Laboratory and Clinical Studies of Cancer Chemoprevention by Antioxidants in Berries

50 Pages·2008·0.4 MB·English

Carcinogenesisvol.29no.9pp.1665–1674,2008
doi:10.1093/carcin/bgn142
AdvanceAccesspublicationJune9,2008
REVIEW
Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries
Gary David Stoner(cid:1),Li-Shu Wangand Bruce Cordell investigated polyphenolic compounds, with demonstrated cancer-
Casto1 preventative effects, such as, the catechins in green tea, theaflavins
inblacktea,curcumininturmeric,resveratrolinredwine,quercitinin
DepartmentofInternalMedicine,CollegeofMedicineand1Divisionof apples and the anthocyanins in berries, are antioxidants that exhibit
EnvironmentalHealthSciences,CollegeofPublicHealth,TheOhioState significantradicalscavengingactivity(12,15–18).
University,Columbus,OH43210,USA
Berriesareamongthemostwidelyconsumedfruitsinthehuman
(cid:1)Towhomcorrespondenceshouldbeaddressed.Tel:þ6142933268; diet.Theyarerichlyabundantinantioxidantflavonoidsandphenolic
Fax:þ6142935952; acids, as well as antioxidant vitamins, carotenoids, tannins, lignans
Email:[email protected] andstilbenes(19).Commerciallythemostimportantberriesinclude Do
w
Reactive oxygen species (ROS) are a majorcause of cellular in- members ofthegenusVaccinium (blueberry,lingonberry,cranberry, n
juryinanincreasingnumberofdiseases,includingcancer.Most bilberry), Rubus (blackberry, black raspberry, red raspberry, arctic loa
ROSarecreatedinthecellthroughnormalcellularmetabolism. raspberry/bramble,cloudberry),Fragaria(strawberry)andSambucus de
d
Tlighhetyacnadnbtoexpicrocdhuemceidcablys,eansvwireolnlmasenbtyalthinesiunlfltsasmumchaatosruyltprraovcioelsest. (reields,etrhbeeirrryu,serefdorenlduetrribtieornrya)l.aRndecmenetdliyc,inthaelpduifrfpeorseenstavnadrifeatcietosrosfinbfleur-- from
Interception of ROS or limiting their cellular effects is a major encingtheirchemicalcompositionhavebeensummarized(20).The h
roleofantioxidants.Duetotheircontentofphenolicandflavonoid presentreviewwilldiscussresearchprogressintheidentificationof ttps
compounds,berriesexhibithighantioxidantpotential,exceeding theactivecancer-inhibitoryconstituentsofberriesandindetermining ://a
that of manyother foodstuffs.Throughtheir ability to scavenge their mechanisms of action. In addition, the available clinical evi- ca
ROS and reduce oxidative DNA damage, stimulate antioxidant dence to suggest that berries might exhibit anticarcinogenic effects de
enzymes, inhibit carcinogen-induced DNA adduct formation inhumanswillbesummarized. mic
andenhanceDNArepair,berrycompoundshavebeenshownto .o
u
inhibitmutagenesisandcancerinitiation.Berryconstituentsalso p
Bioactivephytochemicalsinberries .c
influence cellular processes associated with cancer progression o
m
including signaling pathways associated with cell proliferation, Berries contain a wide range of bioactive phytochemicals, the most /c
differentiation, apoptosis and angiogenesis. This review article prominent of whicharephenolicinnature;i.e.theyhavehydroxyl arc
summarizes laboratory and human studies, demonstrating the ((cid:2)OH) groups on aromatic rings. Phytochemicals are non-nutritive in
/a
panrodteoctthievreceeflfleucltasropfrboecrerssieesslaenaddibnegrtroyccaonncsetritudeenvtesloopnmoexnitd.ative ckonnoswtintuteontdsepferonddupcleadntbsyasgeacinosntdaprryedmateotrasb,omlisicmroibniapllainnftes.ctTiohnesyaanrde rticle
ultravioletlight,toregulatemetabolicpathwaysandprovidecolorand -a
b
flavortotheplant(20).Thebioactivephytochemicalsinberriesfall s
Introduction i(nhtyodrsoexvyercainlnstarmucictuaranldahnyddcrohxeymbiecnazlocilcasasceisdsin),cflluadvionngopidhsen(aonlitchoaccyidas- tract/2
9
Mostchronicdiseases,includingcardiovasculardisease,arthritis,di- nins,flavanols,flavonols),condensedtannins(proanthocyanins),hy- /9
abetesandmanytypesofcancerarecaused,inpart,bytheconversion drolyzable tannins (ellagitannins and gallotannins), stilbenoids, /16
ofcellularmacromoleculestospecificreactiveoxygenspecies(ROS) lignans, triterpenes and sterols (19). The structures of these berry 65
duringnormalcellularmetabolism(1).ROSproducedbyothermeans bioactivesareshowninFigure1,andtheirchemicalfeatures,biolog- /24
including the metabolism of environmental toxins and carcinogens, icalactivitiesandcontentindifferentberrytypeshavebeendescribed 76
byionizingradiationandbyphagocyticcellsinvolvedintheinflam- in detail by Seeram (19). Among the most prevalent compounds in 79
8
matoryresponsealsocontributetothesediseases.Therearefiverec- berries are the anthocyanins and ellagitannins that, collectively, are b
(oOgn.i2ze),dhtyydpreosgeonf RpeOroSx;iid.ee.(sHinOgle)t,ohxyydgroexny(l#rOad2)i,caslup(OerHox.)idanedanniiotrnics rtoestphoensbiibolaecftoivremsulicshteodfitnheFiirgaunrteio1x,idbaenrrtiaecsticvointyta(i2n1m–2a4n)y.Ionthaedrdictoionn- y gue
2 2 2 s
oxide (NO(cid:2)). Although some types are more injurious to cells than stituents known to exhibit cancer-preventive effects including vita- t o
others,thereisnoquestionthat,collectively,theseROScausesignif- mins,suchasA,C,Eandfolicacid,andminerals,suchascalcium, n 3
itchaendteovxeildoaptmiveendtaomfacgherotonicceldluisleaarsDesNiAnc,lpurdoitnegincsaanncderlip(2id).sleadingto ssetilteuneinutmsaarnedduzien,cat(2le5a).stTihnepianrht,ibtoitothryeierfafenctitosxoidfamntosctapoafctihtye.secon- 0 Ma
Epidemiologicalstudiesindicatethatpersonswhoconsumeadiet rch
richinfruitsandvegetableshaveareducedriskofcanceratmultiple Laboratorystudiesoftheanticancereffectsofberrybioactives 20
sites (3–7), although the evidence may not be as strong as reported 19
earlier (8). Since fruits and vegetables are major sources of antiox- Berrybioactiveshavemanyrolesincancerprevention.Theseinclude
idants(amongotherfactors),itishypothesizedthattheseantioxidants protectionagainstoxidativeDNAdamagebythescavengingofROS,
are largely responsible for their cancer-preventive effects. This hy- inhibitionoftheformationofcarcinogen-inducedDNAadducts,en-
pothesis is supported by both epidemiological data, implicating mi- hancementofDNArepair,inhibitionofcarcinogen-inducedtumori-
cronutrient antioxidants in reduction of cancer risk and by genesis in animals and modulation of signaling pathways involved
experimentaldatainvitroandinvivo(2,9–11).Theantioxidanteffects withcellularproliferation,apoptosis,inflammation,angiogenesisand
of fruits and vegetables are due, in significant part, to their content cell cycle arrest. The following sections will summarize laboratory
of flavonoids and phenolic acids (12–14). Some of the extensively studies, both in vitro and in vivo, to determine the mechanisms by
whichberriesandberrycomponentsexertanticarcinogeniceffects.
