Research Article Long Term Exposure to Polyphenols of Artichoke (Cynara scolymus L.) Exerts ...

12 Pages·2015·2.09 MB·English

Hindawi Publishing Corporation
Oxidative Medicine and Cellular Longevity
Volume 2015, Article ID 363827, 11 pages
http://dx.doi.org/10.1155/2015/363827
Research Article
Cynara
Long Term Exposure to Polyphenols of Artichoke (
scolymus
L.) Exerts Induction of Senescence Driven Growth
Arrest in the MDA-MB231 Human Breast Cancer Cell Line
AnnaMariaMileo,1DonatoDiVenere,2ClaudiaAbbruzzese,1andStefaniaMiccadei1
1ReginaElenaNationalCancerInstitute,ViaElioChianesi53,00144Rome,Italy
2CNR,InstituteofSciencesofFoodProduction(ISPA),ViaAmendola122/O,70126Bari,Italy
CorrespondenceshouldbeaddressedtoStefaniaMiccadei;[email protected]
Received8July2014;Accepted8September2014
AcademicEditor:TulliaMaraldi
Copyright©2015AnnaMariaMileoetal. This is an open access article distributed under the Creative Commons Attribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Polyphenolicextractsfromtheediblepartofartichoke(CynarascolymusL.)havebeenshowntobepotentialchemopreventiveand
anticancerdietarycompounds.Highdosesofpolyphenolicextracts(AEs)induceapoptosisanddecreasetheinvasivepotentialof
thehumanbreastcancercellline,MDA-MB231.However,themolecularmechanismunderlyingAEsantiproliferativeeffectsisnot
completelyunderstood.WedemonstratethatchronicandlowdosesofAEstreatmentatsublethalconcentrationssuppresshuman
breastcancercellgrowthviaacaspases-independentmechanism.Furthermore,AEsexposureinducesasignificantincreaseof
senescence-associated𝛽-galactosidase(SA-𝛽-gal)stainingandupregulationoftumoursuppressorgenes,p16INK4aandp21Cip1/Waf1
inMDA-MB231cells.AEstreatmentleadstoepigeneticalterationsincancercells,modulatingDNAhypomethylationandlysine
acetylationlevelsintotalproteins.Cellgrowtharrestcorrelateswithincreasedreactiveoxygenspecies(ROS)productioninAEs
treatedbreastcancercells.InhibitionofROSgenerationbyN-acetylcysteine(NAC)attenuatestheantiproliferativeeffect.These
findingsdemonstratethatchronicAEstreatmentinhibitsbreastcancercellgrowthviatheinductionofprematuresenescence
throughepigeneticandROS-mediatedmechanisms.Ourresultssuggestthatartichokepolyphenolscouldbeapromisingdietary
tooleitherincancerchemopreventionor/andincancertreatmentasanonconventional,adjuvanttherapy.
1.Introduction several clinical trials testing the efficacy of natural com-
poundsareunderway,http://www.clinicaltrials.gov/;someof
Cancerisoneofthemaincausesofdeathworldwide,claiming
themarealreadyconcludedanddemonstratethechemopre-
over six million people each year [1]. According to current
ventiveactivityofdietarypolyphenols[15–17].
estimates, cancer is 30–40% preventable over time with
appropriatenutrition,regularphysicalactivity,andavoidance The current growing interest for dietary plants has led
ofobesity[2]. to a renewed attention for artichoke because of its high
Chemoprevention is a promising strategy which uses polyphenoliccontent.Intheediblepart,itmainlyconsistsof
natural dietary compounds and/or synthetic substances to hydroxycinnamic derivatives, in particular chlorogenic and
block,inhibit,reverse,ordelaytheprocessofcarcinogenesis. dicaffeoylquinicacids.Ourstudiesofbioavailabilitydemon-
Importantpreventivemechanismsincludesuppressionofcell strate that ferulic acid is one of the metabolites present
proliferation and apoptosis and modulation of epigenetic in human plasma after an artichoke meal [18]. Preclinical
processes[3–6]. reports indicate that ferulic acid has antiproliferative and
Manyepidemiologicalstudiessuggestthatdietsparticu- chemopreventive activities in vitro and in vivo [19–21] and
larlyrichinfruitsandvegetableshavecancerpreventivepro- proposedthepotentialuseofferulicacidasanadjuvantagent
perties[7–9].Thebeneficialeffectofdietsisattributable,at duringchemo-and/orradiotherapy[22,23].
leastinpart,topolyphenolswhichhaveantitumouractivities Our previous findings indicate that polyphenolic arti-
bothinanimalmodelsandinhumans[8–14].Furthermore, choke extracts (AEs) protected hepatocytes from oxidative
2 OxidativeMedicineandCellularLongevity
stress and exhibited cancer chemopreventive properties, in 2.MaterialsandMethods
part,bytriggeringapoptosisonhumanhepatomacellsHep
2.1.ArtichokeExtractPreparation. Theediblepart(head)of
G2 [24] and on the human breast cancer cell line, MDA-
freshartichoke(CynarascolymusL.cvViolettodiProvenza)
MB231[25].
buds was used for extract preparation and the analysis of
Despite the growing scientific results regarding chemo-
polyphenols contained in the extracts was performed by
preventive activities of natural dietary compounds [8], the
HPLCaspreviouslydescribed[25].
cellular mechanisms underlying antitumour property of
polyphenolsareyettobeelucidated.
2.2. Cell Lines and Cultured Conditions. Cell lines were
Cellular senescence, a state of cell cycle arrest, can be maintained in a humidified incubator with 5% CO2 and
consideredarelevantmechanismoftumoursuppression[26– 95%airat37∘C.HCT116cells,humancoloncarcinomacell
28].Furthermore,emergingevidencehasdemonstratedthat line,MDA-MB231,oestrogenreceptor-negativebreastcancer
therapy-inducedsenescenceisacriticalmechanismthrough cells, HEY cells, human ovarian cell line, and K-562 cells,
which many anticancer agents inhibit tumour progression human erythromyeloblastoid leukaemia cell line (kindly
[29–31]. Importantly, therapy-induced senescence can be suppliedbyDr.MaurizioFanciulli,Dr.PaolaNistico`,andDr.
achievedinadministeringagentsatlowdoses.Thisapproach Maria Giulia Rizzo, Regina Elena National Cancer Institute
can significantly reduce the side effects of conventional Rome)weregrowninRPMImedium(InvitrogenLifeTech-
anticancer therapy, thus improving the quality of life for nologies,Milan,Italy)supplementedwith10%FBS,10IU/mL
cancerpatients[29,30].Innovativesenescencetherapieswill penicillin,and10𝜇g/mLstreptomycin.GTL-16cells,human
bedevelopedthroughimprovedknowledgeofthemolecular gastric carcinoma cell line, DU 145 cells, human prostate
pathwayscontrollingpermanentgrowtharrestbyspecifically carcinoma cell line, A549 cells, human lung carcinoma cell
screeningforsenescenceeffectors. line, M14 cells, human melanoma cell line, U-373 MG
cells, human astrocytic cell line, and Saos-2 cells, human
Scientificevidencefrominvitrostudiesindicatesthatthe
osteosarcoma cell line (kindly supplied by Dr. Giovanni
cancerpreventionactivityinvolvedmodulationofepigenetic
Blandino, Regina Elena National Cancer Institute, and Dr.
