Effect of diterpenoid kaurenoic acid on genotoxicity and cell cycle progression in gastric cancer cell lines

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Effect

of

diterpenoid

kaurenoic

acid

on

genotoxicity

and

cell

cycle

progression

in

gastric

cancer

cell

lines

Plínio

Cerqueira

dos

Santos

Cardoso

a,1

_,

_Carlos

_Alberto

_Machado

_da

_Rocha

b,

_*

,1

_,

Mariana

Ferreira

Leal

c

_,

_Marcelo

_de

_Oliveira

_Bahia

a

_,

_Diego

_Di

_Felipe

_Ávila

_Alcântara

a

_,

Raquel

Alves

dos

Santos

d

_,

_Natália

_dos

_Santos

_Gonçalves

d

_,

_Sérgio

_Ricardo

_Ambrósio

e

_,

Bruno

Coêlho

Cavalcanti

f

_,

_Caroline

_Aquino

_{Moreira-Nunes}

a,g

_,

_Claudia

_do

_Ó

_Pessoa

f

_,

Rommel

Mário

Rodríguez

Burbano

a,h,

_**

a_Human_Cytogenetic_Laboratory,_Biological_Science_Institute,_Federal_University_of_Pará_(UFPA),_Belém,_Pará,_Brazil

b_Federal_Institute_of_Education,_Science_and_Technology_of_Pará_(IFPA),_Av._Almirante_Barroso,₁₁₅₅_(Marco),_CEP_66093-020,_Belém,_Pará,_Brazil c_Department_of_Morphology_and_Genetics,_Federal_University_of_São_Paulo_(UNIFESP),_São_Paulo,_São_Paulo,_Brazil

d_Laboratory_of_Genetics_and_Molecular_Biology,_University_of_Franca_(UNIFRAN),_Franca,_São_Paulo,_Brazil e_Laboratory_of_{Biotransformation,}_University_of_Franca_(UNIFRAN),_Franca,_São_Paulo,_Brazil

f_Department_of_Physiology_and_{Pharmacology,}_Federal_University_of_Ceará_(UFC),_Fortaleza,_Ceará,_Brazil

g_Laboratory_of_Genetics_of_{Hemoglobinopathies}_and_Hematologic_Diseases,_Federal_University_of_Ceará_(UFC),_Fortaleza,_Ceará,_Brazil h_Hospital_Ophir_Loyola_(HOL),_Belém,_Pará,_Brazil

ARTICLE INFO

Articlehistory:

Received24November2016

Receivedinrevisedform22February2017 Accepted22February2017

Keywords:

Cellcycleprogression Cometassay

Gastriccancercelllines Genetranscriptions Kaurenoicacid Micronuclei

ABSTRACT

Thegoalofourstudywastoevaluatetheeffectofkaurenoicacid,obtainedfromcopaibaoilresin,in gastriccancer(GC)andanormalmucosaofstomach(MNP01)celllines.Thecompoundwastestedat concentrationsof2.5,5,10,30and60mg/mL.Cometandmicronucleusassayswereusedtoaccessits

potential genotoxicity invitro. Moreover, we evaluated the effect of kaurenoic acid in cell cycle progressionandinthetranscriptionofgenesinvolvedinthecontrolofthecellcycle:MYC,CCND1,BCL2, CASP3,ATM,CHK2andTP53.KaurenoicacidinducedanincreaseoncellDNAdamageormicronucleus frequenciesonGCcelllinesinadose-dependentmanner.TheGCandMNP01celllinesenteringDNA synthesisand mitosisdecreasedsigniﬁcantlywithkaurenoicacid treatment,andhad anincreased growthphasecomparedwithnon-treatedcells.Thetreatmentinducedapoptosis(ornecrosis)evenata concentrationof2.5mg/mLinrelationtonon-treatedcells.GCcelllinespresentedreducedMYC,CCND1,

BCL2andCASP3transcriptionwhileATM,CHK2andTP53increasedintranscriptioninrelationto non-treatedcells,especiallyataconcentrationabove10mg/mL.ThegenetranscriptionintheMNP01

(non-treatednon-cancercellline)wasdesignatedasacalibratorforalltheGCcelllines.Inconclusion,our resultsshowedthatkaurenoicacidobtainedfromCopaiferainducesDNAdamageandincreasesthe micronucleifrequencyinadose-dependentmannerinGCcells,withasigniﬁcantgenotoxicityobserved abovetheconcentrationof5mg/mL.Moreover,thiscompoundseemstobeabletoinducecellcyclearrest

andapoptosisinGCcells.

