Broccoli Consumption Interacts with GSTM1 to Perturb Oncogenic Signalling Pathways in the ...

14 Pages·2008·0.46 MB·English

Broccoli Consumption Interacts with GSTM1to Perturb
Oncogenic Signalling Pathways in the Prostate
MariaTraka1,Amy V. Gasper1,Antonietta Melchini1,2,James R.Bacon1,Paul W.Needs1,Victoria Frost3,
Andrew Chantry3, Alexandra M. E. Jones4, Catharine A. Ortori5, David A. Barrett5, Richard Y. Ball6,
Robert D. Mills6, Richard F. Mithen1*
1PhytochemicalsandHealthProgramme,InstituteofFoodResearch,NorwichResearchPark,Norwich,UnitedKingdom,2DepartmentFarmaco-Biologico,Schoolof
Pharmacy,UniversityofMessina,Messina,Italy,3SchoolofBiologicalSciences,UniversityofEastAnglia,Norwich,UnitedKingdom,4TheSainsburyLaboratory,Norwich
ResearchPark,Norwich,UnitedKingdom,5CentreforAnalyticalBioscience,SchoolofPharmacy,UniversityofNottingham,Nottingham,UnitedKingdom,6Department
ofHistopathology/Cytopathology,NorfolkandNorwichUniversityHospital,Norwich,UnitedKingdom,7DepartmentofUrology,NorfolkandNorwichUniversityHospital,
Norwich,UnitedKingdom
Abstract
Background:Epidemiologicalstudiessuggestthatpeoplewhoconsumemorethanoneportionofcruciferousvegetables
perweekareatlowerriskofboththeincidenceofprostatecancerandofdevelopingaggressiveprostatecancerbutthere
is little understanding of the underlying mechanisms. In this study, we quantify and interpret changes in global gene
expression patternsin thehuman prostategland before, duringand after a12monthbroccoli-rich diet.
MethodsandFindings:Volunteers were randomly assigned to either a broccoli-rich or a pea-rich diet. After six months
therewerenodifferencesingeneexpressionbetweenglutathioneS-transferasemu1(GSTM1)positiveandnullindividuals
on the pea-rich diet but significant differences between GSTM1 genotypes on the broccoli-rich diet, associated with
transforminggrowthfactorbeta1(TGFb1)andepidermalgrowthfactor(EGF)signallingpathways.Comparisonofbiopsies
obtainedpreandpostinterventionrevealedmorechangesingeneexpressionoccurredinindividualsonabroccoli-richdiet
thaninthoseonapea-richdiet.Whiletherewerechangesinandrogensignalling,regardlessofdiet,menonthebroccoli
diet had additional changes to mRNA processing, and TGFb1, EGF and insulin signalling. We also provide evidence that
sulforaphane(theisothiocyanatederivedfrom4-methylsuphinylbutylglucosinolatethataccumulatesinbroccoli)chemically
interacts with TGFb1,EGF and insulinpeptides to form thioureas, andenhances TGFb1/Smad-mediated transcription.
Conclusions:ThesefindingssuggestthatconsumingbroccoliinteractswithGSTM1genotypetoresultincomplexchanges
to signalling pathways associated with inflammation and carcinogenesis in the prostate. We propose that these changes
may be mediated through the chemical interaction of isothiocyanates with signalling peptides in the plasma. This study
provides, for the firsttime, experimental evidenceobtained in humansto support observational studiesthat dietsrichin
cruciferous vegetables mayreduce the risk ofprostatecancer and otherchronicdisease.
TrialRegistration:ClinicalTrials.govNCT00535977
Citation: TrakaM,GasperAV,Melchini A,Bacon JR,NeedsPW,etal.(2008)Broccoli Consumption InteractswithGSTM1 toPerturb Oncogenic Signalling
PathwaysintheProstate.PLoSONE3(7):e2568.doi:10.1371/journal.pone.0002568
Editor:PeterNelson,FredHutchinsonCancerResearchCenter,UnitedStatesofAmerica
ReceivedFebruary20,2008;AcceptedMay19,2008;PublishedJuly2,2008
Copyright: (cid:1) 2008 Traka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwasfundedbytheBiotechnologyandBiologicalSciencesResearchCouncil,UK.Thesponsorshadnoroleinthedesignandconductofthe
study,analysisandinterpretationoftheresultsanddidnotreviewthemanuscriptpriortopublication.
CompetingInterests:RichardMithenisaco-inventoronpatents(US:6340784andEuropeP1069819B1)associatedwithhighglucosinolatebroccoli.
*E-mail:[email protected]
Introduction glutathione S-transferase mu 1 (GSTM1) genotype, with individ-
uals who possess at least one GSTM1 allele (i.e. approximately
Prostatecanceristhemostfrequentlydiagnosednon-cutaneous 50%ofthepopulation)gainingmorebenefitthanthosewhohave
cancer within the male population of western countries [1]. ahomozygousdeletionofGSTM1[11].Ourstudyinvestigatesthe
Epidemiological studies have suggested that diets rich in mechanistic basis to the protective effect of broccoli and the
cruciferous vegetables, such as broccoli, may reduce the risk of interaction withGSTM1 genotype.
prostatecancer[2],inadditiontocancersatothersites[3–9]and Broccoli accumulates 4-methylsulphinylbutyl and 3-methylsul-
myocardial infarction [10]. Some studies have specifically phinylpropyl glucosinolates in its florets, which are converted to
demonstrated that consuming one or more portions of broccoli the isothiocyanates (ITCs) sulforaphane (SF) and iberin (IB),
per week can reduce the incidence of prostate cancer [11], and respectively, either by plant thioglucosidases (‘myrosinases’)
alsotheprogressionfromlocalizedtoaggressiveformsofprostate following tissue damage or, if the myrosinases have been
cancer [12]. The reduction in risk may be modulated by denaturedbycookingorblanchingpriortofreezing,bymicrobial
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thioglucosidases in the colon (Figure 1) [13]. These ITCs do not hours[14].ItwasalsoshownthatGSTM1nullindividualsexcrete
have the pungent flavour qualities associated with other dietary a higher proportion of SF via mercapturic acid metabolism than
ITCs,suchasthosefrommustards,rocketsandwatercress.SFand GSTM1 positive individuals, and it was speculated that the
IB are passively absorbed by enterocytes, conjugated with remainingSFmaybemetabolizedviaanunknownpathway,and
glutathione and transported into the systemic circulation to be thatthismayaccountfortheanticarcinogenicactivityofbroccoli.