processes. Epigenetics is defined as heritable changes in
Antonella Farsetti, CNR Rome, purchased from American
gene expression that are not accompanied by alterations in
Type Culture Collection, Rockville, MD, USA) were grown
DNAsequence[32].ThemainepigeneticprocessesareDNA
inD-MEMsupplementedwith10%FBS,10IU/mLpenicillin,
methylation, histone modifications, and chromatin remod-
and10𝜇g/mLstreptomycin.
eling. Aberrant patterns of gene expression are key features
of cancer and both genetic and epigenetic abnormalities
2.3. Reagents. Artichoke extracts were dissolved in PBS
are implicated in this molecular deregulation. In contrast
and0.1%dimethylsulfoxide(Me2SO,Sigma-Aldrich,Milan,
to genetic modifications, epigenetic alterations are poten-
Italy).Paclitaxel(Ptx,Sigma-Aldrich)wasdissolvedinPBS.
tiallyreversibleandstrategiestargetingtheepigenomehave 5-Bromo-4-chloro-3-indolyl-𝛽-d-galactopyranoside (X-gal)
beenproposedforbothcancerpreventionandtherapeutics
was purchased from IBI Scientific (Peosta IA, USA) and
[33].
used as previouslydescribed [38]. N-Acetyl-cysteine (NAC,
Induction of premature senescence and modulation of
Sigma-Aldrich) was dissolved in PBS. Dihydroethidium
epigenetic processes have been identified as relevant anti-
(DHE, Molecular Probes-Invitrogen UK) was dissolved in
cancer features of dietary polyphenolic compounds [34].
Me2SO.
Therearebothinvitroandinvivodatashowingthatseveral
bioactive food components can interfere with DNA methy-
2.4.CytotoxicityandCellProliferationAssays. Humancancer
lationandhistonemodificationsandaffecttheexpressionof cells were seeded at density of 2.0 × 105 in 6-well plates
genesinvolvedinthecarcinogenicprocess[35].Inparticular,
(Sarstedt, Numbrecht, Germany) in triplicate and cultured
phenolicacids,includingchlorogenicacid,havebeenshown
for 24h before adding either the vehicle (0.1% Me2SO) or
toaffectDNAmethylation[36].
AEs. Cytotoxicity experiments were assessed in serum-free
Sincetheinductionofsenescencerequiresmoderatecon- mediumandcellsweretreatedwithAEs(200,400,600,and
centrationsofanticanceragentsandproducesalmostnoside 800𝜇M)for24h.
effects[37],wesoughttoinvestigatewhetheralowdoseand Forthecellproliferationexperiment,MDA-MB231were
chronictreatmentofAEscouldinhibitthegrowthofbreast seeded at a density of 5 × 103 in 6-well plates in triplicate.
cancercellsthroughtheinductionofprematuresenescence. After 24h the cells were exposed to AEs (10 and 30𝜇M)
In the present study, we demonstrate that a moderate and for10 days. To determine therole ofROS in AEs-mediated
chronic treatment of AEs causes a significant increase in senescence,cellswerepretreatedwith10mMNACfor2hand
senescence-associated 𝛽-galactosidase (SA-𝛽-gal) detection thenexposedtoAEs(10and30𝜇M)for10days.
andupregulationofp21Cip1/Waf1andp16INK4aproteinexpres- Cellswereharvestedbytrypsinization,stainedwith0.4%
sion in MDA-MB231 cells. Such a premature senescence is trypanblue(Sigma-Aldrich),andcountedusingahaemocy-
tometer.
via ROS-mediated DNA damage and is associated with the
modulationofepigeneticmachinery.Altogether,theseresults
highlightasignificantcontributionofsenescenceinduction 2.5.Senescence-Associated𝛽-Galactosidase(SA-𝛽-gal)Assay.
toAEsanticancereffects. MDA-MB231 cells were seeded at a density of 5 × 103 in
OxidativeMedicineandCellularLongevity 3
6-wellplatesintriplicateandculturedfor24hbeforeadding For the immunostaining analysis, cells (1 × 105) were
eitherthevehicleorAEs(10and30𝜇M).After10daysofa cytospun using a Shandon Cytospin 3 (Thermo Scientific,
chronic treatment, with every 48h renewal of medium and Waltham, MA, USA) at 1000rpm for 10 minutes and then
treatment, cultured cells were washed in PBS, fixed in 2% processed for 5-mC immunostaining. Cells were permeabi-
formaldehyde/0.2% glutaraldehyde for 15 minutes at room lized with 0.4% triton X-100 in PBS for 20 minutes. Cells
temperature. Cells were then washed and incubated with were washed with PBS for 10 minutesand then blocked for
fresh SA-𝛽-gal staining solution containing 1mg/mL X-gal, 30 minutes with 3% preimmune goat serum in PBS. After
40mMcitricacid/sodiumphosphate(pH6.0),5mMpotas- 20minutesofincubationwith3%H2O2 inordertoquench
sium ferrocyanide, 5mM potassium ferricyanide, 150mM endogenous peroxidase the cells were washed with PBS
∘
NaCl, and 2mM MgCl2 for 16–18h at 37 C. Blue-stained and incubated with 5-mC specific antibody (1:500vol/vol)
senescentcellswerecountedusingalightmicroscopy(Olym- for 2h. Cells were sequentially incubated with biotiny-
pusIX71-OlympusAmerica,CenterValley,PA,USA)andthe latedpredilutedantibody,horseradishperoxidase-conjugated
imageswerecapturedbydigitalcamera(Olympus-Camedia streptavidin(UltraTekHRP,ScyTekLaboratoriesInc,Logan,
C-5060). UT, USA) and AEC substrate (AEC substrate kit, ScyTek
LaboratoriesInc.)andcounterstainedwithhaematoxylin.
2.6.Western-BlotAnalysis. Topreparethewhole-cellextract,
cellswerewashedwithPBSandsuspendedinicecoldRIPA 2.8. Intracellular ROS Detection. MDA-MB231 cells were
lysis buffer (50mM Tris-HCl pH 8, 1mM EDTA, 150mM treatedwitheitherthevehicleorAEs(10and30𝜇M).After
NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS) with 10 days, the oxidation-sensitive fluorescent probe DHE was
protease inhibitors. After 30 minutes of mixing at 4∘C, the usedtoassesstheproductionofcytosolicsuperoxideanions.
mixture was centrifuged (10,000×g) for 10 minutes and Briefly, at the end of exposure, cells were incubated with
the supernatants were collected as whole-cell extracts. The 5𝜇M DHE for 40 minutes at 37∘C in the dark and then
proteincontentwasdeterminedwithaproteinassayreagent rinsed twice with PBS. The cell-permeant DHE entered the
(Bio-Rad, Milan, Italy) using bovine serum albumin as a cells, was oxidized by superoxide anions to form ethidium
standard. An equal protein content of total lysates (25𝜇g) (ETH) which binds to DNA, and produced the fluorescent
fromthecontrolandfromAEstreatedsampleswasresolved ETH-DNA. The fluorescent signals were obtained exciting
on 10% SDS-PAGE with molecular weight markers (Bio- the cultured cells at 𝜆ex 300nm and 𝜆em 610nm. Cells
Rad). Proteins were then transferred to the PVDF mem- were visualized and counted by fluorescence microscope
brane (EMD Millipore Billerica, MA, USA) and reacted (OlympusIX71-OlympusAmerica).
with anti-p16INK4a and anti-p21Cip1/Waf1 antibodies (Santa The role of ROS on AEs-treated cell proliferation was
evaluated using the antioxidant NAC. Cells were preincu-
CruzBiotechnology,CA,USA),acetylated-lysinepolyclonal
batedwith10mMNACfor2handthenexposedtoAEs.After
antibody (Cell Signalling Technology Inc., Danvers, MA,
10days,cellswereharvestedbytrypsinization,stainedwith
USA),andanti-actinantibody(Calbiochem-EMDMillipore
0.4%trypanblue,andcountedusingahaemocytometer.