Abbreviations:AOM,azoxymethane;AP-1,activatorprotein-1;B(a)P,ben-
zo(a)pyrene; BE, Barrett’s esophagus; BPDE, benzo(a)pyrene diol-epoxide;
ScavengingROSandinhibitionofoxidativeDNAadductformation
COX-2, cyclooxygenase-2; DMBA, 7,12-dimethylbenz(a)anthracene; LBR,
lyophilized black raspberry; NF-jB, nuclear factor kappa B; NMBA, Theabilityofdifferentberryjuices(blackberry,strawberry,raspberry,
N-nitrosomethylbenzylamine; 8-OHdG, 8-hydroxy-2#-deoxyguanosine; cranberry andblueberry) toscavenge specific ROS includingsuper-
ROS,reactiveoxygenspecies;VEGF,vascularendothelialgrowthfactor. oxide radicals (O.(cid:2)), hydrogen peroxide (H O ), hydroxyl radicals
2 2
(cid:1)TheAuthor2008.PublishedbyOxfordUniversityPress.Allrightsreserved.ForPermissions,pleaseemail:[email protected] 1665
G.D.Stoneretal.
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Fig.1. Structuresofbioactivesfoundinberries:anthocyanins,flavonol,flavanols,proanthocyanidins,hydroxycinnamicandhydroxybenzoicacids,stilbenoids
andellagitannins.
(OH.) and singlet oxygen (#O ) was investigated by Wang and Jiao lowest antioxidant capacity against OH. and #O Thus, there were
2 2.
(Table I) (15). They found that the scavenging activity of berries significantdifferencesamongthedifferentberrytypesintheirabili-
varied as a function of berry type and cultivar, as well as the types tiestoscavengedifferentROS.
ofROSthatwerescavenged.Ingeneral,blackberrieshadthehighest Inanotherstudy,theantioxidantcapacityofOregongrownblack-
antioxidantcapacityforinhibitionofO .2,H O andOH..Strawber- berries,boysenberries,marionberries,blackraspberriesandredrasp-
2 2 2
ries were second best for these same three radicals; however, they berrieswascomparedusingtheoxygenradicalabsorbance capacity
were superior to blackberries in the scavenging of #O Cranberries (ORAC)assay(26).Antioxidantcapacity,expressedasTroloxequiv-
2.
hadthelowestinhibitionofH O activity,whereasblueberrieshadthe alents/gram of fresh berries, was greatest in black raspberries,
2 2
1666
Cancerchemopreventionwithberries
followed by boysenberries, red raspberries, blackberries and mario- methane(AOM),ledto73,81and83%reductions,respectively,inthe
nberries.Theantioxidantcapacityoftheseberrieswasrelatedtotheir levels of urinary 8-hydroxy-2#-deoxyguanosine (8-OHdG) (33).
contentoftotalphenolicsandanthocyanins.Itshouldbementioned, These results suggest that the berries markedly reduced oxidative
however,thatcautionshouldbeusedindrawingconclusionsregard- DNAdamageinAOM-exposedrats.
ingtheantioxidantcapacityofdifferentberrytypessince,inaddition Incontrasttotheseresults,twohumaninterventionstudiesfailedto
to type and cultivar, the antioxidant capacity of berries canvary as demonstrate aprotective effect ofberrybioactivesagainst oxidative
afunctionoftheclimateandsoilconditionsinwhichtheyaregrown, DNAdamage.Molleretal.(34)placedthreegroupsofhealthysub-
degreeof ripeness,storage temperature,methods usedforindustrial jects on a low flavonoid diet for 3 weeks, supplemented with either
processingandthespecificassayusedtomeasureantioxidantcapacity a black current juice, a black current anthocyanin concentrate or
(27–29). aplacebo.VitaminCincreased inplasmaoftheblackcurrentjuice
InadditiontothedirectscavengingofROS,theantioxidantcapacity group,butthelevelsofendogenousoxidativeDNAdamage,asmea-
ofberriesisalsorelatedtotheircontentofantioxidantenzymes.For suredbytheCometassay,wereunaffectedbyanyofthetreatments.In
example,thejuicesofstrawberriesandblackberrieshavebeenshown a study using cranberry juice (35), 20 healthy female subjects con-
to contain antioxidant enzymes such as superoxide dismutase, cata- sumed250mlofcranberryjuice3(cid:4)/dayfor2weeks.Thistreatment
D
lase,ascorbateperoxidase,glutathioneperoxidaseandglutathionere- led to an increase in vitamin C in the plasma but not in plasma o
ductase, and the activities of these enzymes is positively correlated phenolics. There was no change in strand breakage or resistance/ wn
withantioxidantcapacityoftheberries(30).Thus,theabilityofberry sensitivity to H O in lymphocyte DNA. In addition, there was no lo
2 2 a
bioactivestoscavengeROS,coupledwiththepresenceofantioxidant treatment-relatedreductioninthelevelof8-OHdGeitherinthelym- de
d
emnaziynmtaeinsiannbienrtrriaecse,lmlualyarreosxuildtaitnioantteannudarteiodnucotfioRnO(Srecdoonxc)ebnatrlaatniocention pphootecnyttiealDiNnhAiboitroirnythefefeucrtisneo.fTbheursr,iethsearnedarbeecrroynflciocntisntigtureensutsltosnofotxhie- from
theberriesthemselves(19).Itremainstobedeterminedwhetherthese dativeDNAdamageinhumans.Thishasbeendiscussedinarecent h
berryenzymesexhibitantioxidantactivityinmammalsbecauseitis articlebyFreese(36),whosuggestedthatadditionalwell-controlled ttp
s
notknownwhethertheyremainfunctionalinthegastrointestinaltract. studiesusingvalidatedbiomarkersareneededingroupswithahigh ://a
The prevention of ROS-induced DNA damage is probably a first riskofDNAdamage(e.g.smokers). c
a
lineofdefenseagainstthemultistageprocessofcarcinogenesis.Most d
e
studies with berry-based antioxidants have demonstrated their pro- Inhibitionofcarcinogen-inducedDNAadducts m
ic
tectiveeffectsagainstoxidativeDNAdamageinhumanlymphocytes Dietscomposedofberriesandberryconstituentsthatappeartopro- .o
u
(c3y1te,3s2f)r.oFmonroenx-asmmpolke,eprsrewtriethatqmueenrcteotfinfr(e1s–h1ly00isloMlat)efdohru1mhawnalsymshpohwon- dbueecnersehdouwcntiotonsininhitbhietfthoermfoatrimonatoiofnoxoifdDatNivAeDadNdAucatdsdfurocmtshcahveemaiclsaol p.co
m