metabolized via the mercapturic acid pathway and excreted CellandanimalstudieshaveshownthatSFisapotentinducer
predominantly as N-acetylcysteine conjugates in the urine [14]. of phase 2 enzyme gene transcription, and, at higher concentra-
Wepreviouslydemonstratedthatfollowingbroccoliconsumption, tions, of cell cycle arrest and apoptosis, all consistent with
45% of SF in the plasma occurs as free SF, as opposed to thiol anticarcinogenic activity [15]. However, these phenomena occur
conjugates, and that the peak concentration of SF and its thiol whenculturedcellsareexposedtoconsiderablyhigherlevelsofSF
conjugatesislessthan2 mM,fallingtolow(nM)levelswithinafew (i.e. 10–100mM for 24h) than those found transiently in plasma
Figure1.Metabolismof4-methylsulphinylbutylglucosinolateandsulforaphane.Uponentryintoenterocytessulforaphane(SF)israpidly
conjugated to glutathione, exported into the systemic circulation and metabolized through the mercapturic acid pathway. Within the low
glutathioneenvironmentoftheplasmatheSF-glutathioneconjugatemaybecleaved,possiblymediatedbyGSTM1,leadingtocirculationoffreeSF
intheplasma.ThisfreeSFcanmodifyplasmaproteinsincludingsignallingmolecules,suchasTGFb,EGFandinsulin.
doi:10.1371/journal.pone.0002568.g001
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afterbroccoliconsumption(i.e.,2 mMforlessthanonehour).At
Table1.Volunteer characteristics and plasma PSAlevels.
thesephysiologicalconcentrations,itislikelythatanySFentering
cellswouldbeimmediatelyconjugatedwithglutathioneduetothe
relatively high intracellular glutathione concentration, with no
Age BMI GSTM1 PSA(ng/ml)
effect on, for example, the Keap1-Nrf2 complex [16,17].
Consistent with this hypothesis, there was no evidence for Pre-intervention 6-month 12-month
induction of phase 2 gene transcription in the gastric mucosa Broccoliintervention
after an acute broccoliintervention [18].
68 29 null 4.6 5.4 5.3
In this study, we initially adopt an empirical approach in
68 27 null 3.1 3.2 2.9
humans to elucidate the mechanisms that underlie the beneficial
effects of a broccoli-rich diet, and explore the interaction with 64 26 null 9.4 3.5 2.9
GSTM1.Wecompareandinterpretglobalgeneexpressionprofiles 63 31 null 6.5 7.9 7.2
in human prostate biopsy tissue before, during and after a 12 66 28 null 0.9 1.3 0.9
month broccoli-rich diet and a 12-month pea-rich diet. Subse-
Mean(sd)4.9(3.25) 4.3(2.50) 3.84(2.44)
quently, we provide a mechanistic explanation for how the
57 27 positive 5.5 5.5 5.7
observed changes in gene expression may be induced by normal
66 30 positive 13.6 16.8 13.4
dietary broccoli consumption.
69 27 positive 6.9 3.8 3.7
Methods 62 23 positive 2.2 2.2 1.9
59 29 positive 10.8 10.4 11.2
Subjects and study design
68 25 positive 9.7 12.5 7.2
FulldetailsofthestudyprotocolandthesupportingCONSORT
64 32 positive 6.4 6.1 6.6
checklistandflowdiagramareavailableassupportinginformation
see Protocol S1, Checklist S1 and Figure S1. As stated in the 63 27 positive 7.9 9 10.8
protocol, it was intended to recruit forty men with a previous Mean(sd)7.9(3.5) 8.3(4.85) 7.56(3.95)
diagnosisofhigh-gradeprostaticintraepithelialneoplasia(HGPIN), Peasintervention
the pre-invasive in situ stage of prostatic adenocarcinoma, into a
70 28 null 4.1 4.2 4.4
dietaryinterventiontrialtostudytheeffectsofadietrichinbroccoli
65 24 null 7.5 9.3 8.2
andadietrichinpeasonprostategeneexpression.However,dueto
changesinclinicalpracticeduringtherecruitmentphaseofthestudy 59 24 null 9.3 10.8 8
therewasmarkedreductioninthenumbersofmendiagnosedwith 61 35 null 1.1 1.1 1.1
HGPINand it wasnot possibletorecruit 40men within thetime 57 26 null 5.5 5.4 5.2
constraints of the research programme. Thus, twenty-two male
Mean(sd)5.5(3.15) 6.2(3.92) 5.4(2.92)
volunteersaged57–70years(Table1)wererecruitedbyaconsultant
70 23 positive 8.9 5.2 N/A*
urologistatNorfolk andNorwich University HospitalNHS Trust.
61 29 positive 2.3 2.2 2.5
Histologicaldiagnosiswasmadebytwoconsultanthistopathologists,
whohadaspecialinterestinprostatepathology.Ethicalapprovalfor 57 30 positive 3.5 5.5 4.9
thetrialwasobtainedfromtheNorfolkResearchEthicsCommittee Mean(sd)4.9(3.52) 4.3(1.82) 3.7(1.70)
(reference05/Q0101/9,seeTextS1).Allparticipantsgavewritten,
informedconsent.Volunteerswereexcludediftheywereundergoing Oneofthe22volunteerswasdiagnosedwithprostaticadenocarcinomaatthe
studybaselinebiopsy(pre-intervention)andwasremovedfromthestudy.
chemopreventive therapy, were receiving testosterone replacement *Thisvolunteerdevelopedprostaticadenocarcinomasixmonthsintothe
medication or 5 alpha reductase inhibitor, had active infection interventionandwasremovedfromthestudy.
requiringtreatment,hadabodymassindex(BMI),18.5or.35,or doi:10.1371/journal.pone.0002568.t001
were diabetic. Subjects were allocated into a 12-month, parallel
dietary intervention trial consisting of two dietary intervention a demonstration by the diet cooks at the Institute of Food
groups: (i) consuming 400g broccoli per week or (ii) consuming Researchofhowtocookthevegetables.Portionsofbroccoliwere
400gpeasperweek,inadditiontotheirnormaldiet.Thetrialwas steamedfor4–5minutesandportionsofpeasweresteamedfor2–
conducted from April 2005–April 2007. Plasma prostate specific 3 minutes. Frozen peas (Birds Eye Garden Peas, http://www.
