w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Identification
of
dysregulated
microRNA
expression
and
their
potential
role
in
the
antiproliferative
effect
of
the
essential
oils
from
four
different
Lippia
species
against
the
CT26.WT
colon
tumor
cell
line
Mayna
Gomide
a,∗,
Fernanda
Lemos
b,
Daniele
Reis
c,
Gustavo
José
d,
Miriam
Lopes
b,
Marco
Antônio
Machado
c,
Tânia
Alves
e,
Cíntia
Marques
Coelho
a,d,∗aDepartamentodeBiologia,InstitutodeCiênciasBiológicas,UniversidadeFederaldeJuizdeFora,JuizdeFora,MG,Brazil
bDepartamentodeFarmacologia,InstitutodeCiênciasBiológicas,UniversidadeFederaldeMinasGerais,BeloHorizonte,MG,Brazil
cLaboratóriodeGenéticaMolecular,EmpresaBrasileiradePesquisaAgropecuária,CentroNacionaldePesquisadeGadodeLeite,Brasilia,DF,Brazil
dDepartamentodeGenéticaeMorfologia,InstitutodeCiênciasBiológicas,UniversidadedeBrasília,Brasilia,DF,Brazil
eLaboratóriodeQuímicadeProdutosNaturais,CentrodePesquisasRenéRachou,Fundac¸ãoOswaldoCruz,BeloHorizonte,MG,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received28January2016 Accepted10April2016 Availableonline16June2016
Keywords:
Colorectalcancer CT26.WT
Lippiaessentialoil
MicroRNA Cellcyclephases
a
b
s
t
r
a
c
t
Inspiteofadvancesincolorectalcancertreatments,approximately1.4millionnewglobalcasesare esti-matedfor2015.Inthissense,Brazilianplantdiversityoffersamultiplicityofessentialoilsasprospective novelanticancercompounds.Thisstudyaimedtoevaluatetheantiproliferativeeffectoftheessential oilsfromfourLippiaspeciesinCT26.WTcolontumorcells,asameasurementofcellcyclephase dis-tributionandmicroRNAexpression.CT26.WTshowedcellcyclearrestatG2/Mphaseaftertreatment with100g/mlofLippiaalba(Mill.)N.E.Br.exBritton&P.Wilson,LippiasidoidesCham.,andLippia lacunosaMart.&Schauer,Verbenaceae,essentialoilsand,atthesameconcentration,Lippiarotundifolia
Cham.essentialoilcausedanaugmentofG0/G1phase.ThemiRNAexpressionprofilingshowschange ofexpressioninkeyoncogenicmiRNAsgenesaftertreatment.Ourfindingssuggestgrowthinhibition mechanismsforallfouressentialoilsonCT26.WTcellsinvolvingdirectorindirectinterferenceoncell cyclearrestand/oroncogenicmiRNAsexpression.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
AccordingtotheInternationalAgencyforResearchonCancer, colorectalcancer(CRC)wasthethirdmostcommoncancerinmen andthesecondmostcommoninwomenworldwidein2012(Ferlay etal.,2015).Approximately1.4millionnewglobalCRCcasesand morethan750,000deathswereprojectedfor2015(Ferlayetal., 2015).Colorectal cancertreatments involve the standard tech-niquesofsurgery,radiationandchemotherapyandafewderivative procedureslikecryosurgery,radiofrequencyablationortargeted therapy.Despitetechnologicaladvances,CRCdemonstratesgreat resistanceandresilienceagainsttherapy.Approximately40%ofall patientstreatedforlocalCRCwillhaverecurrence(Siegeletal., 2012)and thus, thesearch for newanticancer agents remains essential.
∗ Correspondingauthor.
E-mails:mayna.gomide@ufjf.edu.br(M.Gomide),cintiacoelhom@unb.br
(C.MarquesCoelho).