toprotectagainstoxidativeDNAdamageinducedbyH2O2asmea- carcinogens(TableII).Krestyetal.(25)reportedthatmaleratsfed /c
sured by the Comet assay (31). Similar findings were reported by a
diets containing either 5 or 10% LBRs or 0.04% ellagic acid for rc
Duthie et al. (32), who treated freshly isolated human lymphocytes 14daysfollowedbyasingleinjectionoftheesophagealcarcinogen, in
awgiethwqauserpcreetviennatenddbmyyqriuceertcine.tiInnhaitbciotinocneonftrHat2ioOn2s-irnadnugciendgDfrNomA1d0amto- Nan-dni3tr8o%so,mreesthpyeclbtievnezlyyl)aomfinOe6-(mNeMthByAlg)u,anhianderaeddduuccetdsilnevtehlesD(7N3A, 8o0f /article
50lMandbymyricetinatconcentrationsof(cid:3)100lM.Inoneanimal theiresophagus.Inlaterexperiments,thefeedingof5and10%LBRs -a
study, the administration of 2.5, 5 and 10% lyophilized black rasp- toNMBA-treatedratsofthesamestrainfor14–21dayswasshownto bs
berry (LBR) diets, which are abundant in anthocyanins and other resultinenhancedmRNAexpressionlevelsofcytochromesP4502a2 trac
antioxidants,giventoratstreatedwiththecoloncarcinogen,azoxy- and3a13intheesophagusandofglutathione-S-transferaseactivityin t/2
9
theliver(37).Itremains tobedetermined whethertheseeffects are /9
responsibleforthereducedlevelsofO6-methylguanineadductsinthe /1
6
esophagus.SimilarresultswerereportedbyCarltonetal.(38),who 65
TableI. Comparisonofthemeanvaluesofthescavengingcapacityofjuice found that diets containing 5 or 10% lyophilized strawberries pro- /24
fromdifferentberryspecies(blackberry,blueberry,cranberry,raspberryand duced similar reductions in O6-methylguanine adducts in the DNA 76
strawberry)onactiveoxygenspecies(O2.(cid:2),H2O2,OH.and#O2) ofesophagifromNMBA-treatedrats.Castoetal.(39),inastudyof 79
8
%Inhibitiona oralcancerusingSyriangoldenhamsters,demonstratedareductionin by
7,12-dimethylbenz(a)anthracene (DMBA)-induced DNA adducts in g
Cultivar O.(cid:2) H O OH. #O thecheekpouchafterfeedinghamstersfor2weekswith5%LBRs. ue
2 2 2 2 s
Blackberryb 64.3 66.3 72.0 12.4 Tanhdea4d8duhc,tsreinspbeecrtrivye-fleyd, ahfatmerstteorpsiwcaelreaprepdliuccaetdiobnyo2f9DanMdB5A5%toatt2h4e t on
Blueberry 60.1 61.2 58.7 7.71 surfaceofthecheekpouch. 30
Cranberry 59.0 59.8 64.2 8.64 M
Raspberry 57.3 60.9 66.9 8.88 arc
Strawberry 64.2 65.3 68.6 15.41 h
LSD0.05 3.02 2.33 1.79 1.16 TableII. Effectsofdietarylyophilizedstrawberriesandblackraspberrieson 20
Significantcultivarc (cid:1)(cid:1) (cid:1)(cid:1) (cid:1)(cid:1) (cid:1)(cid:1) NMBA-inducedDNAadductformationintheratesophagus 19
LSD,leastsignificantdifference. Treatment pmolO6-meGua/mg %Inhibition
DataaretakenfromWangandJiao(15). DNA
aDataexpressedaspercentinhibitionofradical(O.(cid:2),HO ,OH.and’O)
2 2 2 2
productioninthepresenceof0.1mloffruitjuice. NMBA(0.25mg/kg) 4.4±0.9 —
bDataforthethornlessblackberrywerederivedfromthemeanvalue NMBAþ5%STRWs 1.4±0.1a 68
calculatedfromsixcultivars(BlackSatin,ChesterThornless,HullThornless, NMBAþ10%STRWs 1.9±0.7a 57
Smoothstem,Thornfree,TripleCrown);blueberrydatawerederivedfromthe NMBAþ5%BRs 1.2±0.3a 73
meanvaluecalculatedfromfivecultivars(Bluecrop,Elliot,wildblueberry#1, NMBAþ10%BRs 0.9±0.2a 80
wildblueberry#2,wildblueberry#3);cranberrydatawerederivedfromthe
meanvaluecalculatedfromfivecultivars(BenLear,Cropper,EarlyBlack, STRWs,strawberries;BRs,blackraspberries;O6-meGua,O6-methylguanine.
Franklin,Howes);raspberrydatawerederivedfromthemeanvalue Ratswerefed5or10%lyophilizedSTRWsorBRsfor14daysandthengiven
calculatedfromfivecultivars(AutumnBliss,Jewel,Canby,Sentry,Summit); asinglesubcutaneousinjectionofNMBAat0.25mg/kgbodywt.The
strawberrydatawerederivedfromthemeanvaluecalculatedfromsix esophagealDNAwasisolatedandO6-meGuameasured.STRWdataaretaken
cultivars(Allstar,Delmarvel,Earliglow,Latestar,Lester,RedChief). fromCarltonetal.(38)andBRdataarefromKrestyetal.(25).
c(cid:1)(cid:1)NonsignificantorsignificantatP(cid:5)0.05. aSignificantlydifferentfromNMBAcontrols(P,0.05).
1667
G.D.Stoneretal.
Wilms et al. (31) found that quercetin inhibits the formation of berrypowderswouldexhibitinhibitoryeffectsonchemicallyinduced
DNA adducts in human lymphocytes treated with benzo(a)pyrene cancer in animals. The rationale for this stems from the fact that
[B(a)P]. Freshly isolated lymphocytes were treated with quercetin berriesare85–90%water;thus,removalofwaterbythelyophiliza-
(0–100lM),followedbytreatmentwith1lMofB(a)P.Benzo(a)pyrene tionprocesswouldincreasetheconcentrationofellagicacidinberry
diol-epoxide (BPDE)–DNA adducts, evaluated by 32P-post-labeling, powderatleast8-to9-fold.Atthetime,wewereunawareofthemany
decreased in a dose-dependent manner and were undetectable in other chemopreventive agents in berries. In initial tumor bioassays,
cells treated with 100 lM of quercetin. In contrast, when subjects lyophilized berry powders were evaluated for their ability to inhibit
were treated for 4 weeks with a quercetin-rich blueberry/apple NMBA-induced esophageal carcinogenesis in rats when fed to ani-
juice, freshly isolated lymphocytes were not protected from malseitherbefore,duringandafterNMBAtreatment(completepre-
BPDE-adduct formation following B(a)P treatment. Although the ventionprotocol)oronlyaftertreatmentwithNMBA(postinitiation
reasons for this discrepancybetween invitro and invivo results are protocol).Theberrypowderswerefedat5and10%ofsyntheticAIN-
notknown,theratherpoorbioavailabilityofquercetin,andpotentially 76Adiet,which,inhumans,wouldbeequivalenttotheconsumption
othercompoundsinblueberry/applejuice,invivomayberesponsible of2–4cupsoffreshberriesperday.