antigen(PSA)levelswerequantifiedpriortotheinterventionstudy birdseye.co.uk/) were purchased from a local retail outlet. To
andaftersixand12monthsattheNorfolkandNorwichUniversity ensure consistency in glucosinolate content in frozen broccoli
Hospital with the use of a total PSA immunoassay. Volunteers provided to the volunteers, the broccoli required for the
avoidedfoodsknowntocontainglucosinolatesfor48hourspriorto intervention study was grown in one batch at an ADAS
eachbiopsyappointmenttoavoidacuteeffects. experimental farm at Terrington, near King’s Lynn, UK
In addition to the transrectal ultrasound scan (TRUS)-guided (http://www.adas.co.uk/) and processed by Christian Salvesen
needle biopsies of the prostate obtained from the volunteers (Bourne, Lincolnshire, UK, http://www.salvesen.co.uk/). It was
immediately prior to the intervention study, and after six and blanchedat90.1uCfor74s,frozenat230uCandpackagedinto
twelve months, 18 benign and 14 malignant transurethral 100g portions, then stored at 218uC until steamed by the
resection of the prostate (TURP) tissues were also obtained from volunteer. The broccoli was a high glucosinolate variety [19,20].
the Norfolk & Norwich University Hospital Partners in Cancer The levels, mean (SD), of 4-methylsulphinylbutyl and 3-methyl-
Research Human TissueBank. sulphinylpropyl glucosinolates (the precursors of SF and IB,
respectively)were10.6(0.38)and3.6(0.14)mmolesg21dryweight,
Dietary intervention respectively,comparedto4.4(0.12)and0.6(0.01)mmolesg21dry
Vegetablesweredeliveredtothevolunteersonamonthlybasis. weightinbroccolipurchasedfromlocalretailoutlets.Althoughthe
Theywereprovidedwithasteamerandthevolunteersweregiven level of glucosinolates were higher than standard broccoli,
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Broccoli&SignallingPathways
blanching prior to freezing denatured plant myrosinase, thus the minimum information about a microarray experiment (MIAME)
levels of SF and IB derived from the high glucosinolate broccoli criteria and are deposited at Array Express (http://www.ebi.ac.
diet would be similar to or lower than those obtained from fresh uk/microarray-as/aer; AccessionNumberE-MEXP-1243).
broccoli with functional myrosinase. Levels of indole glucosino-
latesweresimilarinbothhighglucosinolateandstandardbroccoli. Microarray data analysis
Rawdatafiles(CEL)wereloadedintotheDNA-ChipAnalyzer
Compliance monitoring and dietary assessment software (dChip, http://biosun1.harvard.edu/complab/dchip/,
Volunteers completed weekly tick sheets during the 12-month build date September 2006) for normalization, generation of
intervention period to identify when the portions of vegetables expressionvaluesandstatisticalanalysis.Followingnormalization
were eaten. Every two weeks, volunteers were contacted by using the Invariant Set Normalization method, probe expression
telephone and asked about adherence to the diet. A seven-day levelswerecalculatedusingthePM-onlymodel.Toidentifygenes
estimated food intake diet diary was completed by volunteers at that were changing between groups, different two-tailed P-value
baseline and after six months using household measures as an thresholdswereappliedcalculatedbyWelchmodifiedtwo-sample
indication of portion size. Food intake from the diaries was t-test in dChip. Paired or unpaired t-tests were performed as
inputtedintoDietCruncherv1.6.1(www.waydownsouthsoftware. appropriate.Tocorrectformultipletesting,FalseDiscoveryRate
com/) and analyzed for differences in nutrient composition (FDR)wasestimatedbypermutationindChipandthemedianof
between the two intervention groups at baseline and six months 100 permutations reported for each of the comparisons (1000
after intervention. permutations on selected samples had little effect on FDR
calculations). Unsupervised clustering was performed on benign
Genotyping and malignant samples using 1-Rank correlation as distance
Genomic DNA wasextracted fromwhole bloodorfromtissue metric on a gene list of 3697 probes. These probes satisfied two
samples using Qiagen QIAamp DNA minikit with RNase criteria:first,thatthecoefficientofvariation(CV)wasbetween0.5
treatment according to the manufacturer’s instructions (http:// and1000;andsecondly,thatthepercentageofPresencecallswas
www.qiagen.co.uk/). GSTM1 (NM_000561) genotype was deter- more than 20%acrossall TURPbenign andmalignant samples.
mined using a real-time PCR procedure based on Covault and For the purpose of sample classification, 19 laser-capture
colleagues, using gene specific primers and probe and quantified microdissected(LCD)epithelialcellmicroarrays(GEOAccession:
relativetoatwo-copygenecontrol,aregioninIVS10ofthebreast GDS1439, http://www.ncbi.nlm.nih.gov/geo/) and 32 TURP
cancer 1, early onset (BRCA1, NM_007294) gene [21]. Primers benignandmalignantmicroarrayswerenormalizedtogetherand
and probes were designed using Applied Biosystems Primer model-based expression was calculated as described above in
Express(http://www.appliedbiosystems.com/)andaregivenwith dChip. The LCD samples were derived from six benign prostate
PCR conditions in Table S1. Data were analyzed with Applied tissue samples, five clinically localized primary prostatic adeno-
Biosystems AbsoluteQuantification software. carcinoma samples, two replicates of the five primary cancer
samples after pooling, four metastatic prostatic adenocarcinoma
RNA extraction and microarray hybridisation samples and two replicates of the four metastatic prostate cancer
Total RNA was isolated from the TURP tissue bank samples samplesafterpooling[22].ClassificationoftheLCDepithelialcell
and the TRUS-guided needle biopsies from the volunteers with samples was then performed using linear discriminant analysis
the use of QIAGENH RNeasy mini kits according to the (LDA) based on the TURP benign and malignant samples as
manufacturer’s instructions (http://www.qiagen.co.uk/). The training samples. LDA wasperformed using 442probes that had
quantity of resulting RNA was measured using a spectrophotom- higherthan100unitsdifferenceinsignalintensitybetweenTURP
eter (Beckman). The RNA quality was determined using the benign and malignant samples and were significantly different at
Agilent 2100 Bioanalyzer (http://www.agilent.co.uk/). RNA P#0.01byWelchmodifiedtwo-samplet-test.Toidentifypathways
samples from TURP biopsies of benign and malignant prostates that are the most over-presented in the lists of differentially
and from TRUS-guided biopsies from both subject groups (peas expressed genes, functional analyses using MAPPFinder and
and broccoli) at baseline, and at six and 12 months after GenMAPPv2.1were performed (http://www.genmapp.org/).
intervention were hybridized onto Affymetrix Human U133 Plus
2.0microarrays(http://www.affymetrix.com/)bytheNottingham
Incubation of peptides with isothiocyanates
Arabidopsis Stock Centre (NASC, http://arabidopsis.info/).
