onewellwasstimulatedwith1μg/mLLPS(K12,Invivogen)andtheotherwasleftuntreated.Cellswereincubatedat37°Cand5%CO2for5h,afterwhichtheywereharvested.Toexcludeculture-inducedeffects,COX-2expressionwasalsoanalyzedonfreshlythawedPBMC.Cellswerestainedwithafixableviabilitydye(FixableViabilityDyeeFluor506,eBioscience)followedbyFcreceptorblockingwith10%normalgoatserum(Sigma-Aldrich).Next,thefollowingantibodieswereadded:CD3-FITC(eBioscience),CD14PerCP-Cy5.5(BDPharmingen),CD56-PE-Cy7(BioLegend)andCD19-eFluor450(eBioscience)for30minat4°C.Afterwashing,sampleswerefixedusingfixation/permeabilizationbuffer(BDBiosciencesCytofixCytopermkit)for20minat4°C.Cellsweresubsequentlyincubatedwitheithernoantibody(unstainedcontrol),mouseIgG1-PEisotypecontrolantibody(BDBiosciences)ormouseanti-HumanCOX-2-PEantibody(BDBiosciences). ForPBMCphenotyping,cellsuspensionswerestainedwithafixableviabilitydye,followedbyFcreceptorblocking.TheantibodystainingpanelsusedtoidentifythedifferentcellularpopulationsinthisstudyaredescribedinTable1.ForTreganalysis,thesampleswerefixedandpermeabilizedusingtheFoxP3StainingBufferSet(eBioscience)andstainedwithFoxP3-APC(eBioscience).ForCD247staining,sampleswerefixedwithPBS/0.5%paraformaldehydefor20minatroomtemperature(RT)inthedark.After2washingstepswithPBS/Tween,100μLcolddigitoninsolution(10μg/mLinPBS)wasadded,followedbyeithermouseIgG1-PEisotype(FMO;BDBiosciences)ormouseanti-humanCD247antibody(BeckmanCoulter)for30minatroomtemperature. TABLE1 Table1.Membraneantigenflowcytometrystainingpanels. MDSCanalysiswasperformedonfreshwholebloodinsteadofcryopreservedPBMC.Bloodwasaliquotedat350μLpertubeand35μLnormalgoatserumwasadded.ForMDSCenumeration,thefollowingantibodycocktailwasadded:CD45-FITC(BioLegend),CD11b-PE(BioLegend),CD14-PerCP-Cy5.5(BDPharmingen),CD3-PE-Cy7(BioLegend),CD19-PE-Cy7(BioLegend),CD56-PE-Cy7(BioLegend),CD15-APC(BioLegend),HLA-DR-APC-H7(BDPharmingen),andCD33-V450(BDHorizon).After30minat4°C,redbloodcelllysiswasperformedbyadding1xPharmLyse(BDBiosciences).After15minincubationatroomtemperatureinthedarkandwashing,thecellswerestainedwithviabilitydye.Analysisofarginase-1expressionbyMDSCwasdonebyreplacingCD45-FITCwithArginase-1-fluorescein(R&DSystems)intheabovementionedMDSCcocktail.Forassessmentofarginase-1expression,cellswerefirststainedformembranemarkersasdescribedabove,subsequentlyfixedandpermeabilizedusingtheFoxP3StainingBufferSet(eBioscience)andstainedwitharginase-1-fluorescein(R&DSystems). AcquisitionwasperformedwithaFACSCantoTMIIusingBDFACSDivaTMsoftware.Forallsamples,between2.5×104and1×105cellswereacquiredinthelivegatepersample.DataanalysiswasdoneusingBDFACSDivaTMsoftware.MDSCweregatedasfollows:first,wegatedoutdeadcellsanddebrisandsubsequentlywegatedonCD45+Lin(CD3-CD19-CD56)−HLA-DRlocells.Withinthisgate,twomajorMDSCsubtypeswereidentifiedasCD11b+CD14−granulocyticMDSCandCD11b+CD14+monocyticMDSC.ForthegranulocyticMDSC,wenextdistinguishedCD15+andCD33+subtypes,whilemonocyticMDSCareCD15−andCD33+.Dendriticcell(DC)gatingstrategywas:afterdeadcellexclusion,cellsweregatedupontheirCD45+andCD14−characteristics.OnthisgateCD11c+CD123−cellsareidentifiedasmDCandCD11c−CD123+arepDC.Onthesesubsets,weassessedtheexpressionofHLA-ABC,HLA-DRandCD54.NKcellswereidentifiedasCD56+CD16−orCD56+CD16+cellsonwhichtheexpressionofCD161,CD69,andHLA-DRwasevaluated.TcellsubsetsweredefinedasCD3+,CD3+CD4+CD8−,CD3+CD4−CD8+orCD3+CD4+CD8−CD25+FoxP3+CD127lo.OnCD4+andCD8+Tcells,wesubsequentlydeterminedexpressionofCD69,CD137,HLA-DR,ICOS,CTLA-4,PD-1andTim-3.ThememoryphenotypeofCD4+andCD8+Tcellswasdeterminedasfollows:Tnaïve(CD45RA+CD45ROCD62L+CCR7+),TCM(CD45RA−CD45RO+CD62L+CCR7+),TEM(CD45RA−CD45RO+CD62L−CCR7−)andTEMRA(CD45RA+CD45RO−CD62L−CCR7−).TCRζexpressionwasmeasuredonCD3+,CD4+,CD8+Tcells,aswellasinCD56+andCD16+NKcells. StatisticalAnalysis ForQoLscoresandplasmacurcuminlevels,weusedthenonparametricWilcoxonmatched-pairssignedranktestbecauseofthesmallsamplesize.Concentrationsofanalyteswerepresentedasmeanvalues±SD.Datawereanalyzedand,whenappropriate,significanceofthedifferencesbetweenmeanvaluesatbaselineincomparisonwithday14valueswasdeterminedbyWilcoxonmatched-pairstestexceptforCA125andCRPwhereitwasdeterminedbyMann-WhitneyU-test.DifferenceswereassumedtobesignificantatP<0.05.One-wayrepeatedmeasureANOVAwasusedtotesttheeffectofcurcumininpatientsatalltimepoints.Allexperimentswereperformedinduplicates.Prism5software(GraphPadSoftwareInc.)wasusedtoperformallstatisticalanalysesandtogenerategraphs. ResultsandDiscussion PatientCharacteristics Weenrolled7patientsbetweenSeptember2013andAugust2015,ofwhich6completedtreatment.PatientcharacteristicsareshowninTable2andFigure1showstheCONSORTflowdiagramtoillustratetheprogressofpatientsthroughthetrial.Supplementationwasadministeredduringanoncologicaltreatment-freeintervaltoavoidimmunomodulatoryeffectsfromstandardoncologicaltreatments.Allpatientshadrecurrentdisease.Inourstudy,wecouldnotassesstheclinicalresponseofthepatients,sincetheyreceivedvariousoncologicaltreatmentsafterthe2-weekCPsupplementationperiod. TABLE2 Table2.Patientcharacteristics. FIGURE1 Figure1.CONSORTflowdiagram. Allpatientsdocumentedtheirdailyconsumptionofcertainfoodsorfoodsupplementsdescribedinadietarylist.Themostfrequentlyconsumedfoodsfromthelistweremushrooms,berries,broccoli,sprouts,watercress,andhorseradish.Onlyonepatientconsumedafoodsupplementcontainingpropolisonadailybasiswhichcouldhaveimmunologicaleffects(31,32). PlasmaLevelsofCurcuminoidsandSolubleInflammatoryMediators Nocurcuminoidsnortheirmetabolites,whichhavealsobeenreportedtoexertimmunomodulatoryeffects(6,33),weredetectableinplasmaatbaseline,i.e.,beforecurcuminintake.Thetwomostabundantcurcuminoids,curcumin,anddemethoxycurcumininfreeform,remainedundetectableinplasmauponcurcuminintake,exceptforafewoutliers.However,itsconjugatedmetabolites,curcuminglucuronideandcurcuminsulfate,becamedetectableaftersupplementation,withslightlyincreasedlevelsafter1–2h(Figure2).ThisprofileissimilartopreviousstudiesandshowsthatCPuptakewasefficient(23,24).Sincecurcuminhasbeenshownpreviouslytoexertanti-inflammatoryeffects(6),weperformedanextensiveinterrogationofabroadsetofinflammatorymediatorsatdifferenttimepointsduringtreatment(atbaseline,onthefirstdayofcurcuminintake,thenonceweeklyduringthe2-weektreatmentperiod,andfinally1weekaftertheendoftreatment).InTable3,wesummarizethelevelsmeasuredatbaselineandatthelastdayofCPsupplementation.Forasubstantialnumberofanalyteshowever,wenotedthatvalueswerebelowthedetectionlimitin>3outof6patients,soweconsideredtheseanalytesasundetectable.Furthermore,wenotedthatabsolutevaluesoftenvariedconsiderablyamongpatients,leadingtohighstandarddeviations.NosignificantchangesfollowingCPforanyofthetestedinflammatorymediatorscouldbenoted. FIGURE2 Figure2.Plasmacurcuminlevels.Plasmalevelsofcurcuminoids(curcuminanddemethoxycurcumin)andtheirmainmetabolites(curcuminglucuronideandcurcuminsulfate)weredeterminedbyLC-ESI-MS/MS.Concentrationsareshowninfmol/mL.Resultsaredepictedasmean+standarderrorofmean.Timepointsareasfollows:T0-baseline,T1-day1oftreatment,T2-day7oftreatment,T3-day14oftreatment,T4,1weekafterlastCPdose. TABLE3 Table3.Solubleinflammatorybiomarkers. COX-2ExpressioninImmuneCells COX-2isawell-knowntargetofcurcumin(34,35).COX-2expressionwasexaminedbyflowcytometryinmonocytes,Bcells,NKcells,andTcells.Boththepercentageandmeanfluorescenceintensity(MFI)ofCOX-2expressionineachcelltypewasmeasured,bothonfreshlythawedPBMCaswellasonPBMCculturedinvitrointhepresenceofLPS.Asexpected,COX-2expressioninfreshlythawedPBMCwasverylow,withhighestpercentageinmonocytes.AfterLPSstimulation,COX-2expressionwasincreased,mainlyinmonocytes.Figure3,showsthepercentageofCOX-2expressingcellsandtheMFIinthemonocytepopulationoffreshlythawedPBMCandLPS-stimulatedPBMC.WewereunabletodemonstratesignificantchangesinCOX-2expressionuponCPsupplementation(Figure3).ThesameanalysiswasperformedforCOX-2expressioninBcells,NKcellsandTcells,withoutsignificantdifferences(datanotshown). FIGURE3 Figure3.EffectofcurcuminsupplementationonCOX-2expression.COX-2expressioninPBMCwasdeterminedbyflowcytometry.Thegraphsshowtheexpressioninthemonocyte(CD14+)gate,eitherexpressedaspercentageofcellsexpressingCOX-2(A,C)orasMFIofCOX-2expressionlevels(B,D).(A,B)ShowtheexpressionofCOX-2inPBMCdirectlyafterthawing,while(C,D)showCOX-expressionlevelsaftera5h-invitrocultureperiodinthepresenceofLPS.Eachlinedepictsonepatient.Timepointsareasfollows:T0-baseline,T1-day1oftreatment,T2-day7oftreatment,T3-day14oftreatment,T4,1weekafterlastCPdose. ImmuneCellSubsets