forthedifferences. The chemopreventive effects of lyophilized strawberries, black
D
raspberriesandblackberriesonNMBA-inducedtumors(papillomas), o
EffectsonDNArepair in therat esophagus, when administered in thecomplete prevention wn
Anumberofreportssuggestthatantioxidantsinfoodsmayhaveadual protocol are given in Table III. Administration of 5 and 10% LBRs lo
a
action;i.e.reducingoxidativeDNAdamagebyscavengingROSand and strawberries had no effect on tumor incidence but significantly de
d
pfrroommoDtiNngAD. NMAosrtepreasireabrychenihnanscuipnpgotrhteorfemthoevalaltotefroxmideacthivaeniasdmduhcatss r(ePd,uce0d.05tu)m(2o5r,3m8)u.lStiuprlpicriitsyingwlhy,en5%codmieptaarryedblwacikthbeNrrMiesBwAerceoenftfreocls- from
revealed that cells or tissues treated with food constituents, after tive in reducing tumor multiplicity, whereas 10% blackberries were h
gammairradiation,exhibitafasterrateofDNArepairthanuntreated ineffective (43). Therewere nosignificant differences in tumor size ttp
s
cellsortissues.Forexample,Mauryaetal.(40)examinedtheeffectof among any of the groups in these studies. One mechanism for the ://a
ferulic acid (a phenolic acid in berries) on the induction of DNA chemopreventiveeffectsofstrawberriesandblackraspberriesinthe c
a
strand breaksin peripheral blood leukocytesand bone marrowcells complete prevention protocol was that of inhibiting the formation d
e
ofmiceexposedtowhole-bodyc-radiation.Intraperitonealadminis- of mutagenic O6-methylguanine adducts in the DNA of esophageal m
ic
tration of ferulic acid (50–100 mg/kg body wt), 1 h prior to 4 Gy epithelium(TableII). .o
c-radiation, resulted in a dose-dependent decrease in DNA strand ThechemopreventiveeffectsoflyophilizedstrawberriesandLBRs up
breaks in both cell types. Administration of 50 mg/kg body wt of whenadministeredtoratsaftertreatmentwithNMBA(postinitiation .co
m
ferulicacid,afterwhole-bodyirradiationofmice,resultedinadisap- protocol) are shown in Table IV. When administered postinitiation,
/c
pearanceofDNAstrandbreaksatafasterratethanwasobservedin LBRsreducedtumorincidencewhereasstrawberrieshadnoeffecton a
rc
irradiatedcontrols,suggestingenhancedDNArepairinferulicacid- tumorincidence(25,38).BothstrawberriesandLBRsproducedsig- in
treatedanimals.Similarresultswereobtainedwithtroxerutin(ade- nificant reductions in tumor multiplicity; however, the 5% dietary /a
rivativeoftheflavonoid,rutin)byMauryaetal.(41).Thus,atleast concentrations were more effectivethan the 10%. The reason(s) for rtic
le
two compounds found in berries are effective in enhancing DNA thisresultisunknown,butdidnotappeartobeduetotoxiceffectsof -a
repair,asevidencedbyamorerapidrejoiningofirradiation-induced 10%berriesforesophagealepithelium.Inthepostinitiationprotocol, bs
DNA strand breaks in berry-treated cells. In contrast, another LBRswerefoundtoinhibittheconversionofpreneoplastic(dysplas- tra
c
investigationfoundthatresveratrolhadonlyamodesteffect.Ellagic tic)lesionstopapillomasandtoreducecellularproliferation,asde- t/2
acid had no effect, on stimulation of the DNA repair protein, termined by measuring the expression of proliferating cell nuclear 9/9
O6-methylguanine-DNA-methyltransferase, in human lymphocytes antigeninesophagealepithelium,atdifferenttimepointsduringthe /1
6
andinglioblastomaandcoloncancercells(42).Theseresultssuggest 6
5
thattheseberrycompoundsmayhavelittleornoeffectonrepairof /2
4
O6-alkylguanineadductsinDNA. 7
6
TableIII. Effectsoflyophilizedstrawberries,blackraspberriesand 7
9
Inhibitionofcarcinogen-inducedtumorsinanimals blackberriesonNMBA-inducedratesophagealtumorigenesiswhen 8
Berry bioactives have been shown to prevent chemically induced administeredinthedietbefore,duringandafterNMBAtreatment(complete by
tumorsin multipleanimal model systems(43). Someoftheearliest preventionprotocol) gu
e
studies were done with ellagic acid [see summary by Stoner and s
Mukhtar (44)] that has been shown to inhibit carcinogen-induced Treatment Rats(n) Tinucmidoernce Tmuumltioprlicity t on
cancerintherodentlung,skin,esophagus,liver,colonandmammary (%inhibition) (%inhibition) 30
gland.Mechanisticstudiesindicatethatellagicacidinhibitscarcinogen- M
inducedDNAadductformation,scavengesthereactivemetabolitesof Vehiclecontrol 15 0 0.0 arc
pooflgylcuytcaltihciohnyed-rSo-ctraarnbsofnesr,assetimanudlatoecsctuhpeieasctsiivtietsyionfDvaNriAoutshaistomfoirgmhst 1N0M%BSATcRoWntsrol 1155 1000 4.10.±00.2 h 20
otherwise react with carcinogens (45). Ellagic acid has also been NMBAþ5%STRWs 15 100 3.1±1.0(24)a 19
NMBAþ10%STRWs 15 80(20) 1.8±1.4(56)a
showntoinhibittheactivityoftheenzyme,ornithinedecarboxylase,
Vehiclecontrol 15 0 0.0
resultinginreducedpolyaminelevelsinmouseskinandaninhibition 10%BRs 14 0 0.0
ofskintumorigenesis(46).Interestinellagicacidasachemopreven- NMBAcontrol 13 100 3.2±0.3
tive agent was significantly reduced, however, by early studies that NMBAþ5%BRs 14 79(21) 1.9±0.4(41)a
demonstrateditspoorbioavailabilityinrodents(47,48). NMBAþ10%BRs 13 92(8) 1.6±0.3(50)a
Whileconductingstudieswithellagicacidinthe1980s,ourlabo- Vehiclecontrol 10 0 0.0
ratorydecidedtoidentifyfoodsinwhichitmightbefound(itwasnot 10%BBs 10 0 0.0
NMBAcontrol 17 82 2.8±0.6
known at that time to be widely present in berries). In examining
NMBAþ5%BBs 20 75(8) 1.5±0.4(46)a
aseriesoffruits,wefoundhighconcentrations(630–1500lg/gdry
NMBAþ10%BBs 18 70(15) 2.3±0.5(18)
wt) of ellagic acid in blackberries, red raspberries and strawberries
andlesseramountsincranberries(49).Theellagicacidwasfarmore
STRWs,strawberries;BRs,blackraspberries;BBs,blackberries.
abundantinthepulpandseedoftheberries;verylittlewasfoundin aStatisticallysignificantrelativetoNMBAcontrols(P,0.05).STRWdata
thejuice.Basedupontheseobservations,wedecidedtotakea‘food- aretakenfromCarltonetal.(38),BRdataarefromKrestyetal.(25)andBB
based’ approach to cancer prevention and determine if lyophilized dataaretakenfromStoneretal.(43).