Incubations of SF or IB with bovine insulin (P01308, Sigma-
Double-strandedcDNAsynthesisandgenerationofbiotin-labeled
Aldrich), recombinant human epidermal growth factor (EGF,
cRNA were performed according to the manufacturer’s protocol
P01133, R&D Systems, http://www.rndsystems.com/) and re-
(Affymetrix, http://www.affymetrix.com/). The final cRNA was
combinant human transforming growth factor beta 1 (TGFb1,
checked for quality before fragmentation and hybridization onto
P01137, R&D Systems) were performed in sodium phosphate-
thearrays.
bufferedsalinesolution(pH7.4)orhumanbloodplasmaat37uC
One of the 22 volunteers was diagnosed with prostatic
for 0.5–24 h. Plasma was pre-treated by ultrafiltration to remove
adenocarcinoma at the study baseline biopsy and was removed
high molecular weight proteins (Microcon Ultracel YM-30 filter,
from the study. Eleven samples from the baseline biopsies, two
MWCO 30,000). Samples were either analyzed directly by LC-
samples from the six-month biopsies and three samples from the
MS/MS or by LC-MS/MS analysis of tryptic digests of gel
12-month biopsies did not produce good quality RNA and/or
electrophoresis bands.
sufficient cRNA and were not hybridized. In addition, one
volunteer showed prostatic adenocarcinoma at the six-month
Direct LC-MS/MS analysis of peptides incubated with
biopsy; subsequent samples were removed from the study.
Fluorescence intensity for each array was captured with a isothiocyanates
GeneChipHScanner30007G.AffymetrixGeneChipHOperating The LC system used was a Shimadzu series 10AD VP
Software (GCOS) was used to quantitate each U133 Plus 2.0 (Shimadzu, http://www.shimadzu.com/). The column was an
array. Microarray data in this paper are compliant to the ACE 300 C18, 15062.1mm (5mm particle size) used at 40uC.
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Mobile phase A was 0.1% formic acid in water, mobile phase B, methionine and carbamidomethyl cysteine residues were allowed
0.1% formic acid in acetonitrile and the flow rate was 0.25 ml/ as variable modifications as wasputative SF. Theerror tolerance
min.Alineargradientwasusedfrom25%Bto35%Bover0to of the parent ion was 61.2Da and the fragment mass tolerance
5 min,thenafurthergradientfrom35%Bto99%Bover6 min was 60.6 Da, one missed cleavage was permitted. Error tolerant
followedby99%Bfor4 min.Thecolumnwasre-equilibratedfor searches in Mascot against TGFb were routinely performed and
a total of 3 min. The injection volume was between 5–20ml. All extracted ion chromatograms and manual inspection of spectra
MSexperimentswereconductedona4000QTRAPhybridtriple- were prepared using Qual Browser and BioWorks 3.3 (Thermo
quadrupole linear ion trap mass spectrometer using Analyst Electron Corporation).
version 1.4.1 software (Applied Biosystems, http://www.applied-
biosystems.com/) equipped with a TurboIon source used in Luciferase reporter gene assay
positive ion electrospray mode. The probe capillary voltage was NIH 3T3 cells stably transfected with a CAGA12-luc plasmid,
optimized at 4200V, desolvation temperature set to 400uC, whichrespondstoSmadactivation[23],wereculturedinDMEM
curtaingas,nebulizingandturbospraygasweresetto40,10and supplementedwith10%fetalcalfserum(FCS),1%penicillin,1%
20, respectively (arbitrary values). Declustering potential was streptomycin, 1% L-glutamine and 0.4 mg/ml geneticin. Cells
ramped between 50–120V. Nitrogen was used for collisionally were seeded into complete growth medium in a six-well tissue
induceddissociation(CID).Thepeak-widthwassetonQ1andQ3 culture dish for 24h, after which the medium was replaced with
at 1.0 Th (measured at half height) for all MS and MS/MS low serum medium (0.5% FCS) containing one of three
experiments.Spectrawereobtainedovertherangem/z800–2000 treatments: (1) TGFb1 (to achieve a final concentration of
with scan times of 1–2sec. Operating in LIT mode Q0 trapping 2 ng ml21) in PBS buffer, (2) TGFb1+10mM DTT in PBS
wasactivatedanddynamicfilltimeused,thescanratewassetto buffer (3) TGFb1+2 mM SF in PBS buffer. To simulate SF
250 Th/s for enhanced product ion (EPI) scans, excitation time pharmacokinetics, all test samples were incubated at 37uC for
was150msec,excitationenergy25Vandentrybarrier4V.For 30minutespriortodialysis,performedinPBSbufferfor4 hours
EPI spectrum acquisition the precursor ions of interest for using Slide-A-Lyzer Dialysis Cassettes MWCO 3.5K (PIERCE,
conjugates of SF with insulin (m/z 1183.9 MH55+), EGF (m/z http://www.piercenet.com/). Dialysis reduced SF concentration
1088.8, MH56+) and TGFb (m/z 1981.9, MH513+) were selected, to 34nM. As additional controls, cells were treated with PBS
the collision energy was ramped between 30–120V and spectra withoutTGFb1andPBSwithSF(34 nM).Theluciferaseactivity
were obtained over the range m/z 100–1500 with a scan time of was determined 16h following treatment using the Luciferase
1.9 sec. MS3 settings were identical to MS2 except that the ReporterGeneassay(RocheAppliedScience,http://www.roche.
collision energy was 50–80 V and declustering potential was 50– com/)inaPerkinElmerWallacVictor21420multilabelcounter
80V. plate reader (http://las.perkinelmer.com/). Briefly, cells were
washedtwicewithPBSandlysedincelllysisbuffersuppliedwith
LC-MS/MSanalysisofTGFb1incubatedwithSFfollowing the assay. Chemiluminescence was immediately quantified
electrophoresis and tryptic digestion following the addition of luciferase assay substrate. Luciferase
1 mgaliquotsoftheTGFb1protein,suppliedwithbovineserum values were normalized to protein concentrationquantified using
albumin as carrier, were incubated with either DMSO or the BCA assay (Sigma-Aldrich, http://www.sigmaaldrich.com/).