1.Introduction

Gastriccancer(GC)remainsthethirdleadingcauseof

cancer-related mortality worldwide, and invasion and metastasis of

gastriccancerrepresentsthemajorreasonforitspoorprognosis

[1].Multimodal therapies haveproven tobeneﬁtpatients with

cancer.However,withoutcurativesurgery,variationsand

combi-nationsofchemotherapyand/orradiationcannotbringclinically

meaningfulsuccessnowadays[2].

*Correspondingauthor.

**Correspondingauthorat:HospitalOphirLoyola(HOL),Av.MagalhãesBarata, 992,CEP66060-281,Belém,Pará,Brazil.

E-mailaddresses:carlos.rocha@ifpa.edu.br(C.A.M.d.Rocha),rommel@ufpa.br (R.M.R.Burbano).

1_These_authors_contributed_equally_to_this_work.

http://dx.doi.org/10.1016/j.biopha.2017.02.085

–

Available

online

at

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One of the main problems in cancer treatment is gradual

resistanceofcancercellsagainsttreatment[3].Therefore,theaim

ofimmunopharmacologicalstudieshasbeentoimprovecancer

treatmentresults[4].Thealternatesolutionfortheharmfuleffects

of synthetic agents is the use of phytotherapy [5]. Bioactive

compoundsfromplantshavealotofbiologicalactivitiesandmay

presentgreatpotentialinthedevelopmentofnewdrugsforthe

treatmentofhumandiseases[6].

Reports about medicinal plants in the Brazilian Amazonian

region have been documented in the literature, especially

concerning the oil resin extracted from the trunk of Copaifera

langsdorfﬁiDesfon(Leguminosae)[7,8].Thisoilresinisareputed

folkremedy used bythe nativesof Brazil for thetreatmentof

several diseases such as sore throat, urinary and pulmonary

infections,andtohastenulcerandwoundhealing[9].Usually,this

oilresinisadministeredorallyinnatura,orappliedinointment

form [10–12]. This substance has great social and economic

representationintheAmazonregion,sinceitrepresentsabout95%

oftheentireoilresinproductioncountrywide[13].

SeveralstudieshavedemonstratedthattheCopaiferaoilresin

hasmanifoldtherapeuticproperties,includinganti-inﬂammatory,

antitumoral,antimelanoma,antiulcerogenic,antilipoperoxidation

and antioxidant [10,12,14–16]. Furthermore, the diterpenes,

kaurenoic and polyaltic acids present in the pure oil seem to

presenthealingandanti-inﬂammatoryproperties[7,8].

Kaurenoicacid(ent-kaur-16-en-19-oicacid)(Fig.1)isclassiﬁed

asaditerpeneandconsideredanintermediateofthegibberellin

plantgrowthhormonebiogenesis[17].Somestudiesshowedthat

kaurenoicacidpresentsinvitroanti-parasiticandanti-microbial

activities[18–23].Moreover,thisditerpenehasanti-proliferative

actioninCEMleukemiccells,MCF-7breastandHCT-8coloncancer

cells[24].Additionally,kaurenoicacidisalsoabletoreducehuman

spermmotilitywhendirectlytestedinspermsamples,butwas

onlyweaklyspermicidal[25].

While Copaifera oil resin has its herbal properties widely

reported, there are few studies in the scientiﬁc literature that

evaluatedtheeffectsofthisoilresincomponents,especiallyinGC

celllines.Thus,thepresentstudyaimstoassessthegenotoxicityof

kaurenoicacidandtheeffectofthiscompoundinthecellcycleand

inthetranscriptionofgenesinvolved,amongotherfunctions,in

GCcelllines.