Evidencesuggeststhecapabilityofcurcumintomodulatethefrequencyandcellularresponseofdifferentcelltypesoftheimmunesystemduringcancer(36–38).Inwholeblood,weverifiedtheeffectofcurcuminontheNeutrophil-to-LymphocyteRatio(NLR)andthefrequencyofMyeloid-DerivedSuppressorCells(MDSC)andtheirexpressionlevelofArginase-1.ThreeMDSCsubtypeswereanalyzed:CD15+granulocytic,CD33+granulocytic,andCD33+monocyticMDSC.Finally,weassessedtheArginase-1expressionlevelforthese3differentMDSCsubtypes.NosignificantdifferenceswereobservedafterCPsupplementation,neitherforNLRnorforMDSCfrequenciesortheirArginase-1levels(datanotshown). WethenisolatedPBMCandinvestigatedthetotalleukocytepopulationandtheirexpressionofMHCmolecules.AsshowninTable4andFigures4A,B,despiteaconstanttotalpercentageofleukocytes,weobservedasignificantdeclineinthefrequencyofHLA-DRexpressingleukocytesandasignificantreductionintheexpressionlevelofHLA-ABCuponCPtreatment(P<0.05).Thiseffectwastransientandlevelswererestored1weekafterdiscontinuationofcurcuminintake. TABLE4 Table4.Effectofcurcuminsupplementationontotalleukocytes. FIGURE4 Figure4.Effectofcurcuminsupplementationonimmunologicalcelltypes.(A)ExpressionofHLA-DRbyCD45+leukocytesdeterminedbyflowcytometry,expressedaspercentageofHLA-DRexpressingCD45+leukocytes.(B)ExpressionofHLA-ABCbyCD45+leukocytesdeterminedbyflowcytometry,shownasmeanfluorescenceintensity(MFI)ofHLA-ABCexpressedbyCD45+leukocytes.(C)PercentageofCD14+monocytesinthetotalPBMCpopulationdeterminedbyflowcytometry.(D)PercentageofCD69-expressingcellswithintheCD56+CD16−NKcellpopulationmeasuredbyflowcytometry.(E)PercentageofICOS-expressingcellswithintheCD3+CD4−CD8+Tcellpopulationmeasuredbyflowcytometry.Eachlinedepictsonepatient.Timepointsareasfollows:T0,baseline,T1-day1oftreatment,T2-day7oftreatment,T3-day14oftreatment,T4,1weekafterlastCPdose.PvaluesweredeterminedusingtheWilcoxonsignedranktest(A,B,D)ortheone-wayrepeatedmeasureanalysisofvariance(ANOVA)test(C,E)withPrismsoftware.*P<0.05. Next,weanalyzedwhetherCPintakeexertedeffectsontheinnateimmunecelltypes(monocytes,dendriticcells,NKcells).ThepercentageofCD14+monocytesdeclinedovertimeuponCPtreatment,buttheirexpressionlevelsofMHCmoleculesremainedunaltered(Figure4C).AnotherimportantinnateimmunecelltypeistheDC,consistingoftwomajorsubsets,myeloidDC(mDC)andplasmacytoidDC(pDC).WealsoassessedtheexpressionofHLA-ABC,HLA-DR,andCD54,asameasureoftheirfunctionality.However,wedidnotfindanychangesintheirfrequencyorexpressionofHLA-ABC,HLA-DR,orCD54(datanotshown).Naturalkiller(NK)cellsconstituteanimportantlineofdefenseintheimmunesystem.CD16+NKcellsareconsideredasthecytotoxicsubsetofNKcells,whileCD16−NKcellsareclassifiedasthecytokine-producingNKcellsubset.Onbothcelltypes,weassessedtheexpressionoftheactivationmarkersCD161,CD69,andHLA-DR.CPsupplementationdidnotresultinsignificantchangesineitherNKcellpopulation(datanotshown).However,thepercentageofCD69expressingCD16−NKcellsincreasedsignificantlyupontreatment(Figure4D,P<0.05). Finally,weassessedtheeffectsofcurcuminontheTlymphocytecompartment.Tlymphocytesplayaprominentroleintumorimmunology,becauseofthecapacityofcytotoxicCD8+TcellstokilltumorcellsortheabilityofTregtosuppresstumor-specificimmunity.NeitheronthegeneralTcellsubsets,CD3+Tcells,CD4+TcellsorCD8+Tcells,noronTreg(CD3+CD4+CD8−CD25+FoxP3+CD127lo),couldweobservechangesfollowingCPsupplementation(datanotshown).Next,weassessedtheexpressionoftheactivationmarkersCD69,CD137,HLA-DR,