1.2 mmolesofSFfor30minutesat37uCandrunontodenaturing The experiment was repeated four times, with three replicates of
4–12% Bis-Tris NuPAGE gels (Invitrogen, http://www.invitro- eachtreatmentperexperiment.Statisticalanalysiswasperformed
gen.com).Bandswereexcisedanddigestedwithtrypsin(Promega, using 1-wayANOVA with thestatistical software, R[24].
http://www.promega.com/) after reduction with dithiothreitol
(DTT)andalklyationwithiodoacetamide(Sigma-Aldrich,http:// Results
www.sigmaaldrich.com/).Extractedpeptideswerelyophilizedand
Comparisonofgeneexpressionofbenignandmalignant
re-dissolved in 1% acetonitrile, 0.1% formic acid for analysis by
mass spectrometry. LC-MS/MS analysis was performed using a TURP tissue samples
LTQ mass spectrometer (Thermo Electron Corporation, http:// We compared global gene expression profiles in surgically
www.thermo.com/) and a nanoflow-HPLC system (Surveyor, resected benign and malignant prostate TURP tissue using RNA
Thermo Electron). Peptides were applied to a precolumn (C18 extractedfromheterogeneoustissue(suchasweintendedtousein
pepmap100,LCPackings,http://www.lcpackings.com/)connect- the intervention study). Unsupervised clustering distinguished
ed to a self-packed C18 8-cm analytical column (BioBasic resin unambiguously the benign and malignant samples (data not
ThermoElectron; Picotip 75mm id, 15mm tip, New Objective, shown).Pathwayanalysesforgenesthatweresignificantlydifferent
http://www.newobjective.com/). Peptides were eluted by a between the two groups were undertaken with the use of
gradient of 2 to 30% acetonitrile in 0.1% formic acid over GenMapp software, and identified pathways that are frequently
40min at a flow rate of approximately 250nLmin21. Data- reportedtobeperturbedduringcarcinogenesis(Tables2aand3a).
dependentacquisitionofMS/MSconsistedofselectionofthefive Tovalidatefurtherourmethodsofdataanalysisandtodetermine
mostabundantionsineachcycle:MSmass-to-chargeratio(m/z) whether microarray data from gross heterogeneous tissue are
300 to 2000, minimum signal 1000, collision energy 25, 5 repeat comparable to data generated from LCD epithelial cells, we
hits, 300sec exclusion. In all cases the mass spectrometer was analyzed independent data sets of LCD epithelial cells (GEO
operated in positive ion mode with a nano-spray source and a Accession: GDS1439) from benign, localized and metastatic
capillarytemperatureof200uC,nosheathgaswasemployed;the prostate cancer. We used our benign and malignant samples as
source voltage and focusing voltages were optimized for the a training set for linear discriminant analyses (LDA) and the
transmission of angiotensin. Raw data were processed using independent data as test sets, and found that the LDA model
BioWorks 3.3 (Thermo Electron Corporation). Searches were correctly distinguished the benign, localized and metastatic LCD
performed with Mascot (Matrix Science, http://www. epithelial cell samples (Figure 2). Thus, this preliminary study
matrixscience.com/) against SPtrEMBL (4719335 sequences) provides validation for our approach to the statistical analyses of
restrictedbytaxonomytoHomosapiens(68982sequences),oxidized array data.
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Broccoli&SignallingPathways
Table2. Pathwayanalysesofprostate biopsy tissue. Table3.Differentially expressed probes inprostate tissue.
Geneschanged/ Adjusted FoldchangeP,0.05* P,0.005* P,0.0005*
Pathway GenesonMAPP P-value*
(a) DifferencesbetweenbenignandmalignantTURPtissue
a. BenigncomparedwithmalignantTURPtissue
BenignvMalignant .1.0 3810(353) 683(7) 124(0)
Focaladhesion 57/187 ,0.001
.1.5 1081(59) 400(2) 104(0)
TGFbreceptor 47/151 0.002
.2.0 277(7) 140(0) 54(0)
Circadianexercise 20/48 0.006
(b) DifferencesbetweenGSTM1positiveandnullgenotypes
Fattyacidmetabolism 24/80 0.012
Benign(TURP) .1.0 661(538) 19(17) 0(0)
Prostaglandinsynthesisregulation 14/31 0.026
.1.5 160(186) 7(14) 0(0)
Actinbinding 53/213 0.028
.2.0 50(40) 1(3) 0(0)
GPCRsClassArhodopsin-like 15/262 0.05
Malignant(TURP) .1.0 686(431) 8(13) 0(0)
b. GSTM1positivescomparedwithGSTM1nullspostsixmonth
.1.5 244(152) 4(8) 0(0)
broccoliintervention
.2.0 44(33) 1(3) 0(0)
EGFR1 76/177 ,0.001
Pre-intervention .1.0 730(484) 26(9) 0(0)
Adipogenesis 52/130 0.026
.1.5 252(79) 16(3) 0(0)
TGF-betareceptor 58/151 0.039
.2.0 43(9) 8(1) 0(0)
c. Pairedsamplespreandpost12monthdietaryintervention
6monthbroccoli .1.0 7976(351) 434(4) 17(0)
0–12monthpeas
.1.5 2790(91) 268(2) 14(0)
Androgenreceptor 18/112 0.042
.2.0 316(8) 31(0) 1(0)
0–12monthbroccoli**
6monthpeas .1.0 220(220) 6(4) 0(0)
mRNAprocessing 40/125 ,0.001
.1.5 33(41) 5(3) 0(0)
Androgenreceptor 33/112 ,0.001
.2.0 5(10) 1(1) 0(0)
TGFbreceptor 39/151 0.004
(c) Differencesbetweenpairedsamples
Insulinsignalling 38/159 0.014
0–12monthsbroccoli .1.0 2857(96) 151(0) 1(0)
Delta-notch 24/85 0.019
.1.5 1243(12) 141(0) 1(0)
Wntsignalling 28/109 0.02
.2.0 213(0) 62(0) 1(0)
EGFR1 40/177 0.02
0–12monthspeas .1.0 1199(42) 19(0) 0(0)
IL-2 21/76 0.036
.1.5 495(18) 19(0) 0(0)
EGFR,epidermalgrowthfactorreceptor;GPCRs,G-proteincoupledreceptors;IL- .2.0 81(1) 4(0) 0(0)
2,interleukin2;TGFb,transforminggrowthfactorbeta;TURP,transurethral
resectionoftheprostate;Wnt,wingless-typeMMTVintegrationsite.Pathwaysin
ProbenumbersthathavesatisfiedthecomparisoncriteriaoffoldchangeandP-
GenMAPPthatareenrichedinthegeneliststhatdifferentiategroupsareshown.
valuecutoffsareshown.Numbersinparenthesesrepresentthemedianfalse
OnlypathwayswithadjustedPvalues#0.05areshown.Also,thenumberof
discoveryratecalculatedindChipafter100permutationsofthesamples.
geneschangingbetweengroupsthatbelongtothesepathwaysisshown *P-valueswerecalculatedindChipbyaWelchmodifiedtwo-samplet-test.
alongsidethetotalnumberofgenesthatconstitutethepathway.Pathway
n=18forBenign,n=14forMalignant;n=4forGSTM1(+)Benign,n=14for
analysiswasperformedongenelistsgeneratedindChipthatwerestatistically
GSTM1(2)Benign;n=5forGSTM1(+)Malignant,n=9forGSTM1(2)
significant(P#0.05,Welchmodifiedtwo-samplepairedorunpairedt-test)
Malignant;n=7forGSTM1(+)Pre-intervention,n=3forGSTM1(2)Pre-
betweenthetwogroups.Nofoldcutoffwasused.Fordetailsongenelistssee
intervention;n=6forGSTM1(+)6-monthbroccoli,n=5forGSTM1(2)6-month
Table3.
broccoli;n=3forGSTM1(+)6-monthpeaandn=5forGSTM1(2)6-monthpea
*P-valueswerecalculatedinGenMAPPusinganon-parametricstatisticbasedon
intervention.