Plantcompoundsfeatureimportantsourcesoftherapeutic com-poundsforcancertreatment.Countrieswithrichflorabiodiversity asBrazilhaveawide rangeofplantspeciesandtherehasbeen aglobalefforttoprospectforbiomoleculeswithpharmacological propertiesintheseregions.Amongthese,monoterpeneshavebeen suggestedasarelevantclassofagentsthatarefoundinseveral plantspecies,includingspeciesoftheLippiagenus,whose phar-macologicalpropertieshavebeenrelatedtosecondarymetabolites, specificallytotheiressentialoils(Pascualetal.,2001).Amongthe beststudiedLippiaspeciesareL.alba(Mill.)N.E.Br.exBritton&P. Wilson,andL.sidoidesCham.,andforbothofthemprevious stud-iesreportedantioxidantactivityindicatingthattheseplantsmight bepotentialtargetstosearchfor antitumorogenicbiomolecules (Ramosetal., 2003;Monteiroet al.,2007).Recently, ourgroup investigated theantiproliferative effectoffive Lippiaspecies on tumorcells,asdeterminedbyMTTassay.Theresultsofthisstudy demonstrated thatL.sidoidesand L.salviifoliaessential oilshad anantiproliferativeeffectonCT26.WTcolontumorcells(Gomide etal.,2013).Monoterpeneslikegeraniolfoundinvegetalessential oilshadalreadybeenshowedtoreducethegrowthofleukemia
http://dx.doi.org/10.1016/j.bjp.2016.04.003
andmelanomacells(Shoffetal.,1991).Othershavealso demon-stratedthatsyntheticgeranioliseffectiveinvitroandinvivoagainst avarietyofcancertypes,includinghepatoma,pancreaticandeven coloncancer,whichishighlyresistanttochemotherapy(Yuetal., 1995;Burkeetal.,1997;Carnesecchietal.,2001,2002;Duncan etal.,2004;Ongetal.,2006;Wisemanetal.,2007).The monoter-pene limonene is another that has exerted antitumor activity, specificallyagainstbreast, skin,liver,lung andstomachcancers inrodents(Elegbedeetal.,1986;WattenbergandCoccia,1991; CrowellandGould,1994;Millsetal.,1995;Kawamorietal.,1996; Crowell,1999).Additionally,anti-tumoractivityhasbeenreported tomonoterpeneslikecarvone,carveol,mentolandperillylalcohol (Heetal.,1997).
Themoleculardrivingforces of CRCcanbecategorized into genomicinstability,genomicmodificationsandepigenetic alter-ations(Kanthanetal.,2012).Morerecently,severalstudieshave observedthatanimbalanceinmiRNAregulatingcellcycle onco-genescouldalsobelinkedtocancerdevelopment.InCRC,studies havedemonstratedanassociationofaberrantmiRNAexpression andcancerdevelopmentwheresomemiRNAhavebeenreported tobeconsistentlydysregulatedinthisdisease(Huangetal.,2010). Theaimofthepresentstudywastoevaluatethe antiprolifera-tiveeffectoftheessentialoilsextractedfromfourdifferentLippia
speciesinCT26.WTcolontumorcellsasameasurementofcellcycle phasedistributionandmiRNAsexpression.
Materialsandmethods
Plantmaterial
FreshleaveswerecollectedfromLippiaalba(Mill.)N.E.Br.ex Britton&P.Wilson,L.sidoidesCham.,L.rotundifoliaCham.andL. lacunosaMart.&Schauer,Verbenaceae,attheExperimentalStation locatedonthecampusoftheFederalUniversityofJuizdeFora, JuizdeFora,Brazil(21◦46′48.4′′S43◦22′24.4′′W).Eachoneofthe
LippiaspecieswascollectedfromNovembertoDecember2010.
ThevoucherspecimensoftheLippiaspeciesevaluatedinthisstudy aredepositedattheHerbariumof theBotanyDepartmentfrom theFederalUniversityofJuizdeForaandthevoucherspecimens numbersare:L.alba:48374,L.sidoides:49007,L.rotundifolia:31376 andL.lacunosa:51920.
Extractionofessentialoils
TheessentialoilsfromleavesoftheLippiaspecieswereobtained byhydrodistillationinaClevenger-typeapparatusfor2h.Theoils wereweighedandaliquotsof5mgofeachoneofthemwerestored at−80◦Cinsealedvialscoveredwithaluminumfoiluntiluse.For eachoneoftheassaysdescribedonealiquotwasthawedand dis-solvedin4%dimethylsulfoxide–DMSO(Sigma,St.Louis,MO,USA) andpurifiedwatermakingupaworkingsolutionof1mg/ml.