Billerica, MA, USA) for protein normalization. The protein
bands were revealed by chemiluminescence using an ECL
detection kit (Amersham Bioscience, Cologno Monzese, 2.9. Statistical Analysis. Statistical analyses were performed
Milan, Italy). Autoradiograms were quantified with ImageJ by Student’s 𝑡-test using GraphPad 5.1 software. For all
1.43 software (National Institutes of Health, Bethesda, MD, statistical tests, a two-tailed 𝑝 value <0.05 was considered
USA). significant. All data reported were verified at least in three
independentexperimentsandexpressedasmean±SD.
2.7. Dot Blot Analysis of DNA 5-Methylcytosine and 5-mC
Immunostaining. MDA-MB231cellswereseededatadensity 3.Results
of2.5×103in6-wellplatesintriplicateandculturedfor24h
3.1. Phenolic Composition of Artichoke Extracts. The arti-
before adding either the vehicle or various concentrations
of AEs (2.5, 5, 10, and 30𝜇M) for 10 days and then har- choke extracts were found to contain monocaffeoylquinic
acids (MCQA), dicaffeoylquinic acids (DCQA), and small
vested. Genomic DNA was isolated using the Wizard DNA
amounts of a luteolin and an apigenin glycoside. The main
purificationkit(Promega,Madison,WI,USA)accordingto
phenoliccomponentsoftheAEsfoundwerechlorogenicacid
the manufacturer’s instructions, transferred onto positively
and two dicaffeoylquinic acids (3,5-DCQA and 1,5-DCQA)
charged Hybond N+ membranes (Amersham Pharmacia
at a ratioofabout 1:1:1. The concentrationsof chlorogenic
Biotech,Piscataway,NJ,USA)usingdotfiltrationapparatus
∘ acid, 3,5-DCQA, and 1,5-DCQA, determined by HPLC, as
and fixed by baking the membrane for 30 minutes at 80 C.
After baking, membranes were incubated with 5𝜇g/mL previouslydescribed[25],werefoundtobe725±70,738±58,
of 5-methylcytosine (5-mC) antibody (Calbiochem, San and632±48mgL−1,respectively.
Diego,CA,USA)followedbyincubationwithahorseradish
peroxidase-conjugatedsecondaryantibody.Membraneswere 3.2.EffectsofAEsonHumanCancerCellLinesViability. We
thentreatedwithchemiluminescencedetectionreagentsand have previouslyreportedthat AEs exhibited cancer chemo-
exposedtoKodakautoradiographfilms.Theintensityofeach preventiveactivitiesonahumanhepatomacellline,HepG2
dotwasquantifiedwithImageJ1.43software. [24], and on a human breast cancer cell line, MDA-MB231
4 OxidativeMedicineandCellularLongevity
Table1:CytotoxicityofAEsinhumancancercelllines.
AEs𝜇M
NT 200 400 600 800
Mean SD Mean SD Mean SD Mean SD Mean SD
HCT116 84.5 3.7 77.2 6.1 84.0 2.7 79.0 4.0 77.0 4.5
∗∗ ∗∗∗
GLT-16 93.5 1.3 89.2 0.9 89.5 4.5 83.2 2.7 69.7 3.6
∗ ∗∗∗ ∗∗∗
MDA-MB231 90.0 2.6 83.7 1.5 81.7 1.0 43.2 1.0 16.0 1.4
∗ ∗∗∗
HEY 91.7 2.2 91.0 3.6 90.0 2.1 73.5 4.9 27.7 1.7
∗∗ ∗∗∗
DU145 92.7 2.3 93.0 2.9 94.0 2.4 80.0 3.4 51.0 2.9
∗∗
A549 86.5 5.9 86.2 5.5 82.5 11.2 70.0 2.0 28.3 3.5
∗∗
M14 88.2 5.6 83.0 2.9 77.7 10.9 74.3 2.5 39.6 3.2
∗∗∗
U-373MG 93.7 3.7 95.0 1.4 90.2 6.3 91.5 1.7 16.5 2.0
∗∗∗ ∗∗∗
Saos-2 89.8 2.3 86.5 6.5 85.3 4.6 37.3 6.9 18.3 1.5
∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗
K-562 89.7 4.3 15.2 4.3 2.7 1.2 3.7 1.5 1.5 0.6
TentumourcelllinesderivedfromvarioushumantissuesaretreatedwithincreasingconcentrationsofAEs(from200to800𝜇M)for24h.Theresultsarethe
mean±SDofatleastthreeindependentexperiments.ThestatisticalsignificancebetweengroupswascalculatedusingStudent’s𝑡-test.Significantdifferences
areindicatedbyasterisks.
GLT-16:∗∗𝑝=0.0092,∗∗∗𝑝=0.0009.
MDA-MB231:∗𝑝=0.0129,∗∗∗𝑝<0.0001.
HEY:∗𝑝=0.0119,∗∗∗𝑝<0.0001.
DU145:∗∗𝑝=0.0041,∗∗∗𝑝<0.0001.
A549:∗∗𝑝=0.0029.
M14:∗∗𝑝=0.0023.
U-373MG:∗∗∗𝑝=0.0001.
Saos-2:∗∗∗𝑝<0.0002.
K-562:∗∗∗𝑝<0.0002.
[25]. To investigate whether the antiproliferative activity of a relevant inhibition of cell proliferation in viable cells
AEs could be extended to other tumours, we describe the (about 90%); the highest concentration (60𝜇M) induced a
effect of AEs on 10 cancer cell lines derived from various dramatic growth arrest and was slightly cytotoxic (about
human tissues, as shown in Table1. This panel provides a 70% viability). Based on these data, we focused on two
wayofpresentingthecellularsensitivityorresistanceatthree noncytotoxic concentrations of AEs, namely 10 and 30𝜇M,
levels of effect. After 24h, 800𝜇M AEs caused about 50% that modulate cell growth. Furthermore, we investigated
ofdeathinprostateandmelanomacells;morethan50%of whetherthesetreatmentscausedareductioninthenumber
toxicitywasdetectedinbreast,ovary,lung,brain,bone,and of living cells through the activation of caspases pathways.
leukaemia cells. Less than 50% of dead cells were observed As shown in Figure1(b), in these experimental conditions
incolonandgastriccancers.Themoreresistantcancercells, protein expression analysis revealed that caspase-9 was not
suchasgastricandcolon,showedsensitivityto1200𝜇MAEs activated. Conversely, high concentration of AEs (400𝜇M),
(unpublishedresults).Thesedataindicatedthathighdosesof used as positive control [25], triggers a significant activa-
AEsarecytotoxicforalltestedcancercelllineswithoutany tion/cleavage of caspase-9 after 24h treatment. In addition,
effectonuntreatedcounterparts. longtermexposuretolowconcentrationsofAEsdidnotacti-
vate caspase-8 (unpublished results). Altogether our results
3.3. Low Doses of AEs Inhibit Breast Cancer Cell Growth stronglysuggestedthatlowdosesofAEsinhibitbreastcancer
viaaCaspases-IndependentMechanism. Wehavepreviously cellgrowthviaacaspases-independentmechanism.