2000permutationsofthedataandfurtheradjustedformultipletestingby **P-valueswerecalculatedindChipbyaWelchmodifiedtwo-samplepairedt-
Westfall-Youngadjustment.
test,n=4foreachofthedietinterventions.
**GSTM1positivevolunteers,n=4.
doi:10.1371/journal.pone.0002568.t003
doi:10.1371/journal.pone.0002568.t002
discoveryrates(Figure3a,Table3b).Likewise,wecomparedgene
Variation in plasma PSA levels
expression profiles obtained from needle biopsies of the prostate
PSAlevelspriortotheinterventionwereinsimilarrangetothat from GSTM1 positive and null men who had previously been
previouslyreportedformenofanequivalentagerangediagnosed diagnosedwithHGPINandfoundfewdifferences.
with HGPIN [25]. There was no significant association with We then compared gene expression profiles between GSTM1
GSTM1 genotype, and no consistent changes in PSA levels after genotypesinneedlebiopsytissueoftwenty-onemenwhohadbeen
six or 12 months within either arm of the intervention study recruited into the dietary intervention study. Eight of the men
(Table 1). within this study had been asked to consume 400g of steamed
frozen peas per week, and the other thirteen were requested to
Differences in global gene expression between GSTM1 consume400gofsteamedfrozenbroccoliperweek,butotherwise
positive and null individuals to consume their normal diet. Diet was assessed with seven-day
We initially genotyped the resected TURP tissue samples and diet diaries prior to the intervention and after six months. No
compared gene expression profiles between GSTM1 positive and significantdifferenceswerefoundindietcomponents,apartfrom
nullgenotypeswithinthebenignandmalignantsamples.Wefound theconsumption ofbroccoli andpeas (Table 4).We foundmany
fewdifferences between genotypes,with similar high medianfalse differences in the prostate gene expression between GSTM1
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Broccoli&SignallingPathways
the broccoli arm, we restricted analyses to GSTM1 positive
individuals. We found after both 6 months and 12 months there
weremorechangesinexpressionwithinthebroccoli-richarmthan
the pea-rich arm (Figure 3b, Table 3c). Pathway analyses with
genes that changed in expression between 0 and 12 months
identified changes only in the androgen receptor pathway in the
pea-rich arm, while in the broccoli-rich arm androgen receptor
pathway was identified, along with several other signalling
pathways, including insulin signalling, TGFb and EGF receptor
pathways (Table 2c, Table S4 and S5). Analyses with genes that
changedinexpressionbetween0and6monthsinthebroccoliarm
also identified changes in TGFb receptor pathway (adjusted
P=0.001), insulin signalling (adjusted P=0.035) and EGF
receptor signalling(adjusted P=0.068).
Figure2.LDAofanindependentprostatemicroarraydataset. Thus, evidence for the effect of broccoli consumption on
Lineardiscriminantanalysis(LDA)usingthebenign(B)andmalignant modulationofTGFbandEGFsignallinghasbeenobtainedintwo
(M)TURPprostatetissueforthisstudyastrainingsamplestoclassifythe independent analyses: Firstly, the comparison of gene expression
laser-capturemicrodissected(LCD)epithelialprostatecellsamples(GEO profiles of GSTM1 positive and null individuals who had
Accession:GDS1439), consisting of benign (Be), primary cancer (PCa)
consumed the broccoli-rich diet for six months, and, secondly,
andmetastaticcancer(MCa)samples.LDAwasperformedonagenelist
the paired analyses of gene expression profiles from biopsies
that distinguished the benign and malignant TURP samples as
describedinMethods.Here,thefirstlineardiscriminant(LD1)isshown. obtained at 0 and 12 months from GSTM1 positive individuals
doi:10.1371/journal.pone.0002568.g002 who had consumed the broccoli-rich diet. It is important to note
that these analyses donot shareany arraydata sets.
positive and null men who had been on the broccoli diet for six
Chemical interactions between TGFb1, insulin and EGF
months, but few, if any, differences in gene expression between
peptides and broccoli isothiocyanates
GSTM1 positive and null men who had been on the pea diet
Having demonstrated that broccoli consumption modulates
(Figure3a,Table3b).Toinvestigatethepotentialconsequencesof
several cell signalling pathways, we sought a mechanistic
the differences in gene expression between GSTM1 genotypes
explanation. Incubation of insulin, EGF and TGFb1 peptides
following the broccoli-rich diet, we analyzed these data via
with the isothiocyanates SF or IB in PBS pH 7.4 at 37uC for a
GenMapp. Three pathways, EGF receptor, adipogenesis and
periodof0.5to24hoursgaveconsistentevidenceoftheformation
TGFb receptor, were identified in which genes occurred at a
ofacovalentlyboundconjugateoftherespectivepeptideandthe
higher frequency than they would by chance (Table 2b). The
ITC. This was further investigated for physiological relevance by
relativeexpressionofgenesbetweentheGSTM1positiveandnull
performing the same incubations in human plasma depleted of
menintheEGFreceptorandTGFbreceptorpathwaysaregiven
high MW proteins. LC-MS/MS analysis showed the appearance
as supporting information(Table S2andS3).
of one or more additional LC-MS peaks when SF or IB were
incubatedwiththepeptides.Forexample,inFigure4anextracted
Changes ingene expressionbefore and after the dietary
ion chromatogram (m/z 1183.9, corresponding to insulin- SF
intervention MH 5+) shows the appearance of two insulin-SF conjugates
5
We used paired t-tests to identify genes that had changed in compared with the control incubation. MS2 analysis of these
expression between 0 and 6 months and 0 and 12 months in peaks(Figure5)confirmedthepresenceoftwodiagnosticfragment
biopsy samples from individuals within each arm of the ionsatm/z235andm/z325correspondingtotheadditionofSF
intervention to quantify changes in expression with time. Within tothetwoN-terminalaminoacidsofinsulinGly-SFandPhe-SF.