2.Materialsandmethods

2.1.Generalexperimentalprocedures

TheACP02,ACP03andAGP01celllines,previouslyestablished

andcharacterizedbyourresearchgroup,wereusedinthepresent

study[26,27].TheACP02celllinewasestablishedfroma

diffuse-typeGC,andACP03andAGP01werefromanintestinal-typeGC.

ACP03isabletostartatumorigenesisprocessinCebuspaella[28].

WealsousedtheGCcelllinePG100obtainedfromRiodeJaneiro

CellBank,Brazil,thatwaspreviouslycytogeneticlycharacterized

byourgroup[29].

Cells lines were cultivated under standard conditions in

Modiﬁed Eagle’s medium salts, supplemented with 10% fetal

bovine serum, 2mM L-glutamine and antibiotics (100IU/mL

penicillin and 100

m

g/mL streptomycin [30]). Cells were

main-tained in 25cm2 _tissue _culture

ﬂasks (TPP, Trasadingen,

Switzerland)at37_C _in_a_humidi

ﬁedatmospherecontaining5%

CO2andwereharvestedbytreatmentwith0.15%trypsinand0.08%

EDTA in phosphate-buffered saline (PBS). Cells (3105) were

seededin5mLofcompletemediumandgrownfor2dayspriorto

treatmentwiththetestsubstance.

Cellsweretreatedwithdifferentconcentrationsofkaurenoic

acid(2.5,5,10,30and60

m

g/mL)for3h.Afterthis,thecellswere

washed withice-cold PBS and trypsinizedwith 100

m

L trypsin

(0.15%) and were resuspended in complete medium. The ﬁnal

concentrationofDMSOintheculturemediumwaskeptconstant,

below 0.1% (v/v). The negative control cell cultures were not

exposed tokaurenoic acid, while themethyl methanesulfonate

(Sigma Aldrich Co, St. Louis, MO, USA) was used as a positive

control (410 5_M). _All _cell _treatments _were _carried _out _with

threereplicates.

2.2.Chemicalisolation

Inthisstudy,kaurenoicacid(CAS6730-83-2)(SigmaChemical

Co., St.Louis,MO, USA) was dilutedundersterileconditions in

dimethyl sulfoxide (DMSO)and theconcentrationsused inthis

studywerebasedonpreliminarycytotoxicityassaysperformedby

our research group (unpublished data). Kaurenoic acid was

obtainedfromtheoilresinofC.langsdorfﬁithatgrowsabundantly

intheAmazonregionofNorthernBrazil[31].

2.3.Genotoxicityassay

TheCometassay(alkalineversion)wasperformedasdescribed

by Singhet al. [32] withsomemodiﬁcations[33,34].Aftercell

treatment,thecellswerewashedwithice-coldPBSandtrypsinized

with100

m

L trypsin(0.15%)and wereresuspendedin complete

medium. An aliquot (450

m

L)of thecell suspension fromeach

experimental groupwas takenandcentrifugedat1.000rpmfor

5mininamicrocentrifuge(Eppendorf).Theresultingpelletwas

homogenizedwith300

m

Lofalowmeltingpointagarose(0.8%),

spreadontomicroscopeslidespre-coatedwithanormalmelting

pointagarose(1.5%)andcoveredwithacoverslip.After5minat

4_C,_the_cover_slip_was_removed_and_the_slides_were_immersed_in

cold lysissolution (2.5MNaCl;100mM EDTA; 10mM Tris,10%

DMSOand1%Triton-X,pH:10)foroneweek.Afterlysis,theslides

were placed in an electrophoresis chamber and covered with

freshlymadeelectrophoresisbuffer(300mMNaOH;1mMEDTA,

pH>13).

The electrophoresis was run for 25min (34V and 300mA).

Afterward,theslideswereneutralizedbysubmersionindistilled

water(4_C)_for₅_min_and_ﬁ_xed_in_100%_ethanol_for₃_min._Staining

oftheslideswasperformedimmediatelybeforetheanalysesusing

ethidium bromide dye (20

m

g/mL). Slides were prepared in

duplicate,and100cellswereanalyzedpersample(50cellsfrom

eachslide)usingaﬂuorescentmicroscope(OlympusBX41)at40

magniﬁcation.