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Cancerchemopreventionwithberries
TableIV. Effectsoflyophilizedstrawberriesandblackraspberrieson TableV. EffectsoflyophilizedblackraspberriesonAOM-inducedratcolon
NMBA-inducedratesophagealtumorigenesiswhenadministeredinthediet tumorswhenadministeredinthedietafterAOMtreatment(postinitiation
afterNMBAtreatment(postinitiationprotocol) protocol)
Tumormultiplicity Tumormultiplicity
Treatment Rat(n) Tumor Tumors/rat %Inhibition Treatment Rat(n) Tumor Tumors/rat Inhibition
incidence (mean± incidence (mean± (%)
(%) standarderror) (%) standarderror)
Vehiclecontrol 10 0 0 0 Vehiclecontrol 18 0 0 0
10%STRWs 10 0 0 0 5%BRs 18 0 0 0
NMBAcontrol 14 100 4.8±0.6 0 AOMcontrol 18 67 2.33±0.5 0
NMBAþ5%STRWs 15 100 3.0±0.3a 38 AOMþ2.5%BRs 18 78 1.35±0.3a 42
NMBAþ10%STRWs 15 100 3.3±0.4a 31 AOMþ5%BRs 18 89 1.27±0.2a 45
Vehiclecontrol 10 0 0 0 AOMþ10%BRs 18 50 0.67±0.2a 71 D
o
10%BRs 10 0 0 0 w
n
NMBAcontrol 15 87 1.4±0.3 0 BRs,blackraspberries.DataaretakenfromHarrisetal.(33). lo
NMBAþ5%BRs 15 40a 0.5±0.2a 64 aStatisticallysignificantrelativetoAOMcontrol(P,0.05). ad
NMBAþ10%BRs 15 47a 0.8±0.3a 43 ed
fro
STRWs,strawberries;BRs,blackraspberries. m
aStatisticallysignificantrelativetoNMBAcontrols(P,0.05).STRWdata h
aretakenfromCarltonetal.(38)andBRdataarefromKrestyetal.(25). TableVI. EffectoflyophilizedblackraspberriesonDMBA-inducedtumors ttp
inthehamstercheekpouchwhenadministeredinthedietbefore,duringand s
afterDMBAtreatment(completepreventionprotocol) ://a
c
a
Tumormultiplicity de
bioassay(25).Inthesestudies,theinhibitoryeffectsofalllyophilized m
berrytypesontumormultiplicitywerenotdoserelated;i.e.10%berry Treatment Rat(n) Tumor Tumors/rat %Inhibition ic.o
dietswerenottwiceaseffectiveinreducingthenumberofNMBA- incidence(%) up
inducedtumorsas5%diets.Similarly,theinhibitoryeffectsofellagic DMBAcontrol 15 100 3.20 0 .co
m
acid on NMBA-induced tumors in the rat esophagus were not dose DMBAþ5%BRs 14 93 1.93 40 /c
related (50). These results suggest that there may be a maximum DMBAþ10%BRs 15 100 2.60 19 arc
thresholdlevelintheuptakeofdietaryberryphenolicsintotheesoph- in
agus,beyondwhichadditionalamountsofdietaryphenolicshaveno BRs,blackraspberries.DataaretakenfromCastoetal.(39). /a
addedbenefit. aStatisticallysignificantrelativetoDMBAcontrol(P,0.05). rtic
le
LBRswerealsofoundtoinhibitAOM-inducedcoloncarcinogenesis -a
inrats,whengiveninapostinitiationprotocol(TableV)(33).When bs
fedat2.5,5.0and10%ofthediet,LBRsdidnotreducetumorincidence; tra
mino)-1-(3-pyridyl)-1-butanoneorB(a)P.Micewereadministeredthe c
however,theyreducedthetotaltumor(adenomaþadenocarcinoma) strawberrydietbeginning1weekbeforeinitialcarcinogentreatment t/2
multiplicityby42,45and71%(P,0.05inallgroups),respectively, and throughout the study. 4-(methylnitrosamino)-1-(3-pyridyl)-1- 9/9
relative to AOM controls. Inhibition of oral cancer by the dietary butanoneandB(a)Pwereadministeredintraperitoniallyovera2week /16
administrationofLBRswasdemonstratedinthehamstercheekpouch 6
periodbeginningatweek2ofthebioassay.At24weeks,therewere 5
byCastoetal.(39)(TableVI).Tumorswereinducedbypaintingboth no differences in lung tumor incidence or multiplicity in any of the /24
cheekpouchesofhamsterswithDMBA.Hamsterswerefedeither5or 7
groups. The successesinreducing carcinogen-inducedcancers in the 6
10%LBRsinAIN-76Adiet,priorto,duringandaftertreatmentwith 7
oralcavity,esophagusandcolon,whereberrycomponentscomeinto 9
DMBA. Treatment with 5% LBRs caused a significant reduction in 8
directcontactwiththetissues,andthefailuretoinhibitlungtumorsin b
tumor multiplicity (P , 0.05), but the reduction with 10% berries y
micesuggestthat(i)eitherlungtumorsfailtorespondtoberrycompo- g
was not significant. The reasons for the lack of inhibitory effect in u
nentsor(ii)theactivecomponentsinberriesfailtoreachthelungin e
animalsadministered10%LBRswerenotdetermined. s
ricAhfrreaccetinotn,stduedryivefrdofmromourLBlaRbosraantodraynsahlocwoheodl:twhaattearn(8a0n:2th0o)ceyxatnrainc-t saumffiocrieenlotgaimcaoluenxtpslatonaetiloicnitsipnrcoetesctutidoine.sTohnethleatuteprtackoenoclfuasniothnomcyaaynibnes t on 3
and ellagitannins from berry juices in rodents have shown that these 0
of LBRs, were equally as effective as a 5% LBR diet in reducing M
NMBA-induced tumors in the rat esophagus (51). The content of c(5o3m,5p4o)u.ndsarepoorlyabsorbedandtheirplasmalevelsdeclinerapidly arc
anthocyanins in the anthocyanin-rich diet and in the alcohol:water h 2
extract diet was essentially identical to that in the 5% LBR diet 0
1
((cid:6)3.5lmol/gdiet).Sinceallthreedietscontainedthesameamounts Modulationofsignalingpathwaysassociatedwithcellproliferation, 9
ofanthocyaninsandwereequallyeffectiveinpreventingesophageal apoptosis,inflammation,angiogenesis,cellcyclearrestandcell–cell
cancer, these data suggest the importance of the anthocyanins as communication
chemopreventive agents inblack raspberries. However,in this same Numerousstudieshaveinvestigatedtheeffectsofberryphytochem-
study,anotherdietcontaininganalcohol-insoluble(residue)fraction icalsonsignalingpathwaysassociatedwithcancerdevelopment(19).
of LBRs, that contained only trace amounts of anthocyanins, was Theseincludeinvitrostudiesoftheabilityofberryextractsandtheir
nearlyaseffectiveastheanthocyanin-richandalcohol:waterextract purifiedderivativesto inhibitcell growth and tostimulate apoptosis
dietsinpreventingNMBA-inducedesophagealtumorigenesis.Recent anddifferentiation.Althoughmanyofthesestudieshavefocusedon
results suggest that the residue fraction contains high levels of ella- the effects of berry extracts, some of them have involved bioassay-
gitannins (S.S. Hecht, S. Carmella, L-S Wang and G.D. Stoner, un- directedfractionationofextractstoidentifytheactiveconstituentsin
publisheddata).Thus,boththeanthocyaninsandellagitanninsappear berries.Resultsfrominvitrostudieshavealsobeenconfirmedinvivo
tobeimportantforthechemopreventiveeffectsofberries. in a limited number of investigations. This review will attempt to
Incontrasttotheaboveobservations,Carltonetal.(52),foundthat discuss only some of the in vitro and in vivo results on the effects
adietcontaining10%lyophilizedstrawberrieswasineffectiveinre- ofberryconstituentsoncellularfunctionsandsignalingpathwaysin
ducing lung tumors in mice that were induced by 4-(methylnitrosa- theparagraphsthatfollow.
1669
G.D.Stoneretal.