demonstrated[25]thathighconcentrationsofAEs(from200
to800𝜇Mfor24h)areabletoactivateanapoptoticprogram 3.4.AEsInducePrematureSenescenceinBreastCancerCells.
inMDA-MB231tohalttumourprogression.Sincebioactive We investigated whether the cell growth arrest, in response
compounds concentrations required to induce apoptosis in tolowdosesofAEs,wascausedbytheinductionofcellular
tumour cell lines might not be reachable in target tissues, senescenceasdemonstratedforchronictreatmentofseveral
we asked whether AEs chronically administered at low and polyphenols in cancer cells [39–42]. The detection of 𝛽-
sublethaldosescanaffectthegrowthoftumourcellsaswell. galactosidase positive cells reflects an increase in lysosomal
Tothispurpose,wefocusedonMDA-MB231,whichprovide massinagingcellsanditiswidelyregardedasamarkerfor
the scientific rationale for testing AEs as an antitumour senescence [43]. MDA-MB231 cells incubated without AEs
agent against invasive and hormone resistant breast cancer showednodetectableSA-𝛽-galactivity,whereascellstreated
phenotype. After 10-day treatment (from 2.5𝜇M to 60𝜇M) with10and30𝜇MAEsrevealedamarkedX-galstainingina
direct cell count assay indicated that the cellular growth dose-dependentmannerafter10days,asdepictedinFigure2.
wassignificantlyinhibitedbychronicexposuretolowdoses MostoftheSA-𝛽-galpositivecellsshowedanenlargedand
of AEs (Figure1(a)). Treatments up to 30𝜇M resulted in flattenedmorphologywithincreasedvolumeandgranularity
OxidativeMedicineandCellularLongevity 5
Effect of AEs on cell growth forimmunocytostainingofDNAmethylationusingananti-
140
40 ∗∗∗ bodyspecificto5-methylcytosine(5-mC).TreatmentofAEs
×1 120 ∗∗∗ resultedinareducednumberof5-mC-positivecellsinadose-
ells100 dependentmannercomparedtountreatedcells(Figures4(a)
c
ng 80 and4(b)).
er of livi 4600 genoTmoifcurDthNeArvweraisfyistohleateeffdecfrtoomfAcEelslsoannDdNaAnamlyseethdyblaytidoont,
b blotassayusinganti-5mCantibody.AsshowninFigure4(c),
m 20
u AEs treatment of MDA-MB231 cells significantly decreased
N
0
C 2.5𝜇M AEs 10𝜇M AEs 30𝜇M AEs 60𝜇M AEs the DNA global methylation level as quantified by densito-
metric values. These results demonstrated that AEs have a
(a)
relevanteffectonregulationofDNAmethylationmachinery.
10days 24h
M
M M 3.6.EffectofAEsonAcetylationofTotalProteinsonHuman
𝜇
0𝜇 0𝜇 00 Breast Cancer Cells. Protein acetylation of lysine residues
C 1 3 4
is an important reversible modification controlling cellular
47kDa
Caspase-9 protein expression [48]. We investigated the effect of AEs
37/35kDa
treatment on acetylation of total proteins in breast cancer
𝛽-Actin cells.MDA-MB231cellswereculturedfor24hbeforeadding
either the vehicle or various concentrations of AEs (10 and
(b) 30𝜇M)for10daysandthenharvested.AsshowninFigure5,
the level of protein acetylation is markedly increased in
Figure1:LowdosesofAEssuppressbreastcancercellgrowthvia
treated cells as indicated by reported densitometric values.
acaspases-independentmechanism.(a)Cellgrowthinhibitionby
AEs.MDA-MB231cellsweretreatedwithincreasingconcentrations These results provide evidence that long term exposure to
ofAEs(from2.5to60𝜇M)for10days.Theresultsarethemean± lowconcentrationsofAEsisassociatedwithincreasedlevel
SDofatleastthreeindependentexperiments.Significantstatistical oflysinesacetylationoftotalproteins.
differences are indicated by asterisks: ∗∗∗𝑝 < 0.0001. (b) Effects
ofAEsoncaspasespathway.Thecellsweretreatedwithlowdoses 3.7.AEsTreatmentIncreasesROSProductioninBreastCancer
of AEs (10 and 30𝜇M) for 10 days or with a high concentration Cells. Since reactive oxygen species (ROS) are well-known
ofAEs(400𝜇M)for24h,asapositivecontrol.Wholecelllysates inducers of cellular senescence, we tested whether AEs
(25𝜇g/lane)weretestedforactivationandcleavageofcaspase-9.𝛽- increaseoxidativestressinbreastcancercells.Toaccomplish
Actinwasusedasaproteinloadingcontrol.
this,cellswereincubatedwithDHEwhichisanindicatorof
thepresenceofsuperoxideanion,keyradicalinROSgener-
ation. As shown in Figures 6(a) and 6(b) AEs-treated cells,
thatwereconsistentwithcellularsenescencestatus,asshown accordingtotheincreasednumberofbrightredfluorescent
inmagnificationareaofFigure2. cells,showedenhancedlevelofsuperoxideanionscompared
To further investigate this antiproliferative mechanism, tocontrolcells.
we examined the expression levels of p21Cip1/Waf1 and To determine the role of ROS in AEs-induced growth
p16INK4a,twopivotalcellcycleregulatorsinvolvedincellular arrest, we sought to examine whether inhibition of ROS
production by antioxidant NAC has any impact on senes-
senescence[44,45].Westernblottingdatademonstratedthat
theexpressionlevelsofp21Cip1/Waf1andp16INK4aweresignifi- cence in breast cancer cells. As shown in Figure6(c), the
presence of NAC significantly reduced the antiproliferative
cantlyincreasedinadose-dependingmannerinAEs-treated
effect of 30𝜇M AEs for long term exposure. Furthermore,
cells (Figures 3(a) and 3(b)). Optical density measurements
NAC treatment decreased percentage of SA-𝛽-gal positive
wereperformedusingImageJsoftwaretoobtainquantitative
cells (unpublished results). According to data in literature
valuesfortheproteinexpressions.Altogether,thesefindings
regarding the natural products modulation of ROS expres-
suggested that low doses and chronic exposure to AEs
sioninsenescence[39,41,49],ourfindingsstronglysupport
inducedsenescenceinMDA-MB231breastcancercells.
thehypothesisthatanoxidativepathwayisinvolvedinAEs-
inducedgrowtharrestinbreastcancercells.
3.5. Effect of AEs on Global Methylation Level in Breast
Cancer Cells. DNA hypermethylation is a major epigenetic 4.Discussion
modification that leads to silencing of tumour suppressor
genes which may contribute to cancer development and In this report, we provide evidence demonstrating that low
progression [46]. Recent investigations suggest that some doses and chronic AEs-treatments exert anticancer activity
dietary phytochemicals may prevent cancer by modulating throughinductionofprematuresenescenceinMDA-MB231,
epigenetic processes [35, 47]. To examine whether chronic a triple negative and highly aggressive breast cancer cell
treatment has epigenetic effects on the DNA methylation line.Experimentaldatademonstratethatmoderatedosesof
level,cellswereexposedtoincreasingconcentrationsofAEs, AEsinhibitthegrowthofbreastcancercellsviaacaspases-
as shown in Figure4. After 10 days, cells were harvested independentmechanism.