Gaschromatography/massspectrometryanalysis
The chemical composition of the essential oil of each Lip-pia specie was determined by gas chromatography coupled to mass spectrometry performed on a Shimadzu QP5050A GC/MS instrument, equipped with a PTE-5 Supelco column (30m×0.25mm×0.25m),asperformedbyGomideetal.(2013). Retentionindexes(RI)werecalculatedfromretentiontimes gen-eratedfromtheanalysisofeachoilincomparisonwithastandard n-alkanessolution,C8-C20,andusedtodeterminethecomponents ofeachoneoftheessentialoils,accordingtoAdams(1995).The amountofcompoundswasdeterminedbypeaksareaintegration ofthespectra.
Celllinesandculturecondition
Mouse colon carcinoma CT26.WT cells were obtained from ATCC(CRL-2638)andweregrownat37◦Cwith5%CO2inRPMI 1640mediumpH7.4(Cultilab,Campinas,SP,Brazil)supplemented with10%fetalbovineserum(FBS),0.1mg/mlampicillin,0.1mg/ml kanamycin, 0.005mg/ml amphotericin, 0.2% NaHCO3 and 0.2% HEPES(Sigma,St.Louis,MO,USA).
Cellcycleanalysis
CT26.WTcellswereseededonto24-wellplatesatadensityof 2×104cells/wellinRPMIsupplementedwith10%FBS.Afterthe cells visiblyreachedaround50%confluence theywereexposed for 12or 24hwithRPMI supplemented with10%FBS contain-ingtheworkingsolutionwiththeessentialoilsofthefourLippia
speciesattheconcentrationsof10,50and100g/ml.The nega-tivecontrolsamplescontained0.4%DMSO,whichisequivalentto thepercentagefoundinthehighestconcentrationevaluated.Then, thecellswerecollectedandresuspendedin300lofHFSsolution (0.05%propidiumiodide,1%sodiumcitrateand0.5%TritonX-100) (Sigma,St.Louis,MO, USA).Cellswereincubatedfor2hat4◦C. TheDNAcontentofthestainedcellswasanalyzedusingFACScan andCellQuestprograms(BDBioscience,SanJose,CA,USA).The his-togramsshowingcellcyclephasedistributionsinG0/G1,S,G2/M andsub-G1cells(usedasmeasureofdeadcells)wereanalyzed usingFlowJoversion7.6.4(Treestar,Inc.,SanCarlos,CA).Allassays wereperformedatleastthreetimes,andatleast15,000events persamplewereanalyzed.Toverifytheexistenceofstatistical dif-ferencesamongthesamplesANOVAfollowedbyBonferronitest wasperformed.Differencesbellow0.05(p<0.05)wereconsidered significant.
MicroRNAanalysis
CT26.WTcellswereseededinthreedifferent25cm2flaskseach oneatadensityof2×104cells/wellinRPMIsupplementedwith 10%FBS.Afterthecellsvisiblyreachedaround50%confluencethey weretreatedwithRPMIsupplementedwith10%FBScontainingthe essentialoilofL.alba,L.rotundifolia,L.sidoidesorL.lacunosaata finalconcentrationof100g/ml.Cellswereincubatedforaperiod of12h.
fixedreallocationrandomizationtest(Pfaffletal.,2002)andCt method(LivakandSchmittgen,2001).Relativeexpressionvalues areshownasmean±standarderror(SEM)anddifferencesbellow 0.05(p<0.05)wereconsideredsignificant.