Invitrostudies. Mostinvitrostudiesoftheantiproliferativeeffectsof Theeffectsofberryextractsonsignaltransductionpathwayshave
berry extracts and components have been conducted with human alsobeendiscussed(62).IninitialstudieswiththeJB-6Cl41mouse
tumorcelllines.Forexample,extractsfromstrawberrycultivarshave epidermalcellmodel,Huangetal.(63)demonstratedtheinhibitory
beenshowntoreducetheproliferationofhumanHepG livertumor effectsofamethanolfractionofblackraspberriesandofotherpuri-
2
cellsandofhumancolon(HT29)andbreast(MCF-7)cancercellsin fiedfractionsontransactivationofAP-1andNF-jBinducedbyBPDE
a dose-dependent manner (55). However, there was no correlation inJB-6Cl41cells.Theinhibitoryeffectsappearedtobemediatedvia
between the antioxidant capacity of the cultivars or the levels of inhibition of mitogen-activated protein kinase activation and inhibi-
anthocyaninsandflavonolsintheextractsandtheirantiproliferative tory subunit jB phosphorylation, respectively. Pretreatment of cells
effectsforthethreecelllines.Jurinacetal.(56)comparedtheeffects with the methanol fraction did not result in an inhibition of BPDE
of a red raspberry extract with ellagic acid alone on the growth of binding to DNA; thus, this was not a mechanism of reduced AP-1
humancoloncancerLS174cellsandnormalimmunecompetentcells. and NF-jB activation. None of the tested fractions affected p53-
Resultsfromthiscomparisonindicatedthattheextractproducedase- dependent transcription activity. The ability of black raspberries to
lectiveinhibitoryeffectonthegrowthofthecoloncancercells,which inhibit tumor development may be mediated by impairing signal
correlatedpositivelywithitscontentofellagicacid. transductionpathwaysleadingtoactivationofAP-1andNF-jB.This
D
Seerametal.(57)evaluatedtheantiproliferativeeffectsofmetha- concept is supported by the observation that the methanol fraction o
nolextractsofblackberry,blackraspberry,blueberry,cranberry,red markedlyinhibitedactivationofPI-3K,Aktandp70S6kinase,sug- wn
raspberry and strawberry for human oral (KB and CAL27), breast gestingthatanothermechanismforthechemopreventiveactivitymay lo
a
(MCF-7),colon(HT29,HCT116)andprostate(LNCaP)cancercell bethroughinhibitionofthePI-3K/Akt/AP-1/VEGFpathway(64). de
d
lminaersk.eTdlhyewexitthenbtoothftihnehitbyiptieonofobfecreryllepxrtorlaicfetraantidonthweaspsefcoiufincdcteollvlainrye ofThhuemeafnfeccatsncoefrincdeilvlidliunaelsbhearrvyebailosaoctbiveeensoinnvgersotiwgtahteadn.dQaupeorpcteotsinis, from
studied. The effects of the different berry extracts on cell apoptosis aflavonolfoundinseveralvarietiesofberriesandblackcurrents,at h
was assessed using HT29 cells that were treated with 200 lg/ml of concentrationsupto70–80mg/kgdrywt,wasfoundtoinhibitgrowth ttp
s
eachberryextract(asignificantly higherlevelthancanbeachieved and stimulate apoptosis of lung cancer cells (65). Survivalofalung ://a
invivo).Onlyblackraspberryandstrawberryextractsproducedasig- cancercellline(NCI-H209)wasdecreasedinaconcentration-dependent c
a
nificantinductionofapoptosis.Theauthorsconcludedthatthereare manner after treatment with quercetin glucuronide. Importantly, d
e
severalfactorsthatmustbeconsideredwhenusingcellculturestudies quercetin was effective invitroatdoses(1–10lM)thatarepharma- m
ic
torankthechemopreventiveactivitiesofberryextracts.Theseinclude cologically relevant in vivo. Quercetin treatment resulted in an in- .o
u
thecelllinebeingused,theartificiallyhighconcentrationsofextract, crease in cell cycle arrest with the cells accumulating in the S and p
stabilityofextractcomponentsindifferentmedia,lengthoftreatment G2/MphasesofthecellcycleandinsubG0whileundergoingapopto- .com
time,differentialuptakeofphenolicsandgenerationofartifactssuch sis.Apoptosiswasassociatedwithdecreasesinmitochondrialmem-
/c
asH O thatisknowntoinduceapoptosis. branepotentialandintheantiapoptoticprotein,Bcl-2,andincreases a
Bi2oac2tivity-guidedfractionationhasbeenusedtoidentifytheactive in the proapoptotic proteins, p21CIP1/WAF1, Bak, Bax, cytochrome rcin
constituents in different berry types. Using bioactivity-guided frac- candcaspase3.Nguyenetal.(66)treatedA549humanlungepithelial /a
tionation,Murphyetal.(58)identifiedtriterpenoidestersincranber- cellswithquercetinandfoundasimilardose-dependentreductionin rtic
le
riesasimportantforinhibitionofthegrowthofhumanMCF-7breast, cellviabilityandinductionofapoptosis.Mostofthereductionincell -a
ME180 cervical and PC3 prostate tumor cell lines. Similarly, Han viabilityatthetwohighestdoses(43.5and58lM)wasattributedto bs
et al. (59) used bioactivity-guided fractionation to identify chemo- apoptosis.Inaddition,quercetintreatmentledtophosphorylationof tra
c
preventive phytochemicals in black raspberries. They compared the theextracellularsignal-relatedkinase,ERK1/2,andERK1/2activa- t/2
effectsofseveralextractfractionsofblackraspberrieswiththoseof tion was accompanied by phosphorylation of the mitogen-activated 9/9
theberryconstituents:ellagicacid,ferulicacidandb-sitosterolonthe kinase,MEK. /1
6
growthandcellcycleofnormal,premalignantandmalignanthuman Individualanthocyaninsinberrieshavealsobeenshowntoexhibit 6
5
oralcavitycells.Anethanolextractwasfoundtoinhibitgrowthofthe chemopreventive effects in vitro. Ding et al. (67) investigated the /2
4
premalignantandmalignantbutnotthenormaloralcelllines.Ellagic effectsofcyanidin-3-O-glucoside,isolatedfromblackberries,ongene 7
6
acidinhibitedthegrowthofnormal,premalignantandmalignantcell expressioninJB-6cells.Cyanidin-3-O-glucosidepretreatmentledto 7
9
lines,whereasferulicacidandb-sitosterolexhibitedselectivityforthe a dose-dependent decrease in the expression of cyclooxygenase-2 8
b
premalignantandmalignantlines.Thus,theseworkersconcludedthat (COX-2) and activities of AP-1, NF-jB and tumor necrosis factor y
g
ferulic acid and b-sitosterol were effective bioactives in black rasp- (TNF)a, when the cells were treated with 12-O-tetradecanolyphorbol- u
e
berries. Using western blot analysis, all treatments were found to 13-acetate or ultraviolet. Finally, an anthocyanin-rich preparation, s
affectdifferentcyclinsandcyclin-dependentkinasesinthepremalig- fromberries(wildblueberry,bilberry,cranberry,elderberry,raspberry t on
nantandmalignantlines.Thisledtotheconclusionthatthegrowth- and strawberry), was found to reduce VEGF expression in a sponta- 3
0
inhibitoryeffectsofblackraspberriesonpremalignantandmalignant neously immortalized human keratinocyte cell line (HaCaT) and in- M
a
oral epithelial cells reside in the specific effects of different berry hibit endothelial tube formation in a Matrigel assay (68). rc
componentsonsignalingpathwaysregulatingcellcycleprogression. Itshouldbementionedthatthereisgrowingconcernthatobserva- h 2
Rodrigoetal.(60)alsoevaluatedthepreventativeeffectsofaneth- tionsoftheeffectsofphenolicantioxidantsinvitroasameasureof 0
1
anolfractionofblackraspberriesonhumanoralcancercelllinesand their chemopreventive activity may be deceptive and should not be 9
foundthattheextractinhibitedcellproliferation,vascularendothelial extrapolatedtoactivityinvivo(69–72).Longetal.(70)suggestedthat
growth factor (VEGF) production and nitric oxide synthase activity the effects of several antioxidants in vitro are artifacts due to the
and stimulated apoptosis and terminal cell differentiation. In an ex- production of hydrogen peroxide generated from these antioxidants
tensionofthesestudies,anotherethanolfractionofblackraspberries intheculturemedium.TheypointedoutthatH O canproducemany
2 2
wasextensivelysubfractionatedbyhigh-performanceliquidchroma- oftheeffectsobservedinvitrowithphenolics.Theseeffectsinclude
tographyandtheindividualsubfractionscomparedfortheirabilityto elevationofintracellularCaþþlevels,activationoftranscriptionfac-
downregulatetheexpressionlevelsofnuclearfactorkappaB(NF-jB) tors,inhibitionofcellproliferation,cytotoxicity,alterationofcertain
and activator protein-1 (AP-1) in mouseepidermal JB-6 Cl 41cells signaltransductionpathways,stimulationofapoptosis,expressionof
(61).Interestingly,themajorconstituentsofthemostactivesubfrac- adhesion molecules such as intercellular adhesion molecule 1, pro-
tionswerethethreemostprevalentanthocyaninsinblackraspberries: motionofdifferentiationorcellsenescence,downregulationofacti-
cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and cyanidin-3-O- vation of AP-1 and suppression of protein kinase activation. These
(2G-xylosylrutinoside).Theseresultsfurthersuggestthatthecyanidin findingsaresupportedbytheworkofotherinvestigators(69,71,72),
glucosides (anthocyanins) account for at least some of the chemo- whoalsosuggestcautionininterpretingtheresultsofstudiesofthe
preventiveactivityofblackraspberries. effectsofantioxidantsonnormalandtumorcellsinvitro.