Thedamageindex(DI)isbasedonthelengthofmigrationand

theamountofDNAinthetail.Itisconsideredasensitivemeasure

ofDNA.Fivecategories(0–4)wereusedhere:class0(nodamage),

class1(slightdamagewithataillengthshorterthanthediameter

ofthenucleus),class2(mediumdamagewithataillengthoneor

twotimesthediameterofthenucleus),class3(signiﬁcantdamage

with a tail length greater than two times the diameter of the

Fig.1.Molecularstructureofkaurenoicacid(ent-kaur-16-en-19-oicacid).

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nucleus)andclass4(signiﬁcantdamagewithtaillengthgreater

thanthreetimesthediameterofthenucleus).Damagedindexthus

ranged from 0 (no damage: 100 cells0) to 4 (completely

damaged:100cells4)[35,36].

2.4.Mutagenicassay

The micronucleusassaywas performedaccording toFenech

[37].Threehoursaftertreatment,thecultureswerewashedtwice

withthe medium and Cytochalasin B(Sigma Chemical Co., St.

Louis,MO, USA)wasadded ataﬁnalconcentrationof2

m

g/mL.

Cultureswereharvested 21hafterCytochalasinBaddition.The

cells wereseparated fromthe bottle bytrypsinization and the

cellularsuspensionwascentrifugedat1000rpmfor5min.Then,

cellswereresuspendedin aKCl0.075Msolutionmaintainedat

4_C _for ₃_min _(mild _hypotonic _treatment). _Cell _cultures _were

harvested72haftertheincubation,centrifuged(5minat800rpm)

andsubmittedtohypotonicsolution(KCL0.075M).Afterward,GC

celllineswerewashedoncewith5mLofacoldmethanol:acetic

acid(5:1) (v/v) ﬁxative and washed againwith 5mL of a cold

methanol:aceticacid(3:1)(v/v)ﬁxative.Thecellsuspensionwas

droppedontoslidesandstainedinasolutionof5%Giemsadye

(SigmaChemicalCo.,St.Louis,MO,USA)inaphosphatebuffer(pH

6.8)for5min.Micronucleiwerescoredin2000binucleatedcells

using the criteria according to Fenech [37]. The analysis was

performedusinganopticalmicroscope(BIOVALL2000A)at100

magniﬁcation.

2.5.Cellcycleanalysis

Cell cycle distribution followed the procedure described by

Nicolettietal.[38].GClineswereincubatedat37_C_for₃_h_in_the

darkinalysissolutioncontaining0.1%,citrate,0.1%tritonX-100

and50

m

g/mLpropidiumiodide.Cellﬂuorescencewasdetermined

byﬂowcytometryinaGuava1_EasyCyteTM_Mini_System_cytometer

(GuavaTechnologiesInc.,Hayward,CA,USA),usingCytoSoft4.1

software.

2.6.Genestranscriptionanalysis

Weanalyzedthetranscriptionofgenesinvolvedinthecellcycle

regulationtoevaluatetheeffectof kaurenoicacidtreatmentin

ACP02,ACP03,AGP01andPG100celllines(Table1).TotalmRNA

was isolated from all cell line samples using Trizol Reagent

(Invitrogen,Carlsbad,CA,EUA).TheextractedRNAwaspuriﬁed

usingRNeasyMiniKit(Qiagen,Valencia,CA,EUA),accordingtothe

recommended protocol. The concentration and quality were

determined using a NanoDrop spectrophotometer (Kisker,

Germany)and1%agarosegels,respectively.Sampleswerestored

at 80_C_until_use.