Resultsanddiscussion
CompositionoftheessentialoilsobtainedfromfourLippiaspecies
Since previous studies have demonstrated that the quan-tity of the main components of the essential oils obtained fromLippia speciesvariesaccordingtothesamplingperiod,gas chromatography–massspectrometryanalysis(GC–MS)was per-formedtoquantifyeachoilcomposition.Themostconcentrated compounds(above6%oftotal composition)wereidentifiedand areshowninTable1.Atotalofninemaincompoundswerefound for allspecies. In L. alba oil(Alb), geranial and citral predomi-nate.ForL.sidoidesoil(Sid),thymolando-cymenewerethemost concentrated. The major constituent of L. rotundifolia essential oil(Rot)was-myrceneandfinally,-myrceneandmyrcenone werethemostabundantinL.lacunosaoil(Lac).Thisdataagrees withpreviousresultsfromGomideetal.(2013),andtheobserved chemotypesvalidatetheidentitiesoftheLippiaspeciesusedinthis study.
TheantiproliferativeeffectofessentialoilsfromLippiaspeciesas determinedbythedistributionofCT26.WTcolontumorcellcycle phases
Several studies have shown that terpenes present chemo-preventive and therapeutics properties against human cancers (Kinghornetal.,2003).Amongtheterpenes,theclassof monoter-peneshasemergedas anadvantageousagentto beusedasan anticancer drug for treatment of tumors that are resistant to chemotherapyandtominimizethesideeffectsofcurrent treat-ments(Shoffetal.,1991;Yuetal.,1995;Burkeetal.,1997;He etal.,1997;Crowelletal.,1999;Duncanetal.,2004;Wisemanetal., 2007;Paduchetal.,2007).Severalmonoterpenesareidentifiedin
LippiaspeciesBrazilbeingoneofthelargestcentersofdiversityof thisgenus,comprising70–75%ofallknownspecies.
A previous study showed potent antiproliferative effects in CT26.WTcells treated withLippia essential oils (Gomide et al., 2013).Inthisstudy,CT26.WTcellsweretreatedfor12and24h withtheessentialoilsextractedfromL.alba,L.sidoides,L. rotundi-foliaandL.lacunosa.ItwasobservedthatallfourLippiaessential oilsaffectedtheCT26.WTcelllinebyinducingcellcyclearrests eitherinG0/G1orinG2/Mphases.Therepresentativehistograms ofthecellcyclephasedistributionofCT26.WTcellsareshownin Fig.1.Table2presentsthepercentagesofsub-G1,G0/G1,Sand
G2/MCT26.WTtreatedcellsasmeasuredbyflowcytometryafter PIstaining.
At50and100g/ml,resultsshowthattreatmentwithAlblead toasignificantincreaseofG2/Mphaseafter12and24h.Decreasing theconcentrationsto10g/mlstillshowsanincreaseofG0/G1 phasecellsforthesametimes(Table2andFig.1).Inagreementwith ourresults,theantiproliferativeeffectofgeranial,themajor com-poundofAlb(Table1),hadalreadybeendemonstrated.Carnesecchi etal.(2001)andWisemanetal.(2007)observedgeraniol(its oxi-dizeformisgeranial)cellcyclearrestingeffects.Thefirstreported geraniolaffectedprogressionthroughtheSphaseofthecellcycle oncoloncancercells,whilethesecondreportedaG0/G1cellcycle arrestonpancreaticcancercells.Inaddition,Wisemanetal.(2007) reportedaroleforp21Cip1andp27Kip1asmediatorsofG0/G1 cellcyclearrestinpancreaticadenocarcinoma,andreduced lev-elsofexpressionofcyclinsA,B1andtheCDK2.Inagreementwith thesepreviousstudies,oursresultsalsoindicatedthatthe antipro-liferativeeffectsofAlbmightrelatetoitsabilitytoaffectthecell cycle,specificallyatG2/Mphase.Similarresultswereobservedby Chaoukietal.,2009whenMCF-7breastcancercellsweretreated for48and72hwithcitral,anothermajorcompoundfoundinAlb (Table1).
SidalsoaffectedCT26.WTcellcycle.Treatmentwith100g/ml showedanincreasedpercentageofG2/MphaseanddecreasedS phasecellsafter12h,whileanincreaseinG0/G1wasobservedafter 24h.Theantiproliferativeeffectofthymol,themajorcompound ofSid,hadbeenpreviouslydemonstrated.Recently,Jaafarietal. (2012)andDebetal.(2011)obtainedcellcyclearrestatsubG0/G1 afterthymoltreatmentinleukemiccells.