ICOS,CTLA-4,PD-1andTim-3onCD4+andCD8+TcellsandweobservedasignificantdeclineinICOSexpressionbyCD8+TcellsafterCPsupplementation(Figure4E).ForCD4+Tcells,thismarkeralsodeclinedbutnotsignificantly.Theotheractivationmarkersremainedunaltered(datanotshown).WealsoinvestigatedtheeffectofcurcuminonthecompositionofthememoryTcellrepertoire,butfoundnosignificantdifferences.TheTcellantigenreceptor(TCR)zeta(TCRζ)chainisanessentialcomponentoftheTCRcomplex.LossofTCRζisfrequentlyobservedincancerandindicatesimmunosuppressionbyMDSC(39).TCRζexpressionwasmeasuredasTCRζMFIindex(40)inCD3+,CD4+,CD8+Tcells,aswellasinCD56+andCD16+NKcells,butwedidnotfindchangesuponCPtreatment(datanotshown). QualityofLifeScores CompleteEORTCQLQ-C30andEQ-5Dscoresfrombaselineandthelastdayofcurcuminintakewereavailablefor5outof7patients.OnepatientdidnotcompletetheEORTCQLQ-C30questionnaireonthelastdayofcurcuminintakeandonepatientpartiallycompletedtheEQ-5Dquestionnaireonthelastdayofcurcuminintake.ChangesinQoLscoresuponCPsupplementationareshowninTable5.NosignificantchangesinQoLcouldbenoted. TABLE5 Table5.Qualityoflifescores. Discussion Insummary,althoughtheCPformulationwastakenupinthebloodofthepatients,weonlydetectedminorimmunologicaleffects.WeobservedadownregulationofMHCexpressionbyleukocytes,areductioninthefrequencyofmonocytesandadecreasedICOSexpressionbyCD8+TcellsuponCPintake,whilethelevelofCD69onCD16-NKcellswasupregulated.Wedidnotfindsignificantchangesininflammatorybiomarkerlevels,frequenciesofotherimmunecelltypes,TcellactivationandCOX-2expression.Anon-significanttrendtoimprovedQoLwasobserved. Amajorshortcomingofourstudyisthesmallpopulationsizeandahighinter-patientvariability,whichmightmasksmalleffectsizes.Furthermore,thesupplementationperiodwasonly2weeks,whichmightbetooshorttorevealsmallchanges. Severalstudieshaveobservedchangesininflammatorybiomarkersuponcurcuminintake.TheabsenceofchangesininflammatorybiomarkerlevelsseemscontradictorytootherstudieswhereCPtreatmenthasshowntodecreasethelevelsofseveralinflammatorymarkerssuchasCRP(25),IL-1β(41),IL-6(8,41,42),IL-22(43),sCD40L(41).However,inourstudy,thelevelsofasubstantialnumberofanalyteswerebelowthedetectionlimit,sofurtherdecreasescannotbedetected.Togetherwiththeabove-mentionedsmallpopulationsizeandhighvariability,thismightexplainthisdiscrepancy. WewereunabletodemonstratesignificantchangesinCOX-2expressionuponCPsupplementation,whichisincontrasttofindingsinpancreaticcancer(34).DespitethehigherdosingofthecurcumincomplexusedbyDhillonetal.,theplasmaconcentrationsofcurcuminmetabolitesuponcurcuminintakeweresimilartothelevelsobservedinourstudy.Moreover,COX-2reductionbyDhillonetalwasmeasuredafteronly8daysofsupplementation,whilewemeasuredCOX-2expressiononthefirstdayofintake,after1weekandafter2weeksanddidnotobserveareductionatanyofthetimepoints.Bothstudieshowever,useadifferentmethodtoassessCOX-2expression,whichcouldhaveadifferentsensitivity.ThismightexplainthehigherpercentageofCOX-2positivityinPBMCobservedbyDhillonetal