Figure3.Effectofdietaryinterventionongenetranscription.a,NumberofprobesthatdifferbetweenGSTM1positiveandnullgenotypes
(P#0.005,Welchmodifiedtwo-samplet-test)inTURPtissuefrombenign(Ben)andmalignant(Mal)prostates,andTRUS-guidedbiopsytissuefrom
volunteersatpre-intervention(Pre),post6monthsbroccoli-richdiet(Broc)andpost6monthspea-richdiet(Peas).b,Numberofprobesthatdiffer
betweenpre-interventionTRUS-guidedbiopsysamplesandafter6monthsbroccoli(6B)-,6monthpea(6P)-,12monthbroccoli(12B)-and12month
pea(12P)-richdiets(P#0.005,Welchmodifiedtwo-samplepairedt-test).Shadingcorrespondtodifferentfoldcutoffsapplied.SeeTable2forfull
detailsofprobenumbers,P-valuesandmedianfalsediscoveryrates.
doi:10.1371/journal.pone.0002568.g003
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Broccoli&SignallingPathways
corresponding to the active dimer. The N-terminal peptide
Table4.Dietaryanalysisofaveragedailyintakesofnutrients.
ALDTNYCFSSTEK was identified from parent ion m/z 768.5
in both DMSO (control) and SF-treated samples (Figure 6). A
precursorionm/z877.2wasobservedonlyinSFtreatedsamples.
Variable Baseline 6months P-value*
MS/MSanalysisofbothprecursorionsrevealedastrongseriesof
Pea-richdiet(n=7) fragment peaks that were common to both (Figure 7) precursor
Fat(g) 93.76(32.97) 90.99(25.83) 0.831 ions. These fragmentation patterns are consistent with the
Protein(g) 87.50(15.15) 95.77(33.31) 0.526 unmodified y ion series for the peptide ALDTNYCFSSTEK
(including carbamidomethyl cysteine +57) and a b ion series
CHO(g) 240.04(79.85) 242.94(72.97) 0.872
shifted by 217.460.8Da in the SF-treated sample. These results
Energy(KJ) 9081.88(2480.80) 9506.00(2405.38) 0.738
strongly support an N-terminal modification to TGFb1 by SF.
Alcohol(g) 12.65(9.87) 25.07(35.42) 0.415
AdditionofSFwouldresultinamassadditionof177,asobserved
Cholesterol(mg) 353.57(74.43) 345.29(201.06) 0.930 with LC-MS analyses of intact TGFb1, as described above. It is
VitaminC(mg) 81.00(66.05) 79.43(62.58) 0.846 highlylikelythattheadditionof217,asopposedto177,isdueto
VitaminE(mg) 8.24(5.64) 7.15(3.60) 0.395 subsequentreactionofthethioureawithiodoacetamide,addedto
the reaction mixture to alkylate reduced disulphide linkages, to
VitaminD(mg) 3.77(3.25) 3.84(1.76) 0.949
result in a mixture of isomeric carbamimidoylsulfanylacetamides,
b-Carotene(mg) 1.94(1.57) 3.22(2.29) 0.184
which undergo cyclisation and loss of NH to give the
3
Folate(mg) 261.00(89.36) 332.86(214.50) 0.489 corresponding iminothiazolidinones(Figure S2).
Iron(mg) 11.73(4.36) 13.32(4.92) 0.555
Selenium(mg) 50.14(13.01) 49.14(23.08) 0.923 Enhancement of TGFb1 signalling after pre-incubation
Peas(g) 8.57(10.23) 57.41(18.86) 0.001 with sulforaphane
Broccoli(g) 18.49(30.89) 9.90(13.61) 0.431 Asthioureaderivativesofproteinsbyisothiocyanateshavebeen
shown to modify physicochemical and enzymatic properties
EstimatedGSL(mmol) 9.36(15.63) 5.01(6.88) 0.431
[26,27], we sought to assess whether SF modification of
Broccoli-richdiet(n=11)
extracellular signalling proteins had functional consequences. We
Fat(g) 90.93(29.27) 91.57(33.70) 0.929 focussed on TGFb1 signalling due to its profound role in
Protein(g) 96.99(20.02) 96.97(21.26) 0.996 maintaining tissue homoeostasis through controlling cell prolifer-
CHO(g) 276.28(76.03) 296.18(72.99) 0.305 ation and behaviour [28,29]. TGFb1-induced Smad-mediated
transcription was quantified in NIH3T3 cells stably transfected
Energy(KJ) 9633.45(2311.35) 9980.73(2286.62) 0.488
with a CAGA12-luc plasmid, in which luciferase activity can be
Alcohol(g) 9.75(7.20) 10.48(9.70) 0.841
measureduponactivationofSmadproteins[30].Exposureofcells
Cholesterol(mg) 337.27(168.29) 298.46(123.99) 0.211
toTGFb1inducedluciferaseactivityasexpected.Whencellswere
VitaminC(mg) 262.55(175.83) 303.00(188.52) 0.590 exposed to TGFb1 that had been pre-incubated with physiolog-
VitaminE(mg) 11.31(5.73) 11.14(4.82) 0.924 ically appropriate concentrations of SF (2 mM) for 30minutes
VitaminD(mg) 5.22(3.17) 3.65(1.08) 0.076 followedbydialysis,tosimulateSFplasmapharmacokinetics[14],
there was an increase in Smad-mediated transcription compared
BetaCarotene(mg) 4.07(3.01) 3.63(2.53) 0.667
to exposure to TGFb1 alone (Figure 8). Exposure of cells to the
Folate(mg) 477.82(188.20) 491.36(193.47) 0.762
residual SF (34 nM) did not result in enhanced transcription
Iron(mg) 14.29(2.09) 14.09(2.25) 0.916 suggesting that SF induces Smad activation indirectly, consistent
Selenium(mg) 77.09(29.89) 68.82(26.22) 0.304 withourpreviousobservationthatSFbindstotheliganditself.Itis
Peas(g) 4.16(5.51) 7.60(8.46) 0.227 also conceivable that SF may interact with the extracellular
domain ofthereceptor toalterdownstream signalling.