TheRotcausedanincreaseofCT26.WTcellsonG0/G1phase at50and100g/mlafter12and24hoftreatment(Table2and Fig.1).Treatmentwith50and100g/mlofLacleadtoaG2/Mphase increaseafter12and24haswellasaconsiderabledecreaseinS phase.At100g/mltherewasalsoanincreaseinG0/G1phaseafter 24hoftreatment(Table2andFig.1).AbdallahandEzzat(2011) showedthattheessential oilextractedfromPituranthos tortuo-sus,whichcontains-myrcene(majorcompoundofRotandLac) showedcytotoxicityagainstcolon,liverandbreastcancercelllines. Therewereveryfewstudiesreportingeffectsofotheridentified compoundsfromthoseLippiaoils.
IdentificationofdifferentialexpressionofmiRNAsinLippia
essentialoilCT26.WTtreatedcells
SeveralstudiesshowthatmiRNAsaremasterregulatorsofcell cyclegenesindifferentcancerandtherefore,wedecidedto inves-tigateifmiRNAsdysregulationwereinvolvedintheobservedcell cycle interference caused by essential oils from Lippia species. ToidentifydifferentialexpressionpatternsofmiRNAsintreated
Table1
PercentagechemicalcompositionofthemajoritycompoundsoftheessentialoilsextractedfromleavesofLippiaalba,L.sidoides,L.rotundifoliaandL.lacunosa,asdetermined bygas-chromatographyfollowedbymassspectrometry.
Compounds RIa L.alba L.sidoides L.rotundifolia L.lacunosa
-Pinene 980 16.30
-Myrcene 991 52.39 53.52
␣-Phellandrene 1005 13.27
o-Cymene 1022 30.08
Limonene 1031 22.43 10.94
Myrcenone 1148 25.41
Citral 1240 20.54
Geranial 1270 29.24
Thymol 1290 38.42
Total 72.21 68.50 92.90 78.93
Numberofretainedcompoundsselected 7 8 5 4
0
100 101 M1
102
FL3-H
103 104 80 160 Counts 240 320 400 L. alba Control – 12h
A
B
C
D
L. alba Control – 24h
L. sidoides Control – 24h
L. rotundifolia Control – 12h
L. lacunosa Control – 24h
L. lacunosa 50 µg/ml – 24h
L. lacunosa 100 µg/ml – 24h L. rotundifolia
100 µg/ml – 12h L. sidoides 100 µg/ml – 24h L. alba 10 µg/ml – 24h L. alba 10 µg/ml – 12h
L. alba 100 µg/ml – 12h
L. alba 100 µg/ml – 24h
0
100 101 M1
102
FL3-H
103 104 80 160 Counts 240 320 400 0
100 101 M1
102
FL3-H
103 104 80 160 Counts 240 320 400 0
100 101
M1
102
FL3-H
103 104
80 160 Counts 240 320 400 0
100 101
M1
102
FL3-H
103 104
80 160 Counts 240 320 400 0
100 101 M1
102
FL3-H
103 104 80 160 Counts 240 320 400 0
100 101 M1
102
FL3-H
103 104 80 160 Counts 240 320 400 0
100 101 M1
102
FL3-H
103 104 80 160 Counts 240 320 400 0
100 101
M1
102
FL3-H
103 104
80 160 Counts 240 320 400 0
100 101 M1
102
FL3-H
103 104 80 160 Counts 240 320 400 0
100 101
M1
102
FL3-H
103 104
80 160 Counts 240 320 400 0
100 101
M1
102
FL3-H
103 104
80 160 Counts 240 320 400 0
100 101 M1
102
FL3-H
103 104 80 160 Counts 240 320 400
Fig.1.RepresentativehistogramsofthecellcyclephasedistributionofCT26.WTcellsafter12and24htreatmentwithdifferentconcentrationsoftheessentialoilsextracted
fromLippiaalba(A),L.sidoides(B),L.rotundifolia(C),andL.lacunosa(D).DNAcontentofthestainedcellswasanalyzedusingFACScanandCellQuestprogram.Thenegative
controlsamplescontained0.4%DMSO,whichisequivalenttothepercentagefoundinthehighestconcentrationevaluated.Allassayswereperformedatleastthreetimes, andatleast15,000eventspersamplewereanalyzed.