RNAwas reverse-transcribed using the High-Capacity cDNA

Archive kit according to the manufacturer’s protocol (Life

Technologies,Pittsburgh,PA,USA).ComplementaryDNAwasthen

ampliﬁedbyreal-timereversetranscriptionquantitativePCR

(RT-qPCR) using TaqMan probes purchased as Assays-on-demand

Productsfor GeneExpression (LifeTechnologies,Pittsburgh,PA,

USA; Table 1) and a 7500 FastReal-Time PCR instrument (Life

Technologies,Pittsburgh,PA,USA).TheGAPDHgenewasselected

as an internal control for RNA input and reverse-transcription

efﬁciency.AllRT-qPCRswereperformedintriplicateforboththe

targetgenesandtheinternalcontrol.Therelativequantiﬁcationof

genetranscriptionwascalculatedaccordingtothemanufacturer’s

software (Life Technologies, Pittsburgh, PA, USA). The gene

Table1

SummaryofSevenTargetGenesandtheReferenceGene.

Gene symbol

Name genefunction assay*

MYC v-mycavianmyelocytomatosisviral oncogenehomolog

progressionofcellcycleprogression,apoptosisandcellulartransformation/oncogene hs00153408_m1

CCND1 cyclinD1 positiveregulationofmitoticcellcycle/oncogene hs00765553_m1

BCL2 b-cellcll/lymphoma2 anti-apoptotic/oncogene hs00608023_m1

CASP3 caspase3,apoptosis-relatedcysteine peptidase

pro-apoptotic hs00234387_m1

ATM atmserine/threoninekinase cellcyclearrestinresponsetoDNAdamageandforgenomestability/tumorsupressor hs01112355_g1 CHK2 checkpointkinase2 cellcyclecheckpointregulatorinresponsetoDNAdamageandtoreplicationblocks/

putativetumorsuppressor

hs00200485_m1

TP53 tumorproteinp53 inductionofcellcyclearrest,apoptosis,senescence,DNArepair,orchangesin metabolism/tumorsupressor

hs01034249_m1

*_TaqMan_probes_were_purchased_as_{assays-on-demand}_products_for_gene_expression_(Life_{Technologies,}_USA).

Fig.2. Genotoxicityofkaurenoicacidingastriccancercellline.A)Damageindexbycometassay;B)micronucleifrequency.DI:damageindex;MN:micronucleifrequency.C-: control,whichwerenon-treatedcells;C+:cellstreatedwithmethylmethanesulfonateon*Signiﬁcantlydifferentfromthecontrol(p<0.05;byKruskal-Wallistestfollowedby Games-Howellpost-hocanalysis).**Signiﬁcantlydifferentfromthecontrol(p0.001;byKruskal-WallistestfollowedbyGames-Howellpost-hocanalysis).

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transcriptionintheMNP01(non-treatednon-cancercellline)was

designatedasacalibratorforalltheGCcelllines.

2.7.Statisticalanalysis

Thedataareshownastheasthemeanstandard deviation

(SD). The Shapiro-Wilk test was used to evaluate the data

distribution and to determine the appropriate subsequent test

for statisticalcomparisons. The Kruskal-Wallistest followedby

Games-Howellpost-hoctestformultiplecomparisonswasusedto

comparethestudiedgroups.Spearmancorrelationtestwasusedto

evaluate the possible correlation between kaurenoic acid

concentration and genotoxicty or gene transcription. A p-value

lessthan0.05wasconsideredsigniﬁcant[39].

3.Resultsanddiscussion

GCcelllinestreatedwithmethylmethanesulphonatepresented

asigniﬁcantincreaseintheDNAdamageindexwhencompared

withcontrolnon-treatedcells[333.5(13.5)versus25.5(7.5),

p<0.001;Fig.2A].Similarphenomenonwasrepeatedfornormal

cellsMNP01(356versus22.3,p<0.001).Morethan70%ofGCcells

treatedwiththisdrugpresentedcometclass4,whichwasdetected

inlessthan2%ofnon-treatedcells(Fig.3).