6 OxidativeMedicineandCellularLongevity
C 10𝜇M AEs 30𝜇M AEs
Figure2:ChronictreatmentofAEsinducescellsenescence.MDA-MB231cellsweretreatedwithlowdosesofAEs(10and30𝜇M)for10
daysandthenanalyzedfortheSA-𝛽-gal,senescence-associated𝛽-galactosidaseactivity.Theimageshownisrepresentativeofatleastthree
independentexperiments.Scalebar:50𝜇m.Thesenescentcellsversustotalcellswerecountedinrandomfieldsunderaninvertedmicroscope
(20x)andthefollowingdataarethemean±SD:C=7.7±0.5,10𝜇MAEs=31±5.6,𝑝 = 0.0044∗∗,30𝜇MAEs=51±6.6,𝑝 = 0.0005∗∗∗.
Significantstatisticaldifferencesareindicatedbyasterisks.Boxedarea,regardingbluecellswithatypicalsenescentflattenedandenlarged
morphology,ismagnified(2x).
AEs Besidecellcycleinhibitors,p21Cip1/Waf1andp16INK4aare
C 10𝜇M 30𝜇M identifiedassenescencemarkers[44,45].
Both p21Cip1/Waf1 and p16INK4a pathway induction can
p16
lead to the inhibition of pRb phosphorylation by inhibit-
1.0 1.7 2.45 ingCDK2/cyclinEandCDK4/cyclinDcomplex,respectively.
𝛽-Actin Furthermore,p21Cip1/Waf1andp16INK4aarelikelytocooperate
to keep pRb in a hypophosphorylated form during senes-
(a) cence [50]. According to data regarding the prosenescence
property of a derivative of caffeic acid [51], we showed that
AEs
low doses of AEs containing mono- and dicaffeoylquinic
C 10𝜇M 30𝜇M acids induce an increased number of SA-𝛽-gal positive and
p21 flattenedcellswithenhancedexpressionofp21Cip1/Waf1 and
p16INK4a.
1.0 1.8 2.74
High doses of AEs treatment induces cell death [25]
𝛽-Actin
in breast cancer as well as in other cell lines derived from
various human cancers (Table1) whereas AEs chronically
(b)
administeredonMDA-MB231exertantiproliferativeactivity
Figure 3: Chronic treatment of AEs induces increased p16INK4a viainductionofacaspases-independentmechanism.
and p21Cip1/Waf1 expression. MDA-MB231 cells were treated with Thisisasignificantfindingsincethemajorchallengefor
low doses of AEs (10 and 30𝜇M) for 10 days. Whole cell lysates anticancertherapeuticstrategyleadingtoapoptosisinvitrois
were tested for (a) p16INK4a and (b) p21Cip1/Waf1 protein expres- that effective concentrationsof an antitumouragent should
sion.Immunoblotsarerepresentativeofatleastthreeindependent betoohightobereachableinvivo[52].
experiments. Intensities of electrophoretic bands relative to the Data in literature are consistent with our results since
immunoblotshownwerequantifiedbydensitometryandthevalues dietary polyphenols [42, 49, 53, 54] activate apoptotic
arereported.
machinery when used at high doses whereas low level
treatmentsinducesenescenceincancercells.
The senescence program involves epigenetic processes
Several reports point to a crucial physiological role for suchasDNAmethylation,histonemodifications,andchro-
cellularsenescenceinfightingtumorigenesis.Cellularaging matin remodeling. DNA hypermethylation is a major epi-
is a state of cell cycle arrest induced at the end of the genetic mechanism in the silencing of tumour suppres-
cellular life span or in response to agents causing oxidative sor genes. Protein lysine acetylation is a posttranslational
stress and DNA damage. Moreover, senescence is regarded change that has long been known to play a prominent
asanimportantmechanismeitherforfightingpremalignant role in the regulation of gene expression via modulation
tumoursand/orfor inducingtumourregression [2] since it of chromatin structure. This epigenetic process involves
limitstheproliferativecapacityofcells. modification of histone and nonhistone proteins through
Increasing evidence has demonstrated that many phy- acetyltransferase (HATs) and deacetylase (HDACs) activi-
tochemicalsexertanticancerandchemopreventiveactivities ties. Abnormal DNA methylation and dysregulated histone
throughtheinductionofsenescencegrowtharrest[39–42]. acetylation are implicated in numerous reported diseases,
OxidativeMedicineandCellularLongevity 7
C 2.5𝜇M AEs 5𝜇M AEs 10𝜇M AEs 30𝜇M AEs
(a)
Mean SD pvalue AEs (𝜇M)
C 47.3 ±2.5 C 2.5 5 10 30
2.5𝜇M AEs 27.3 ±2.1 0.0004∗∗∗
5𝜇M AEs 24.3 ±2.9 0.0005∗∗∗
10𝜇M AEs 1.3 ±0.8 <0.0001∗∗∗
30𝜇M AEs 1.3 ±0.8 <0.0001∗∗∗ 1 0.7 0.2 0.15 0.05
(b) (c)
Figure4:EffectofAEsonglobalDNAmethylationlevelsinMDA-MB231cells.LowdosesofAEs(2.5–30𝜇M)for10daystreatmentdecreased
levelsof5-mC,5-methylcytosine,inadose-dependentmanner.5-mCspecificantibodywasusedfor(a)and(b)cytostainingand(c)fordot
blotanalysisongenomicDNA.(a)Theimageshownisrepresentativeofatleastthreeindependentexperiments.Scalebar:100𝜇m.(b)The
5-methylcytosinepositivecellsversustotalcellswerecountedbyusinganinvertedmicroscope(20x)andtheresultsarereportedasmean±
SD.(c)TheintensityofindividualDNAdotsshownwasquantifiedbydensitometry.
AEs vivodatashowingthatseveralbioactivefoodcomponentscan
(kDa) C 10𝜇M 30𝜇M interfere with DNA methylation and histone modification
affectingtheexpressionofgeneinvolvedinthecarcinogenic
103 process [35]. Epigallocatechin-3-gallate (EGCG), the major
77 polyphenolicconstituentofgreentea,hasbeendemonstrated
50
toinhibitDNAmethyltransferaseinseveralcancercelllines,
includingMDA-MB231cells[60].Thisactivitywasassociated
34.3 with promoter demethylation and reactivation of p16INK4a.
28.8
20 The positive clinical efficacy of EGCG in the treatment or
preventionofchroniclymphocyticleukaemia[16]orprostate
1.0 2.4 2.7 cancer [15] has been evaluated. A synergistic mixture of
𝛽-Actin green tea plus capsicum has been demonstrated to have
a chemopreventive effect versus different types of cancer
Figure 5: Effect of AEs on lysine acetylation of total proteins in in subjects testing positive for ENOX2, (ecto-nicotinamide
MDA-MB231 cells. Cells were treated with low doses of AEs for adenine dinucleotide oxidase disulfide-thiol exchanger 2),
10 days. Cell lysates were analyzed for lysine acetylation of total whichisideallysuitedasatargetforearlydiagnosisofcancer
proteins.Immunoblotisrepresentativeofatleastthreeexperiments.
aswellasforpreventiveintervention[17].Curcuminhasthe
Theintensitiesofelectrophoreticbandsrelativetotheimmunoblot
potentialtotreatawidevarietyofdiseasesincludingcancer
shownwerequantifiedbydensitometry.