1670
Cancerchemopreventionwithberries
Anadditionalconcernofconsiderable importanceintheextrapo-
lationofinvitrodatatotheinvivosituationisthatofdoseextrapo-
lation. Most, if not all, berry bioactives produce chemopreventive
effects in cell cultures when added at micromolar concentrations
((cid:6)10to150lM).Incontrast,pharmacokinetic studiesindicatethat
berry bioactives, such as the anthocyanins and ellagitannins, reach
only nanomolar concentrations ((cid:6)1 to 20 nM) in blood and tissues
when administered in the diet (13,77). Thus, caution must be used
whenextrapolatinginvitroresultstotheinvivosituation;perhaps,the
mostappropriateuseoftheinvitrodataisthatofidentifyingpossible
mechanisticbiomarkersforclinicalinvestigations.
Invivostudies. Theeffectsofdietscontaininglyophilizedberrypow-
dersontheexpressionofgenesinvolvedinsignaltransductionhave D
beeninvestigated.AsindicatedinTableIV,atdietaryconcentrations ow
of 5 and 10%, lyophilized strawberry and black raspberry powders n
lo
significantly reduced tumor multiplicityby30–60%, whenadminis- a
d
tteurmedoripnroagrpeossstiionnitiinattihoenrsactheesmopeh,aignudsic(a2t5in,3g8)t.hTeihreamboillietycutloarimnheicbhi-t Ftriagn.s2d.ucTtihoenepfafethcwtsaoyfs.bAerlrlygbeinoeasctsihvoewsnonarceeldluowlanrrfeugnucltaitoendsbayndbesrigrynal ed fro
anismsofthisinhibitoryeffectontumorprogressionwereinvestigated m
by Chen et al. (73), who showed that the 5% black raspberry diet Bbiaoxa,cctiyvteoschexrocmepetcfoarnidntcearcspelalsuela3rwadhhiecshioanremuoplreecguulleat1e,dp.21CIP1/WAF1,Bak, http
downregulated the mRNA and protein expression levels of COX-2 s
and c-Jun (a component of AP-1), in preneoplastic tissues of ://a
c
NMBA-treatedesophagus.ThisreductioninCOX-2expressioncor- ongoingorhavebeencompleted.Abriefdiscussionofstudiesfrom ad
relatedwithloweredlevelsofprostaglandinE inesophagealepithe- e
2 whichdatahasbeenreportedisasfollows. m
lium that might be expected to reduce cell proliferation. The ic
inhibition in c-Jun expression might also be expected to reduce the .ou
rate of cell proliferation since this gene is a component of the tran- Barrett’sesophagus p.c
scription activator, AP-1, which is induced by activated Ras onco- Barrett’sesophagus(BE)affectsanestimated700000USadultsand om
genic proteins (74). Nearly all NMBA-induced tumors in the rat remainstheonlyrecognizedprecursorlesiontoesophagealadenocar- /c
a
esophagus have an activated H-ras oncogene (75). The reduction in cinoma, a cancer that has increased dramatically in incidence rc
inducible nitric oxide synthase (iNOS) expression correlated with throughout most of the western world over the last three decades in/a
loweredlevelsofnitrate/nitriteintheesophagusand,alongwithre- (78).Astudywasundertakentodetermineiftheoraladministration rtic
ducedCOX-2,mightbeexpectedtoinhibitinflammatoryeventsinthe of black raspberry powder would influence parameters associated le
tissue.TheloweredlevelsofVEGFexpressionwereaccompaniedby with the progression of Barrett’s lesions (79). Twenty BE patients -a
b
acastiinggnifithcaatntthreebdeurcrtiieosnininhimbiitcerdovaensgsieolgedneenssiisty(7in6)t.hTeheusso,pthheasgeuss,tuinddieis- cboenrrsyumpoewdd3e2rodrai4l5y,gas(feamsalulerryanidnmwaaltee,rrefospre2c6tivweelye)kso.fBbiloacpksierassopf- strac
demonstratedthatlyophilizedberriesreducemolecularmarkersofcell Barrett’slesionsweretakenbeforeandafterberrytreatmentforbio- t/2
9
proliferation,inflammationandangiogenesisincarcinogen-treatedrat marker analysis. Based upon interim results from 10 of the 20 pa- /9
esophagus. A summary of the effects of berry bioactives on cellular tients,berrytreatmentdidnotresultinareductioninsegmentlength /16
6
functionsandsignaltransductionpathwaysisshowninFigure2. of Barrett’s lesions at 26 weeks. Urine was collected from each 5
subject at study baseline, at week 12 of study and at 26 weeks /24
7
and evaluated for the oxidative damage biomarkers; 8-OHdG and 6
7
Clinicalstudiesoftheanticancereffectsofberrybioactives 8-epi-prostaglandin F2a (8-Iso-PGF2). Levels of urinary 8-Iso-PGF2 9
8
were significantly reduced (Figure 3), but there was no significant b
There are relatively few clinical studies of the anticancer effects of y
change in mean levels of urinary 8-OHdG (although five of the 10 g
berries. Most of the available data on berry bioactives, in humans, u
subjects had reduced levels). The authors concluded that the daily e
derive from pharmacokinetic studies of the absorption, distribution, s
mfoeotdasbtoulfifssmoranads epxucrirfieteidoncoomfbpeorurnydcso.mThpeosuendsstu,doibetsaihnaevdeebiteheenrsfruomm- cinontshuemputriionnaryofelxycorpehtiiolinzedofbtlwacok braiospmbaerrkrieerss porfomoxoitdesatirveeduscttrieosnss, t on 3
8-Iso-PGF2, and to a lesser more-variable extent, 8-OHdG, among 0
marized extensively by Seeram (19). Recently, our laboratory con- M
patientswithBE.Oneconcernfromthisstudyisthatthetransittime a
ductedaphaseItrialin11subjectstodeterminethesafety/tolerability of the black raspberry slurry across the esophagus, when consumed rc
ofLBRsandtomeasureanthocyaninsandellagicacidinplasmaand orally,isveryrapidandmaynotpermitsufficientlocalizedabsorption h 2
urine(77).Subjectswerefed45g(equivalenttoa5%berrydietin 0
ofberrycompoundsintoBarrett’slesionstobeeffective. 1
animals) of black raspberry powder as a slurry in water daily for 9