Fig.3.EffectofkaurenoicacidonthescoreofDNAdamagebycometassayingastriccancercelllines.TheDNAdamagewasclassifiedbasedonthelengthofmigrationandon theamountofDNAinthecomettail.Class0:nodamage;Class1:littledamagewithataillengthshorterthandiameterofthenucleus;Class2:mediumdamagewithatail lengthoneortwotimesthediameterofthenucleus;Class3:significantdamagewithataillengthgreaterthantwotimesthediameterofthenucleus;Class4:significant damagewithtaillengthgreaterthanthreetimesthediameterofthenucleus;C-:control,whichwerenon-treatedcells;C+:cellstreatedwithmethylmethanesulfonateon* Significantlydifferentfromthenegativecontrol(p<0.05;byKruskal-WallistestfollowedbyGames-Howellpost-hocanalysis).**Significantlydifferentfromthecontrol (p0.001;byKruskal-WallistestfollowedbyGames-Howellpost-hocanalysis).

Fig.4.Genotoxicityofkaurenoicacidbycometassayineachstudiedgastriccancercellline.A)ACP02;B)ACP03;C)AGP01;D)PG100.DI:damageindexbycometassay.C-: control,whichwerenon-treatedcells;C+:cellstreatedwithmethylmethanesulfonateon*Signiﬁcantlydifferentfromthecontrol(p<0.05;byKruskal-Wallistestfollowedby Games-Howellpost-hocanalysis).**Signiﬁcantlydifferentfromthecontrol(p0.001;byKruskal-WallistestfollowedbyGames-Howellpost-hocanalysis).

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Kaurenoic acid induced DNA damage in a dose-dependent

manner(Fig.2).Atconcentrationsabove5

m

g/mL,kaurenoicacid

induceda signiﬁcantincrease in the DNAdamage index when

comparedwithcontrolvalues:47(25.75;p=0.001),47(42.75;

p=0.006),173(69;p<0.001),205(43;p<0.001)for5, 10,30or

60

m

g/mL, respectively (Fig. 2A). Kaurenoic acid induced DNA

damageclassiﬁedascometclass4(Fig.3);however,lessthan30%

ofcellspresentcometclass4.

Moreover, the methyl methanesulphonate treatment also

inducedasigniﬁcantincreaseofmicronucleicomparedwiththe

control[96.5(12)versus20.5(8),p<0.001].Additionally,GC

cell lines presented a signiﬁcant increase of the frequency of

micronuclei when treated with kaurenoic acid concentrations

above 5

m

g/mL: 30.5 (7.25; p=0.004), 37 (8; p<0.001), 53

(19; p<0.001) and 74.5 (15.75; p<0.001), respectively

(Fig.2B).

The kaurenoic acid concentration treatment was strongly

correlatedwiththeDNAdamageindex(rS=0.912,p<0.001)and

themicronucleifrequency(rS=0.925,p<0.001).Asimilarpattern

wasobservedinresponsetothekaurenoicacidtreatmentineach

cellline(Figs.4and5).

Genotoxiceventsareconsideredcrucialstepsinthe

progres-sion of cancer. In this study, we performed comet assay and

micronucleustests in four GC cell lines treated withdifferent

concentrationsofkaurenoicacid.Toourknowledge,noprevious

studyevaluatedtheeffectofthiscompoundinGCcelllines.Here,

we observed that kaurenoic acid has a genotoxic potential

especially at higher concentrations, evidenced by increased

frequenciesofbothmicronucleiandDNAdamage.Kaurenoicacid

inducedDNAdamageinadose-dependentmanner.Ourcurrent

ﬁndings in GCcelllines areinpartinagreementwithﬁndings

previouslyobservedinChinesehamsterlungﬁbroblastcells(V79

cells)treatedwithkaurenoicacid.Therefore,thiscompoundmay

actasaDNAintercalatingagent[31],whichinduceschromosomic

alterationsandDNAbreaksinmammaliancells[40].

Few studies have evaluated the effect of kaurenoic acid.

However, the genotoxic effect of diterpens was previously

reported. Usingthecometassay,Katoet al.[41]showed thata

pimarane-type diterpene, pimaradienoic acid, induced DNA

damage at concentrations of 2.5 and 5.0

m

g/mL in V79 cells.

Moreover,DNAdamagewasalsoreportedinhepatocytesofSwiss

micetreatedwith80mg/kgofthisditerpene.