[61]. Its epigenetic modulator activity has been a focus in
clinicaltrials(terminatedorcompleted).Phenolderivatives,
including quercetin and resveratrol, have been shown to
including cancer [55]. In contrast to genetic modifications, possessepigeneticpropertiesthroughsirtuinactivation[57].
epigenetic deregulation is potentially reversible and thera- Phenolic acids, including chlorogenic acid, the main com-
peutic strategies, targeting the epigenome, have been pro- ponentofAEs,havebeenshowntoaffectDNAmethylation
posed for both cancer prevention and clinical treatment [36].
[56]. Many epigenetic modulators have been used in clin- According to these data, we observed consistent DNA
ical trials (either completed or terminated) to treat human hypomethylationandincreasedtotalacetylationproteinlev-
cancers(http://www.clinicaltrials.gov/).Accordingtoresults elsinAEs-treatedMDA-MB231cells.
of preclinical studies and clinical trials, epigenetic modula- Ithasbeendemonstratedthatseveralanticancerpolyphe-
tors, administered as monotherapy or in combination with nolic compounds from fruit and vegetables induce tumour
conventionalchemo-andradiotherapies,arepotentiallyvery cellulargrowtharrestlargelythroughthegenerationofROS
useful[57]. [62].Lowdosesofresveratrolinhibitcellgrowthandenhance
Modulationofepigeneticprocesseshasbeenidentifiedas radiosensitization via the induction of ROS-mediated pre-
a relevant anticancer feature of many dietary polyphenolic mature senescence in lung cancer cells [39, 49]. Sin et al.
compounds[33,34,58,59].Therearebothinvitrodataandin suggestthatchronictreatmentwith20(S)-ginsenosideRg3,a
8 OxidativeMedicineandCellularLongevity
C 10𝜇M AEs 30𝜇M AEs
(a)
AEs increase ROS production
ve cells (%) 178900000 ∗∗∗ 4×10cells11802000 NAC reduces the effect of AEs treatmen∗t∗
orescent positi 34560000 mber of living 246000
u 20 u
fl N 0
HE 10 AEs30𝜇M − − + +
D 0
C 10𝜇M AEs 30𝜇M AEs NAC10mM − + − +
(b) (c)
Figure6:ROSproductioninMDA-MB231cellstreatedwithAEs.(a)ThepresenceofROSwasdetectedbyDHEfluorescentstainingafter
10daystreatment.Scalebar:50𝜇m,originalmagnification20x.(b)Redfluorescent-stainedcellsversustotalcellswerecountedusingan
invertedfluorescencemicroscope.Theresultsarethemean±SDofatleastthreeindependentexperiments.Significantstatisticaldifferences
areindicatedbyasterisks:∗∗𝑝=0.0012.(c)NACreducedtheantiproliferativeeffectofAEs(30𝜇Mfor10days).Theresultsarethemean±
SDofatleastthreeindependentexperiments.Significantstatisticaldifferencesareindicatedbyasterisks:∗∗𝑝=0.0012.
compoundextractedfromginseng,atasubapoptoticconcen- celldeath.Apromisingcytostaticapproachisprosenescence
tration caused senescence-like growth arrest and increased therapywhichmayprovidearelevantgrowthinhibitoryeffect
ROS production in human glioma cells [54]. Moreover, inbothearlyandlatestagecancers[29,64].Targetedpros-
bisdemethoxycurcumin, a natural derivative of curcumin, enescence therapies may be of remarkable clinical interest
suppresseshumanbreastcancercellproliferationbyinducing since it might minimizetoxicity and improvequalityof life
oxidativestresssenescence[41].ArelevantroleofROSwas for cancer patients [29, 30]. Over the last few years, it has
also demonstrated for the phenethyl isothiocyanate induc- been demonstrated that several prosenescence polyphenols
tionofapoptosisandsenescenceintumours[53].Altogether, and natural compounds could represent a promising novel
these findings suggest the crucial role of ROS as effectors therapeuticapproachforcancerintervention[39,41,42,54,
of polyphenol-induced prooxidant damage in cancer cells. 65].Thesuppressiveroleofsenescenceincancerprogression
Accordingly,weshowthatAEs-inducedgrowtharrestisasso- has promoted the idea that induced premature cell aging
ciated with increased ROS production, suggesting that AEs could be an alternative or a complement to conventional
mayinducesenescenceviaanoxidative-mediateddamagein anticancertreatments.
breastcancercells.Toconfirmthisimportantcontributionof Inlinewiththis,ourpresentstudydemonstratesforthe
ROS, we demonstrate that antioxidant NAC attenuates AEs firsttimethatAEsmayinduceROSaccumulationinMDA-
proliferativeeffectonMDA-MB231cells. MB231breastcancercellsandmodulatethep21Cip1/Waf1and
Itisimportanttohighlightthatwehavepreviouslyshown p16INK4a pathways to cause a senescence-mediated tumour
thatAEshaveaprooxidantactivityonbreastcancercells[25] suppression.Ourstudyaddsanovelaspectoftheunderlying
andanantioxidanteffectonnormalhepatocyte[24].Given mechanismsoftheanticancerpropertiesofAEs.However,in
that aberrant redox system is frequently observed in many order to provide a solid basis for evaluating the efficacy in
tumour cells [63], we hypothesize that AEs may selectively humanclinicaltrials,furtherstudiesarerequiredonanimal
inhibitthegrowthoftumourcellswithlittleornotoxicityon models. In particular, deep pharmacokinetic and metabolic
normalcellsbasedontheirdifferentialredoxstatus. studiesofAEsareneeded.
Conventional cancer therapy is traditionally based on In conclusion, our findings propose dietary artichoke
theefficacyofcytotoxictreatmentseventhoughsevereside polyphenolsasaverypromisingtooleitherforthemanage-
effects on patientsare a clinical relevant problem. An alter- mentofcancerpreventioninhealthy,highriskbreastcancer
nativestrategyistheinductionofcytostatis,whichdisables women or for the design of innovative, nonconventional,
the proliferative capacity of cells without inducing cancer adjuvanttherapiesincancertreatment.
OxidativeMedicineandCellularLongevity 9
ConflictofInterests [11] S.M.Petiwala,A.G.Puthenveetil,andJ.J.Johnson,“Polyphe-
nolsfromtheMediterraneanherbrosemary(Rosmarinusoffic-
Theauthorshavedeclarednoconflictofinterests. inalis) for prostate cancer,” Frontiers in Pharmacology, vol. 4,
article29,2013.
Authors’Contribution [12] A.S.DarveshandA.Bishayee,“Chemopreventiveandthera-
peutic potential of tea polyphenols in hepatocellular cancer,”
NutritionandCancer,vol.65,no.3,pp.329–344,2013.
AnnaMariaMileo,StefaniaMiccadei,andDonatoDiVenere
conceived,designed,andperformedtheexperiments.Clau- [13] P.Wang,J.V.Vadgama,J.W.Saidetal.,“Enhancedinhibition
of prostate cancer xenograft tumor growth by combining
dia Abbruzzese analyzed the data. Stefania Miccadei and
quercetinandgreentea,”The Journal of Nutritional Biochem-
AnnaMariaMileowrotethepaper.
istry,vol.25,no.1,pp.73–80,2014.
[14] M. Iriti and E. M. Varoni, “Chemopreventive potential of
Acknowledgments flavonoidsinoralsquamouscellcarcinomainhumanstudies,”
Nutrients,vol.5,no.7,pp.2564–2576,2013.