Broccoli(g) 25.89(24.49) 55.84(7.71) 0.002
EstimatedGSLmmol 13.10(12.39) 79.30(10.94) ,0.0001
Discussion
Variablesshownaregiveninmean(sd)unitsperday.GSLreferstothe
glucosinolateprecursorsofsulforaphaneandiberin(ie4-methylsulphinylbutyl Toourknowledge,thisisthefirstdietaryinterventionstudyto
and3-methylsulphinylpropylglucosinolate)respectively.Similaranalysis analyseglobalgeneexpressionprofileswithinatargettissuebefore
betweenGSTM1positiveandnullindividualsshowednodifferenceindietary andaftera12monthintervention,andtostratifygeneexpression
intakesafter6monthswithineitherbroccoli-richorpea-richintervention.
profiles by genotype. While we do not observe any consistent
*P-valueswerecalculatedinMinitabusingapairedt-test.
doi:10.1371/journal.pone.0002568.t004 changes in plasma PSA levels over the 12 month period of the
intervention, we were able to quantify extensive changes in gene
Similar results were obtained to identify Gly-IB (m/z 221) and expression. We find little evidence to support potential mecha-
Phe-IB (m/z 311) from the incubation (data not shown). nisms derived from animal and cell models to explain the
Comparable evidence was obtained for the formation of EGF observational data that consuming broccoli may reduce risk of
conjugates with SF in human plasma corresponding to the cancer, but considerable evidence for the perturbation of several
additionofSFtotheN-terminalasparaginesresidue(m/z309)of signalling pathways that are associated with carcinogenesis and
EGF. inflammation(Table2bandc).Itispossiblethattheneteffectof
To provideadditional information ofmodifications toTGFb1, perturbation of these pathways may reduce the risk of cell
we adopted a complementary approach. 1 mg aliquots of the proliferation,andmaintaincellandtissuehomoeostasis.However,
proteinwereincubatedwitheitherDMSOor1.2mmolesofSFfor whilst quantification of gene expression and pathway analyses
30minutesat37uCandseparatedbySDS-PAGEelectrophoresis. providesinformationconcerningwhichpathwaysmaybemodified
Bands were excised and digested with trypsin before analysis by bytimeordiet,itcanprovidelittleinformationabouttheprecise
LC-MS/MS. TGFb1 was robustly identified in bands of 25kDa natureofhowthesepathwaysareperturbed.Thisrequiresfurther
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Broccoli&SignallingPathways
Figure4.LC-MSofinsulinincubatedwithandwithoutSFinhumanplasma.ExtractedionLC-MSchromatograms(m/z1183.6–1184.1)of
insulin-SFMH 5+in(A)unmodifiedinsulin(20mg/ml)inhumanplasmacontroland(B)humanplasmaincubatedwithinsulin(20mg/ml)and50mM
5
SFfor4hat37uC,showingtheappearanceoftwodifferentinsulin-SFconjugatesatretentiontimesof6.46and7.08min.Theenhancedproduction
(EPI)-MSspectraofthesetwoinsulin-SFconjugatesareshowninFigure5.
doi:10.1371/journal.pone.0002568.g004
analysis of mRNA and protein turnover, and post translational havebeenshowntohaveamultitudeofbiologicalactivitiesincell
protein modifications such as phosphorylation, associated with models consistent with anticarcinogenic activity [15]. However,
components of the signal transduction pathway and downstream thesestudieslargelyinvolveexposingcellstoconcentrationsofSF
targets. Furthermore, the effect of pathway perturbation may andIBfarinexcessofthosewhichoccurtransientlyintheplasma
depend upon the precise cell type, with potentially differential afterbroccoliconsumption,andaremediatedbytheintracellular
effects occurring in epithelial and stromal cells. Despite these activityoftheITCsby,forexample,perturbingintracellularredox
reservations,itisofconsiderableinterestthatbroccoliintervention status, depletion of glutathione and perturbation of the Keap1-
is associated with perturbation of TGFb1, EGF and insulin Nfr2 complex. We question whether these processes would occur
signalling, each of which has been associated with prostate in vivo, as any of the ITCs entering cells would immediately be
carcinogenesis[31–35],inadditiontocarcinogenesisatothersites inactivated through conjugation with glutathione that would be
[28,36,37], and inflammation associated with myocardial infarc- present in relatively high concentration. Thus, we explored
tion [38]. It is noteworthy that broccoli consumption was also whetherthebiologicalactivityofITCsmaybemediatedthrough
associatedwithalterationsinmRNAprocessing,andthisisbeing their chemical interaction with signalling peptides within the
further explored. extracellular environment of the plasma, which has a low
It is likely that the major bioactive products derived from glutathione concentration. We demonstrated that ITCs readily
broccoli are the isothiocyanates, sulforaphane and iberin. These form thioureas with signalling proteins in the plasma through
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Broccoli&SignallingPathways
Figure 5. Enhanced product ion (EPI)-MS spectra of the two insulin-SF conjugates. MS2 product ion spectra of (A) 6.46min and (B)
7.08minretentiontimepeaksfromLC-MSanalysisofhumanplasmaincubatedwithbovineinsulinand50mMSFfor4hat37uC.In(A)and(B)m/z
1183.9correspondstoinsulin-SFMH 5+andin(A)m/z235.0correspondstoGly-SF,theN-terminalaminoacidofinsulinAchainandin(B)m/z325.2
5
correspondstoPhe-SF,theN-terminalaminoacidofinsulinBchain.
doi:10.1371/journal.pone.0002568.g005
covalently bonding with the N-terminal residue. It is likely that enhancement of Smad-mediated transformation by SF would be
ITCs chemically react with other plasma proteins and a global consistent with the anticarcinogenic activity of broccoli, in
analysisofplasmaproteinmodificationsbyITCsiswarranted.Itis addition to reduced risk of myocardial infarction [10,38]. In
also possible that other types of chemical modification of plasma certaincircumstances,enhancementofTGFbsignallinghasbeen
proteins by ITCs may occur, such as covalent bonding through associated with tumour progression within already initiated cells,
cysteine andlysineresidues [39,40]. althoughtheprecisepathwaysbywhichthisismediatedhavenot
Previous studies have shown that isothiocyanate-derived been fully resolved [42].To whatextent a broccoli-richdiet may
thioureas modify the physicochemical and enzymatic properties influence these processes requires further studies. However, we
of the parental proteins [26,27]. Thus, it is possible that the consider it likely that it is the net effect of changes in several
perturbationofsignallingpathwaysintheprostateismediatedby pathways, as opposed to just TGFb1, which may underlie the
proteinmodificationsthatoccurintheextracellularenvironment. observed reduction in both cancer and myocardial infarction
Weprovidefurtherevidenceforthishypothesisbydemonstrating through broccoli/crucifer consumption.
that pre incubation of TGFb1 with a physiological appropriate A previous study has demonstrated that isothiocyanates can
concentrationofSF(2 mMfor30minutes),followedbydialysisfor inhibit EGF signalling, but without a mechanistic explanation
4 h to simulate SF pharmacokinetics, results in enhanced Smad- [43].Inthecurrentstudy,weshowthatSFwillbindtotheEGF
mediated transcription. As TGFb1/Smad-mediated transcription ligand, and this may underlie our results and those reported
inhibits cell proliferation in non-transformed cells [31,41], the previously [43]. Moreover, chemical modification of signalling
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