Paclitaxel(Taxol1₎_is_a_diterpenoid_clinically_effective

antineo-plasticdrugthathasbeenusedforthetreatmentofseveralhuman

cancers,includingGC[42].Thisditerpensuppressthemicrotubule

spindle dynamics, which results in mitosis inhibition and

apoptosis induction [43,44]. Branham et al. [45] previously

described that paclitaxel is able to induce DNA damage in

lymphocytes treatedwith concentrationsof 10

m

gorhigher. In

gastrointestinal cell lines (HT29 and MKN45), 0.01

m

g/mL of

paclitaxelseemstobecytotoxicbyMTTassay

([2-(2-methoxy-4-

nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazo-lium,Monosodiumsalt](WST-8)colorimetricassay)[46].

Themethylmethanesulphonatetreatmentinducedapoptosis

ornecrosis[sub-G1;40.2(4.7)versus1.6(0.8);p<0.001]and

reducedtheGCcellsentering mitosis[G2/M;12.1(1.7)versus

24.1(4.11);p<0.001]inrelationtocontrols.TheGCcellsentering

DNAsynthesisandmitosisdecreasedsigniﬁcantlywithkaurenoic

acidtreatment,alongwithanincreaseinthegrowthphase(G1)

comparedwithcontrols.Inaddition,kaurenoicacidtreatmentwas

able to signiﬁcantly induce apoptosis (or necrosis) even at

concentrations of 2.5

m

g/mL in relation to non-treated cells

Fig.5. Effectofkaurenoicacidonmicronucleifrequencyineachgastriccancercellline.A)ACP02;B)ACP03;C)AGP01;D)PG100.MN:micronucleifrequency.C-:control, whichwerenon-treatedcells;C+:cellstreatedwithmethylmethanesulfonateon*Signiﬁcantlydifferentfromthecontrol(p<0.05;byKruskal-Wallistestfollowedby Games-Howellpost-hocanalysis).**Signiﬁcantlydifferentfromthecontrol(p0.001;byKruskal-WallistestfollowedbyGames-Howellpost-hocanalysis).

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(Table2andFig.6).Asimilarpatternwasobservedinresponseto

thekaurenoicacidtreatmentineachcellline.

TofurtherunderstandtheeffectofkaurenoicacidinGCcells,

weevaluatedthecellcycleprogressionandthetranscriptionof

genes involved, among other functions, in the control of this

process.KaurenoicacidwasabletoinducecellcyclearrestatG1

andinduceapoptosisinadose-dependentmanner,reducingthe

DNA synthesis and mitosis processes. The gene transcription

analysesareinagreementwiththeseﬁndings.

TheGCcelllinespresentedanincreasedtranscriptionofMYC

[fold-change=2.66 (0.42); Fig. 7A], CCND1 [fold-change=1.96

(0.19);Fig.7B],BCL2[fold-change=2.08 (0.21);Fig.7C],and

CASP3[fold-change=1.66(0.10);Fig.7D]inrelationtothe

non-neoplasticMNP01cells that wereusedas a calibratorfor gene

transcription analysis. On the other hand, the GC cell lines

presented a reduced transcription of ATM [fold-change=0.36

(0.14);Fig. 7E], CHK2 [fold-change=0.40 (0.11);Fig. 7F] and

TP53 [fold-change=0.27 (0.11); Fig. 7G] in relationto MNP01

cells.

TheGCcellstreatedwithmethylmethanesulphonatepresented

asigniﬁcantlyreducedtranscriptionofCCND1[1.34(0.11)versus

1.95 (0.19), p<0.001; Fig.7B],BCL2 [1.85 (0.18) versus 2.08

(0.21), p=0.048; Fig. 7C] in relation to non-treated GC cells.