.comparedtoourresults.However,basalCOX-2expressionlevelsinPBMCreportedpreviouslyinliteratureareingeneralmorecomparabletothelevelsobservedinthisstudyandithasbeenshownthatLPSstimulationisneededtoincreaseCOX-2expression(44,45). WeobservedasignificantdeclineinthefrequencyofHLA-DRexpressingleukocytesandasignificantreductionintheexpressionlevelofHLA-ABCuponCPtreatment.IthasbeendescribedpreviouslythatcurcumincandownregulateMHCclassIIgeneexpressionbyinhibitingIFNγsignaling(46).Thismightalsobetheexplanationforourresults,althoughweonlyobservedatrendtowarddecreasedIFNγcontentinpatientplasmauponcurcuminintakewhencomparingthebaselinewiththeendoftreatmentvalue(P=0.0625,Wilcoxonmatched-pairstest;Table2).However,therepeatedmeasuresANOVAthatcomparestheeffectofcurcuminatalltimepointsdidindicateasignificantdecreaseofIFNγconcentrationinplasmauponcurcuminintake(P=0.0189,datanotshown).Sincewedidnothavetumortissueavailableinthisstudy,wecouldnotinvestigatewhetherthedownregulationofHLAmoleculesisalsomeasurableintumorcells. Verylittleevidenceabouttheeffectofcurcuminonimmunologicalcelltypesisavailable.OurdataabouttheabsenceofeffectofcurcuminonMDSCfrequenciesinbloodareincontrastwithareportinthe4T1mousemodelwherecurcuminshowedatrendtowardMDSCreductioninblood(predominantlygranulocyticMDSC),whichbecamesignificantwhencurcuminwascombinedwithalisteriavaccine(42).Asignificantdecreaseinthepercentageofmonocytesuponcurcumintreatmentwasalsoobservedinanasthmamodel(47).OurresultsonactivationofNKcellsareinagreementwitharecentreportontheeffectsofananocurcuminformulation,whereinincreasedNKcellactivitywasfound(48).However,curcuminhasalsobeenreportedtoincreasethefrequencyofNKcellsinclinicalstudiesandanimalmodels(49),whichwedidnotobserve.PreviousdataintheliteratureshowthatcurcumincanincreaseCD4+andCD8+TcellsbutalsoTreg,whichwecouldnotobserve(49).WedidnotobservechangesinthememoryTcellrepertoireuponCPintake,whileanotherstudyshowedthatcurcumincouldrestorecentralmemoryTcell(TCM)andeffectormemoryTcell(TEM)populationsintumor-bearingmice,buttheycompareduntreatedandcurcumin-treatedtumor-bearingmiceanddidnotassesstheeffectofcurcumintreatmentinthesamemouse(50). WeobservedasignificantdeclineinICOSexpressionbyCD8+TcellsafterCPsupplementation(Figure4E).ICOSisaco-stimulatorymoleculeoftheCD28-B7superfamilyanditsroleincanceriscontroversial.Ontheonehand,datasupportaroleofICOS:ICOSLinfacilitatingtheanti-tumorTcellresponsebecauseofobservationsthatdiminishedICOSlevelsinbloodassociatewithworseprognosisincoloncancerandthathighICOSexpressionontumor-infiltratinglymphocytesinmetastaticmelanomalesionswasassociatedwithbetterpost-recurrencesurvival.Ontheotherhand,aninhibitory,pro-tumorrolehasbeenattributedtoICOSsignalingrelatedtoitsfunctioninTreghomeostasis,thusfacilitatingtumorimmuneevasion(51).DataalsoindicatethattheICOS:ICOSLpathwayisrequiredforoptimalantitumorresponsesmediatedbyanti-CTLA-4therapy(52).TheavailabledataabouttheeffectofcurcuminonICOSinliteraturearescarceandcontradictoryandmainlycomefrominvitrosystemsusingsupra-physiologicalcurcuminconcentrations(53,54). TheabsenceofchangesinQoLscoresindicatestheabsenceoftoxicitiesrelatedtocurcuminintake,whichhasalsobeenshowninotherstudies(1,55).However,inarandomizedcontrolledtrial,curcuminoidsupplementationwasassociatedwithasignificantimprovementinQoLcomparedtoplacebo(56).WealsoobservedatrendtoincreasedQoLscoresuponCPsupplementationwithbothquestionnairesused,althoughnotsignificant.SincetheCPdosewastolerable,increasingthedosageofCPcouldalsobeconsideredinfurtherstudies.LackofsignificancemightbeexplainedbythesmallnumberofpatientsinourstudyorbytheshortperiodofsupplementationorbythefactthatinthepaperofPanahietalthepre-treatmentQoLscoreforthecurcuminoidgroupwaslowercomparedtotheplacebogroupwhilethepost-treatmentscoreswereequal,whichmightindicatearandomizationproblemfortheQoLparameter. Inconclusion,weobservedonlyminorimmunomodulatoryeffectsofcurcuminsupplementationinendometrialcancerpatients.TheQoLscoresconfirmedtheabsenceoftoxiceffectsbycurcuminsupplementation,butnoimprovementinQoLisseen.Itremainstobeexploredwhetherdifferentsupplementationregimensorschemescouldinduceimmunologicalbenefitinendometrialcancer. EthicsStatement ThisstudywascarriedoutinaccordancewiththerecommendationsofEthischeCommissieOnderzoekUZLeuvenwithwritteninformedconsentfromallsubjects.AllsubjectsgavewritteninformedconsentinaccordancewiththeDeclarationofHelsinki.TheprotocolwasapprovedbytheEthischeCommissieOnderzoekUZLeuven. AuthorContributions STconceivedtheproject,designedresearch,performedexperiments,interpreteddata,andwrotethepaper.KRdesignedresearchandeditedthepaper.TEperformedtheexperiments.AVNinterpreteddataandeditedthepaper.FAconceivedtheprojectandeditedthepaper. Funding ThisworkwassupportedbytheAnticancerFundthroughtheVerelstUterineCancerFundatKULeuven. ConflictofInterestStatement Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. Acknowledgments WethankRawandAbuusbaforherhelpwiththeexecutionoftheimmunologicalassaysandDr.MagaliVerheeckeforscreeningeligiblepatients.WearegratefultoDr.LynneM.HowellsandDr.RajSingh(UniversityofLeicester,Leicester,UK)forthemeasurementofthelevelsofcurcuminoidsandtheirmetabolitesinpatientsplasma.WewouldalsoliketoexpressourgratitudetoDr.AntonellaRivaandDr.GiovannaPetrangolini(IndenaSpA,Milan,Italy)fortheirscientificsupportoncurcumin,theireditorialsupportandtheprovisionofCP.WewouldalsoliketothankNutrisanNVfortheformulationofCPintocapsules. 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Editedby:TheaMagrone,UniversityofBari,Italy Reviewedby:ZhijingTan,MichiganMedicine,UniversityofMichigan,UnitedStatesLeiShi,GeorgiaStateUniversity,UnitedStates Copyright©2019Tuyaerts,Rombauts,Everaert,VanNuffelandAmant.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)andthecopyrightowner(s)arecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:SandraTuyaerts,[email protected] COMMENTARY ORIGINALARTICLE Peoplealsolookedat SuggestaResearchTopic>