CT26.WTcellsweusedreal-timePCR-basedmiRNAexpression pro-filingarraywithapanelof95cancer-relatedmiRNAsandanU6 transcripttonormalizesignal.Table3showstheproportionof miR-NAsfromCT26.WTcellstreatedwiththeessentialoilsfromLippia
speciesthatshoweddifferentialexpressionfromcellstreatedwith 0.4%dimethylsulfoxide(DMSO).Theresultsshowedthat27.36%of analyzedmicroRNAsweredysregulatedonCT26.WTcellstreated withAlbandLacand36.84%oncellstreatedwithSidandRot.
Fig.2showsa heatmapcomparingdifferentmiRNAs expres-sionafterCT26.WTcellstreatmentwithLippia oils.Somegenes
Table2
Percentagesofsub-G1,G0/G1,SandG2/MofCT26.WTcellsafter12and24htreatmentwiththeessentialoilsextractedfromLippiaalba,L.sidoides,L.rotundifoliaandL.
lacunosa,asdeterminedbyflowcytometryafterPIstaining.Thenegativecontrolsamplescontained0.4%DMSO,whichisequivalenttothepercentagefoundinthehighest
concentrationevaluated.Allassayswereperformedatleastthreetimes,andatleast15,000eventspersamplewereanalyzed.Statisticaldifferencesweredeterminedwith ANOVAfollowedbyBonferronitest(p<0.05).
Essentialoil (solutions)
sub-G1 G0/G1 S G2/M
12h 24h 12h 24h 12h 24h 12h 24h
Lippiaalba Negativecontrol
<4 <5
25.65±1.58a 27.37±1.30a 40.34±2.82a 39.38±1.38a 24.25±4.03a 23.46±1.56a
10g/ml 31.12±2.04b 32.35
±0.75b 39.91
±0.96a 35.29
±1.20a 17.96
±0.83b 23.11
±0.34a
50g/ml 23.69±0.1a 23.07
±0.82c 20.53
±1.40b 17.31
±2.35b 41.89
±0.87c 45.31
±2.75b
100g/ml 21.03±0.61a 21.26
±1.17c 30.98
±2.50c 27.33
±2.27c 32.40
±2.34d 39.03
±1.32c
Lippiasidoides Negativecontrol
<4 <2
28.59±3.44a 29.62
±2.00a 38.35
±3.32a 39.12
±2.48a 18.81
±3.93a 18.32
±1.40a
10g/ml 26.82±3.18a 33.82
±1.50ab 35.50
±3.35a 34.30
±1.91ab 18.86
±1.69a 19.52
±1.77a
50g/ml 27.84±1.69a 31.35
±2.84a 25.98
±2.32b 37.04
±0.90a 26.15
±1.38ab 18.93
±1.59a 100g/ml 23.64±1.76a 38.17±1.92b 19.14±2.66c 30.31±1.15bc 33.25±5.88b 21.23±1.88a
Lippiarotundifolia Negativecontrol
<3 <1
29.76±1.87a 36.55
±0.47a 38.75
±2.44a 29.60
±1.17a 15.57
±2.36a 20.46
±1.19a
10g/ml 33.77±2.74a,b 37.90
±1.80a,c 35.54
±2.33ab 28.04
±1.58ab 15.93
±2.48a 20.98
±2.00a
50g/ml 36.01±1.28b 40.99±1.79bc 33.59±1.26b 24.53±0.42b 15.28±3.06a 20.86±1.91a
100g/ml 41.65±2.11c 38.30±0.80ac 26.01±1.81c 24.71±0.60ab 16.02±1.35a 21.09±1.77a
Lippialacunosa Negativecontrol
<5 <6
33.49±1.51a 28.27±3.27a 35.30±1.99a 34.75±2.11a 17.79±1.34a 26.27±2.53ab 10g/ml 33.32±5.53a 32.78±0.87a 24.49±6.19b,d 30.86±0.54b 19.63±1.52a 23.25±1.49a
50g/ml 19.51±3.21b 19.53±5.35b 9.12±0.44c 20.82±1.00c 33.38±1.76b 29.87±2.32bc
100g/ml 35.99±2.10a 48.93
±1.84c 15.39
±7.05cd 4.98
±1.93d 33.86
±6.82b 34.98
±2.70c Thevaluesrepresenttheaverageofatleastthreereplicates±SD.