The authors thank Dr. Marco Giorgio Paggi (Regina Elena
[15] J. McLarty, R. L. H. Bigelow, M. Smith, D. Elmajian, M.
National Cancer Institute) for his helpful comments and Ankem, and J. A. Cardelli, “Tea polyphenols decrease serum
advice.TheauthorsacknowledgeMs.TaniaMerlinoforher levelsofprostate-specificantigen,hepatocytegrowthfactor,and
grammaticalsuggestionsfortheEnglishpaper.TheContract vascularendothelialgrowthfactorinprostatecancerpatients
grant sponsor is Lega Italiana per la Lotta contro i Tumori andinhibitproductionofhepatocytegrowthfactorandvascular
(LILT),ContractGrantno.08/12/C/73. endothelialgrowthfactorinvitro,”CancerPreventionResearch,
vol.2,no.7,pp.673–682,2009.
[16] T.D.Shanafelt,T.G.Call,C.S.Zentetal.,“Phase2trialofdaily,
References
oralpolyphenoneinpatientswithasymptomatic,Raistage0
toIIchroniclymphocyticleukemia,”Cancer,vol.119,no.2,pp.
[1] A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward, and D.
363–370,2013.
Forman,“Globalcancerstatistics,”CACancerJournalforCli-
[17] C.Hanau,D.J.Morre`,andD.M.Morre`,“Cancerprevention
nicians,vol.61,no.2,pp.69–90,2011.
trial of a synergistic mixture of green tea concentrate plus
[2] T. Norat, D. Aune, D. Chan, and D. Romaguera, “Fruits and Capsicum(CAPSOL-T)inarandompopulationofsubjectsages
vegetables:updatingtheepidemiologicevidencefortheWCRF/ 40–84,”ClinicalProteomics,vol.11,no.1,pp.1–11,2014.
AICRlifestylerecommendationsforcancerprevention,”Cancer
[18] E.Azzini,R.Bugianesi,F.Romanoetal.,“Absorptionandmeta-
TreatmentandResearch,vol.159,pp.35–50,2014.
bolismofbioactivemoleculesafteroralconsumptionofcooked
[3] L. Shu, K.-L. Cheung, T. O. Khor, C. Chen, and A.-N. Kong, edible heads of Cynara scolymus L. (cultivar Violetto di
“Phytochemicals:cancerchemopreventionandsuppressionof Provenza)inhumansubjects:Apilotstudy,”BritishJournalof
tumor onset and metastasis,” Cancer and Metastasis Reviews, Nutrition,vol.97,no.5,pp.963–969,2007.
vol.29,no.3,pp.483–502,2010.
[19] C.MancusoandR.Santangelo,“Ferulicacid:pharmacological
[4] A.C.Tan,I.Konczak,D.M.-Y.Sze,andI.Ramzan,“Molecular andtoxicologicalaspects,”FoodandChemicalToxicology,vol.
pathwaysforcancerchemopreventionbydietaryphytochemi- 65,pp.185–195,2014.
cals,”NutritionandCancer,vol.63,no.4,pp.495–505,2011. [20] B.Janicke,C.Hegardt,M.Kroghetal.,“Theantiproliferative
[5] C.Gerhauser,“Cancerchemopreventionandnutri-epigenetics: effectofdietaryfiberphenoliccompoundsferulicacidandp-
stateoftheartandfuturechallenges,”TopicsinCurrentChemi- coumaricacidonthecellcycleofCaco-2cells,”Nutritionand
stry,vol.329,pp.73–132,2013. Cancer,vol.63,no.4,pp.611–622,2011.
[21] N. Baskaran, S. Manoharan, S. Balakrishnan, and P. Pugal-
[6] V.S.Thakur,G.Deb,M.A.Babcook,andS.Gupta,“Plantphy-
endhi,“Chemopreventivepotentialofferulicacidin7,12-dime-
tochemicalsasepigeneticmodulators:roleincancerchemopre-
thylbenz[a]anthracene-induced mammary carcinogenesis in
vention,”AAPSJournal,vol.16,no.1,pp.151–163,2014.
Sprague-Dawleyrats,”EuropeanJournalofPharmacology,vol.
[7] V. D. Longo and L. Fontana, “Calorie restriction and cancer 637,no.1–3,pp.22–29,2010.
prevention: metabolic and molecular mechanisms,” Trends in
[22] S. Hemaiswarya and M. Doble, “Combination of phenylpro-
PharmacologicalSciences,vol.31,no.2,pp.89–98,2010.
panoidswith5-fluorouracilasanti-canceragentsagainsthuman
[8] M. Gonza´lez-Vallinas, M. Gonza´lez-Castejo´n, A. Rodr´ıguez- cervicalcancer(HeLa)cellline,”Phytomedicine,vol.20,no.2,
Casado, andA. Ram´ırez de Molina, “Dietaryphytochemicals pp.151–158,2013.
incancerpreventionandtherapy:acomplementaryapproach
[23] S.Karthikeyan,G.Kanimozhi,N.R.Prasad,andR.Mahalak-
withpromisingperspectives,”NutritionReviews,vol.71,no.9,
shmi,“Radiosensitizingeffectofferulicacidonhumancervical
pp.585–599,2013.
carcinomacellsinvitro,”ToxicologyinVitro,vol.25,no.7,pp.
[9] N.Namasivayam,“Chemopreventioninexperimentalanimals,” 1366–1375,2011.
AnnalsoftheNewYorkAcademyofSciences,vol.1215,no.1,pp. [24] S.Miccadei,D.DiVenere,A.Cardinalietal.,“Antioxidativeand
60–71,2011. apoptoticpropertiesofpolyphenolicextractsfromediblepartof
[10] T.M.C.M.deKok,P.deWaard,L.C.Wilms,andS.G.J.van artichoke(CynarascolymusL.)onculturedrathepatocytesand
Breda,“Antioxidativeandantigenotoxicpropertiesofvegetables onhumanhepatomacells,”NutritionandCancer,vol.60,no.2,
anddietaryphytochemicals:thevalueofgenomicsbiomarkers pp.276–283,2008.
in molecular epidemiology,” Molecular Nutrition and Food [25] A.M.Mileo,D.DiVenere,V.Linsalata,R.Fraioli,andS.Mic-
Research,vol.54,no.2,pp.208–217,2010. cadei, “Artichoke polyphenols induce apoptosis and decrease
10 OxidativeMedicineandCellularLongevity
theinvasivepotentialofthehumanbreastcancercelllineMDA- [42] W. W. Quitschke, “Curcuminoid binding to embryonal Car-
MB231,”JournalofCellularPhysiology,vol.227,no.9,pp.3301– cinoma cells: reductive metabolism, induction of apoptosis,
3309,2012. senescence,andinhibitionofcellproliferation,”PLoSONE,vol.
[26] M.Braig,S.Lee,C.Loddenkemperetal.,“Oncogene-induced 7,no.6,ArticleIDe39568,2012.
senescence as an initial barrier in lymphoma development,” [43] G.P.Dimri,X.Lee,G.Basileetal.,“Abiomarkerthatidentifies
Nature,vol.436,no.7051,pp.660–665,2005. senescent human cells in culture and in aging skin in vivo,”
[27] X.Guo,W.M.Keyes,C.Papazogluetal.,“TAp63inducessene- Proceedings of the National Academy of Sciencesof the United
scence and suppresses tumorigenesis in vivo,” Nature Cell StatesofAmerica,vol.92,no.20,pp.9363–9367,1995.