7days.Bloodandurinesampleswerecollectedpriortoandafterberry
treatment.Theberrieswerefoundtobewelltolerated;theonlyclin- Colonpolypsandcancer
icalobservationwasalowincidenceofmildormoderateconstipation BaseduponpositiveresultsoftheinhibitoryeffectsofLBRsoncolon
infourofthe11subjects.Maximumconcentrationsofanthocyanins tumordevelopmentinrodents(33),weinitiatedastudyin50subjects
andellagicacidinplasmaoccurredat1–2h,andmaximumquantities withcolorectalcancerand/orpolypstodetermineiftheoraladmin-
inurineappearedfrom0to4h.Theuptakeoftheanthocyaninsinto istration of LBRs would modulate biomarkers of colon cancer de-
plasmawasreflectiveoftheirrelativeconcentrationsinblackraspberry velopment. Biopsies of normal and tumor/polyp tissues are
powder with cyanidin-3-rutinoside . cyanidin-3-xylosylrutinoside . collected at baseline. Subjects consume 20 g of freeze-dried LBR
cyanidin-3-glucoside5cyanidin-3-sambubioside.Overall,theuptake powder, as a slurry in water, three times per day (60 g total), until
ofellagicacidandtheanthocyaninswas,1%oftheadministereddose. theirscheduledsurgerydate,usuallywithin2–4weeks.Posttreatment
Interestingly,themajormetabolitesoftheanthocyaninsinurinewere biopsyspecimensarecollectedduringthesurgery.Pre-andposttreat-
methylatedderivativesandneitherglucuronidenorsulfateconjugates. mentspecimensfrom23patientshavebeenanalyzedtodateforthe
Several investigations of the effects of berry powders on cancer effectsofLBRsoncellproliferationusingKi67,apoptosisbyTUNEL
developmentinthehumanesophagus,colonandoralcavityareeither and angiogenesis by staining for CD105. Proliferation and
1671
G.D.Stoneretal.
that this is the sole mechanism for their inhibitory effects. Invitro
studieshaveclearlydemonstratedtheabilityofberrycompoundsto
entercellsandtobindcovalentlytocellularproteins(84);thiscould
be responsible for at least some of their effects on cell signaling
pathwaysandothercellularfunctions.
Investigationsinanimalsindicatethatdietaryberrybioactivesare
effectiveinpreventingcancerintheoralcavity,esophagusandcolon.
Preliminaryresultssuggestthattheymightalsoexhibitpreventative
effectsinthehumanoralcavity,esophagusandcolon.Inthesetissues,
localizedabsorptionofberrycompoundsappearstobeimportantfor
theirchemopreventiveeffects.Berrieswerenoteffectiveinprevent-
inglungcancerinmicewhenadministeredinthediet(52),presum-
ablybecausetheydonotreachthelunginsufficientconcentrationsfor
chemoprevention.Thisisconsistentwithpharmacokineticstudiesin
D
animals and humans in which it has been shown that the uptake of o
berrybioactives,suchastheanthocyanins,ellagitanninsandquerce- wn
tin,intothebloodstreamislow;i.e.inthenanomolarrange.Thus,the lo
a
Fig.3. Changesofmeanurinary8-epi-prostaglandinF2alevelsinBE future development of whole berry formulations that augment the de
patientsduring26weeksofstudy. d
ainbsmoroprteioenffeocftibveerrpyrebvieonatcitoinveisnitshewoorrathlycaovfitpyu,ressuoipthaangduscoaunlddcroelsounlt, from
aswellasinotherorgansitesinwhichberrieshavenotbeenshownto h
angiogenesis biomarkers were reduced significantly by the berry exhibitchemopreventiveefficacy. ttp
s
treatment,whereasapoptosiswasenhanced(80). ://a
c
Acknowledgements ad
Oralcancer e
m
BaseduponanimaldatademonstratingaprotectiveeffectofLBRson ConflictofInterestStatement:Nonedeclared. ic
.o
chemicallyinducedcancerinthehamstercheekpouch(39),studies u
p
havebeenundertakentodetermineifLBRsmightexertaprotective .c
effect on the development of oral cancer in humans. A preliminary References om
phase I study evaluated the utility of a mucoadhesive gel for the /c
a
localized delivery of berry compounds to human oral mucosa (81). 1.Weisburger,J.H.(1999)Mechanismsofactionofantioxidantsasexempli- rc
fiedinvegetables,tomatoesandtea.FoodChem.Toxicol.,37,943–948. in
Bmlualcakterdatsopbinesruryregtehlessctaobnitlaiitnyionfgth5eaanndth1o0c%yanbienrsr.yMpaoxwimdeurmwsetraebifloitry- 2.Acamnceesr,.BM.Nu.teatt.aRl.e(s1.9,9215)0E,3n–d1o6g.enousmutagensandthecausesofagingand /artic
opfertahteusreecoofm4p(cid:2)Cou.nWdsheinntthheeggeellwwaassoabpspelrievdedtoaptipcaHlly3.5toatnhdeaotraaltmemu-- 3.SCtaenincmereCtza,Kus.Aes.Cetoanlt.ro(l1,929,14)2V7e–g4e4t2a.bles,fruitandcancer.IImechanisms. le-ab
s
cosa of normal subjects, the anthocyanins were found to be readily 4.Block,G.etal.(1992)Fruit,vegetablesandcancerprevention:areviewof tra
absorbedintothetissueasevidencedbydetectablebloodlevelswithin theepidemiologicalevidence.Nutr.Cancer,18,1–29. c
5minaftergelapplication.Inaddition,allfouranthocyaninsinblack 5.Willett,W.C.(1995)Diet,nutrition,andavoidablecancer.Environ.Health t/29
raspberries were found to penetrate into explant tissues of oral mu- Perspect.,103,165–170. /9
6.Vainio,H.etal.(2003)FruitandVegetables.IARCPress,Lyon,France. /1
cosa,invitro,withthegreatestpenetrationbeingfromthe10%berry 6
7.World Cancer Research Fund/American Institute for Cancer Research. 6
gel.Basedupontheseresults,theeffectsoftopicalapplicationofthe 5
(2007) Food,Nutrition,PhysicalActivity,andthePreventionofCancer: /2
10%blackraspberrygeltooralintraepithelialneoplasticlesionsin17 4
aGlobalPerspective.AICR,Washington,DC. 7
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preventive assessments of lyophilized black raspberries: interim results ReceivedMarch29,2008;revisedMay30,2008;acceptedJune1,2008 fro
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