a,b,c,dDifferentlettersindicatesignificantstatisticaldifferencesineachcellcyclephaseandexposuretimecolumnbyessentialoil.
treatmentwithLippiaessentialoils.DataindicatesAlb,RotandSid treatmentcorrelatestoreversedmiR-15bexpressioninCT26.WT coloncancercellline.
ReversionofexpressionwasobservedformiR-92,miR-93, miR-135b,miR-155,miR-191,miR-181aandmiR-186geneswhichwere previouslyfoundtobeupregulatedincoloncancer(Voliniaetal., 2006;Monzoetal.,2008;Schepeleretal.,2008;Arndtetal.,2009; Sarveretal.,2009;Earleetal.,2010;Huangetal.,2010)andwere downregulatedinCT26.WTcellsaftertreatmentwithAlb,Sidand Rot(Fig.2).Incontrast,miR-143appearedupregulatedonCT26.WT cellstreatedwithAlb,RotandLac(Fig.2),buthasbeenshowntobe consistentlydownregulatedincolorectalcancer(Chenetal.,2009; Kuldaetal.,2010).Chenetal.(2009)showedthatmiR-143actsasa tumorsuppressorbyinhibitingtheKRASoncogenetranslation.Alb, RotandLacseemtoincreaseexpressionofmiR-143whichcould possiblycausearecoveryofKRAStumorsuppressorrole.
Inthisstudy,miR-192genewasupregulatedonCT26.WTcells exposedtoAlbandLacanddownregulatedbyRot(Fig.2).Chen etal.(2009)andEarleetal.(2010)demonstratedthat miR-192 wasdownregulatedincolorectalcancer.Theseresultsindicatethat AlbandLacmightberevertingexpressionofmiR-192inthistype ofcancer.Braunetal.(2008)alsodemonstratedthatmiR-192is capableofsuppressingcarcinogenesisbyincreasingthelevelofa G1cellcycleinhibitorp21,leadingtocellcyclearrestinG1phase andinG2/MphaseinHCT116humancolorectalcancercellline. Inagreement, cellcyclearrestatthesephaseswasobservedon CT26.WTcellstreatedwithAlbandLac(Table2andFig.1). Expres-sionofmiR-222wasdecreasedinCT26.WTcellsexposedtoAlb,Sid andRot(Fig.2).Visoneetal.(2007)showedthattheexpressionof miR-222togetherwithmiR-221inhumanthyroidcarcinomacell lineinducedcellcycleprogressiontotheSphaseandreducedthe expressionleveloftheG1cellcycleinhibitorp27KIP1.
AfewmiRNAgenesshowedreversedexpressionaftertreatment exclusivelywithone of thefour Lippia essential oils.The miR-NAsmiR-196a,miR-214,miR-149andmiR-30bwereshowntobe downregulatedincolorectalcancer(Monzoetal.,2008;Schepeler
Table3
TotalmicroRNAsdifferentiallyexpressedafterCT26.WTcelllinetreatedwithLippia
essentialoils.