Biology,vol.11,no.12,pp.1451–1457,2009. [44] C.PapazogluandA.A.Mills,“p53:atthecrossroadbetween
[28] M.Bennecke,L.Kriegl,M.Bajboujetal.,“Ink4a/Arfandonco- cancerandageing,”TheJournalofPathology,vol.211,no.2,pp.
gene-induced senescence prevent tumor progression during 124–133,2007.
alternativecolorectaltumorigenesis,”CancerCell,vol.18,no.2, [45] W.Y.KimandN.E.Sharpless,“TheregulationofINK4/ARFin
pp.135–146,2010. cancerandaging,”Cell,vol.127,no.2,pp.265–275,2006.
[29] J. A. Ewald, J. A. Desotelle, G. Wilding, and D. F. Jarrard, [46] S. Kristiansen, D. Nielsen, and G. So¨le´tormos, “Methylated
“Therapy-inducedsenescenceincancer,”JournaloftheNational DNAformonitoringtumorgrowthandregression:howdowe
CancerInstitute,vol.102,no.20,pp.1536–1546,2010. getthere?”CriticalReviewsinClinicalLaboratorySciences,vol.
51,no.3,pp.149–159,2014.
[30] D.A.Gewirtz,S.E.Holt,andL.W.Elmore,“Acceleratedsenes-
cence:anemergingroleintumorcellresponsetochemotherapy [47] S.M.Henning,P.Wang,C.L.Carpenter,andD.Heber,“Epi-
and radiation,” Biochemical Pharmacology, vol. 76, no. 8, pp. geneticeffectsofgreenteapolyphenolsincancer,”Epigenomics,
947–957,2008. vol.5,no.6,pp.729–741,2013.
[31] J.C.AcostaandJ.Gil,“Senescence:anewweaponforcancer [48] K.E.WellenandC.B.Thompson,“Atwo-waystreet:recipro-
therapy,”TrendsinCellBiology,vol.22,no.4,pp.211–219,2012. cal regulation of metabolism and signalling,” Nature Reviews
MolecularCellBiology,vol.13,no.4,pp.270–276,2012.
[32] V.B.O.Ayissi,A.Ebrahimi,andH.Schluesenner,“Epigenetic
effects of natural polyphenols: a focus on SIRT1-mediated [49] H. Luo, L. Wang, B. A. Schulte, A. Yang, S. Tang, and G. Y.
mechanisms,”MolecularNutrition&FoodResearch,vol.58,no. Wang, “Resveratrolenhances ionizingradiation-inducedpre-
1,pp.22–32,2014. maturesenescenceinlungcancercells,”InternationalJournalof
Oncology,vol.43,no.6,pp.1999–2006,2013.
[33] P. Mummaneni and S. S. Shord, “Epigenetics and oncology,”
Pharmacotherapy,vol.34,no.5,pp.495–505,2014. [50] I. B. Roninson, “Tumor cell senescence in cancer treatment,”
CancerResearch,vol.63,no.11,pp.2705–2715,2003.
[34] T.P.Ong,F.S.Moreno,andS.A.Ross,“Targetingtheepige-
nomewithbioactivefoodcomponentsforcancerprevention,” [51] A.Dong,Y.Fang,L.Zhangetal.,“Caffeicacid3,4-dihydroxy-
JournalofNutrigeneticsandNutrigenomics,vol.4,no.5,pp.275– phenethylesterinducescancercellsenescencebysuppressing
292,2012. twist expression,” Journal of Pharmacology and Experimental
Therapeutics,vol.339,no.1,pp.238–247,2011.
[35] S.Shankar,D.Kumar,andR.K.Srivastava,“Epigeneticmodi-
ficationsbydietaryphytochemicals:Implicationsforpersonal- [52] E.Scott,W.P.Steward,A.J.Gescher,andK.Brown,“Resveratrol
izednutrition,”PharmacologyandTherapeutics,vol.138,no.1, inhumancancerchemoprevention-choosingthe“right”dose,”
pp.1–17,2013. MolecularNutritionandFoodResearch,vol.56,no.1,pp.7–13,
2012.
[36] W. J. Lee and B. T. Zhu, “Inhibition of DNA methylation
by caffeic acid and chlorogenic acid, two common catechol- [53] N.Roy,I.Elangovan,D.Kopanja,S.Bagchi,andP.Raychaud-
containingcoffeepolyphenols,”Carcinogenesis,vol.27,no.2,pp. huri,“Tumorregressionbyphenethylisothiocyanateinvolves
269–277,2006. DDB2,”CancerBiologyandTherapy,vol.14,no.2,pp.108–116,
2013.
[37] M. Lee and J.-S. Lee, “Exploiting tumor cell senescence in
anticancertherapy,”BMBReports,vol.47,no.2,pp.51–59,2014. [54] S. Sin, S. Y. Kim, and S. S. Kim, “Chronic treatment with
ginsenosideRg3inducesAkt-dependentsenescenceinhuman
[38] F. Debacq-Chainiaux, J. D. Erusalimsky, J. Campisi, and O.
gliomacells,”InternationalJournalofOncology,vol.41,no.5,pp.
Toussaint, “Protocols to detect senescence-associated beta-
galactosidase(SA-𝛽gal)activity,abiomarkerofsenescentcells 1669–1674,2012.
incultureandinvivo,”NatureProtocols,vol.4,no.12,pp.1798– [55] J. D. Choi and J. S. Lee, “Interplay between epigenetics and
1806,2009. geneticsincancer,”Genomics&Informatics,vol.11,no.4,pp.
164–173,2013.
[39] H.Luo,A.Yang,B.A.Schulte,M.J.Wargovich,andG.Y.Wang,
“Resveratrolinducesprematuresenescenceinlungcancercells [56] M.ConteandL.Altucci,“Molecularpathways:thecomplexity
via ROS-mediated DNA damage,” PLoS ONE, vol. 8, no. 3, of the epigenome in cancer and recent clinical advances,”
ArticleIDe60065,2013. ClinicalCancerResearch,vol.18,no.20,pp.5526–5534,2012.
[40] L.L.Zamin,E.C.Filippi-Chiela,P.Dillenburg-Pilla,F.Horn, [57] A.Nebbioso,V.Carafa,R.Benedetti,andL.Altucci,“Trialswith
C.Salbego,andG.Lenz,“Resveratrolandquercetincooperate “epigenetic”drugs:anupdate,”MolecularOncology,vol.6,no.6,
toinducesenescence-likegrowtharrestinC6ratgliomacells,” pp.657–682,2012.
CancerScience,vol.100,no.9,pp.1655–1662,2009. [58] M. Schnekenburger, M. Dicato, and M. Diederich, “Plant-
[41] Y.-B. Li, J.-L. Gao, Z.-F. Zhong, P.-M. Hoi, S. M.-Y. Lee, and derivedepigeneticmodulatorsforcancertreatmentandpreven-
Y.-T. Wang, “Bisdemethoxycurcumin suppresses MCF-7 cells tion,”BiotechnologyAdvances,2014.
proliferation by inducing ROS accumulation and modulating [59] H. S. Lee and Z. Herceg, “The epigenome and cancer pre-
senescence-relatedpathways,”PharmacologicalReports,vol.65, vention:acomplexstoryofdietarysupplementation,”Cancer
no.3,pp.700–709,2013. Letters,vol.342,no.2,pp.275–284,2014.