miRNA L.alba L.sidoides L.rotundifolia L.lacunosa
Total 59 59 57 59
Significants(p<0.05) 26 35 35 26
Upregulated 8 13 12 23
Downregulated 18 22 23 3
etal.,2008;Chenetal.,2009;Earleetal.,2010)andourresults demonstratedthatthesemiRNAwereupregulatedafterCT26.WT cellsweretreatedwithLac(Fig.2).Onthecontrary,miR-17-5p andmiR-17-3pweredownregulatedonCT26.WTcellsbyAlband Lacessentialoils,respectively(Fig.2).Bandresetal.(2006)and Voliniaetal.(2006)observedmiR-17-5pupregulationin colorec-talcancertissue.Monzoetal.(2008)reportedthatthismiRNAis acriticalmemberofafunctionalgroupinvolvedinregulatingthe expressionofE2F1,anupstreamregulatorof TP53incolorectal cancercells. Theyalsoshowedthat cells transfected with anti-miR-17-5p,hadanincreasedE2F1expression,reducingcellgrowth inadosedependentmanner.Inanothertransfectionexperiment, Kanaanetal.(2012)transfectedmiR-17incoloncancerlinesHT-29 andHCT-116andshowedthatmiR-17isanE2F1regulator. Fur-thermore,Cloonanetal.(2008)haveshownthatmiR-17-5pacts specificallyonthetransitionfromG1/Scellcyclephases,interfering withmorethan20genes.
Taken together,theseresultssuggestthat apossible mecha-nismforLippiaoilsgrowthinhibitionmightbethroughreversion ofexpressionofmiRNAsregulatingcellcycleinhibitors.
miR-202 miR-142-3p miR-219 miR-143 miR-15a miR-19a+b miR-218 miR-154 miR-106b miR-210 miR-7 miR-107 miR-18a miR-199a+b miR-30b miR-194 miR-22 miR-125a miR-149 miR-150 miR-185 miR-196a miR-92 miR-30a-5p miR-21 miR-191 miR-192 miR-214 miR-27a+b miR-106a miR-15b miR-181a miR-132 miR-296 miR-186 miR-93 miR-16 miR-195 miR-103 miR-145 miR-151 miR-20a miR-24 miR-25 miR-17-3p miR-17-5p miR-222 miR-23a miR-26a miR-125b miR-30c miR-181c miR-9-1 miR-155 miR-30a-3p miR-135b miR-140
Lac Rot
Sid Alb
0 0.25 0.63 1.00 3.25 5.50
Fig.2.HeatmapofdifferentiallyexpressedmicroRNAsinCT26.WTcelllineafter treatmentwithLippiaoils.CT26.WTcellsweretreatedwith100mg/mlofL.alba
(Alb),L.sidoides(Sid),L.rotundifolia(Rot)andL.lacunosa(Lac)essentialoilsfor12h. ApooloftriplicatesofeachtreatmentwassubjectedtomicroRNAsanalysisby real-timePCRtestusingapanelof95cancer-relatedmicroRNA.TheCtvaluesobtained werenormalizedfromtheU6transcribedandanalyzedbytheRESTsoftware (sig-nificancelevelp<0.05).Thevaluesoffoldchangeobtainedabove1,referredto up-regulatedmicroRNAs,weredividedintotwogroupsaswellasthosebetween0 and1correspondingtothedown-regulatedmicroRNA.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.
Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.
Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.
Authors’contributions
MSG (M.Sc.student) contributed in collectingplant sample, extractingandanalyzingtheessentialoil,runningthelaboratory work,analysisofthedataanddraftedthepaper.FOLandMTPL con-tributedtobiologicalstudiesanddatadiscussion.DRLRandMAM contributedtomicroRNAanalysisandtodatadiscussion.GPCJ con-tributedtocriticalreadingofthemanuscript.TMAAcontributedin GC/MSanalysis.CMCdesignedthestudy,supervisedthe labora-toryworkandcontributedtocriticalreadingofthemanuscript. Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
TheauthorsthankFundac¸ãodeAmparoàPesquisadoEstadode MinasGeraisforthefinancialsupportunderGrantAPQ-00722-09. WearealsogratefulforthetechnicalassistancefromLaboratóriode SubstânciasAntitumoraisatUniversidadeFederaldeMinasGerais, fromLaboratóriodeGenéticaMolecularatEmpresaBrasileirade PesquisaAgropecuáriaandfromLaboratóriodeQuímicade Produ-tosNaturaisatFundac¸ãoOswaldoCruz.Finally,wealsothankDra. LucíolaBastosfromtheDepartmentofPhysiologyandBiophysics, BiologicalSciencesInstitute, FederalUniversityofMinasGerais, Brazil,forcollaboratinganddonatingCT26.WTcelllinewhichwas usedinthisstudy.
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