Conversely, CASP3 transcription was increased in the GC cells

treated withmethyl methanesulphonatein relation tocontrols

[1.94(0.15)versus1.66(0.10),p<0.001;Fig.7D].Moreover,we

observed a signiﬁcantly reduced MYC transcription in ACP02,

ACP03, AGP01 and PG100 cell lines treated with methyl

methanesulphonate in relation to non-treated cells (p<0.05),

wheneachcelllinewasevaluatedseparately.Additionally,methyl

methanesulphonate induced a signiﬁcant reduction of ATM

transcriptioninACP02andPG100cells(p<0.05),andanincrease

ofCHK2transcriptioninACP03andPG100cells(p<0.05).

Table 3 shows us how the kaurenoic acid affected the

transcription of genes involvedin the controlof the cellcycle.

MYCtranscriptionwassigniﬁcantlyreducedinGCcelllinestreated

with 30 and 60

m

g/mL of kaurenoic acid compared with

non-treatedcells(Fig.7A).Lowconcentrationsofkaurenoicacidwere

alsoabletoreduceMYCtranscriptioninsomecelllines.Moreover,

GCcelllinestreatedwith10,30and60

m

g/mLofkaurenoicacid

alsopresentedreducedCCND1(Fig.7B),BCL2(Fig.7C),andCASP3

(Fig. 7D) transcription in relation to non-treated cell lines.

Conversely, GC cell lines treated with10, 30 and 60

m

g/mL of

kaurenoicacidpresentedincreasedATM(Fig.7E),CHK2(Fig.7F),

andTP53(Fig.7G)transcriptioninrelationtonon-treatedcelllines.

Inaddition,5

m

g/mLofkaurenoicacidalsoinducedincreasedCHK2

transcriptioninGCcelllinesinrelationtocontrols(Fig.7F).

Moreover, we observed an inverse correlation between

kaurenoic acid concentration treatment and MYC (rS= 0.770,

p<0.001), CCND1 (rS= 0.880, p<0.001), BCL2 (rS= 0.826,

p<0.001) and CASP3 (r_S= 0.828, p<0.001) transcription. On

the other hand, kaurenoic acid concentration treatment was

directly correlated with ATM (rS=0.863, p<0.001), CHK2 (rS=

0.871,p<0.001)andTP53(r_S=0.721,p<0.001)transcription.

Weobservedthat MYC,ATM,CHK2,CCND1,BCL2,CASP3, and

TP53transcriptionslightlyvariedinresponsetothekaurenoicacid

treatmentineachcellline.Inthisanalysis,weﬁrstnormalizedthe

studiedgenestranscriptioninGCcelllinesbytheirtranscriptionin

non-neoplastic cells. As expected, the GC cell lines presented

increasedtranscriptionoftheoncogenesMYC,CCND1andBCL2and

reduced transcription of the ATM, CHK2 and TP53 tumor

suppressors.

TheresponsetokaurenoicacidslightlyvariedamongGCcell

lines,whichwouldbeexpectedconsideringthedifferencesinthe

geneticbackground[26–29].Ingeneral,higherconcentrationsof

thiscompoundinducedlowerMYC,CCND1andBCL2transcription

Table2

Effectofkaurenoicacidoncellcycleprogressionandinductionofapoptosisornecrosis.

Kaurenoicacid(mg/mL) DNAContent(%)

S G2/M G1 Apoptosis/Necrosis

0(Control) 21.5(3.4) 24.1(4.1) 47.1(4.1) 1.6(0.8)

2.5 p=NS p=NS 50.9(4.9)

p=0.021

5.5(4.2)

p<0.001

5 p=NS 17.1(6.1)

p=0.002

52.2(4.8)

p<0.001

7.3(5.6)

p<0.001

10 16.5(3.3)

p<0.001

13.9(5.8)

p<0.001

53.9(5.7)

p<0.001

11.7(9.1)

p<0.001

30 13.8(2.5)

p<0.001

12.3(3.5)

p<0.001

56.9(7.6)

p<0.001

17.8(9.6)

p<0.001

60 12.3(1.8)

p<0.001

11.3(3.6)

p<0.001

p=NS 23.2(9.7)

p<0.001

NS=notstatisticallysigniﬁcant.

Fig.6.Kaurenoicacideffectoncellcycleprogressionofgastriccancercelllines.C-:control,whichwerenon-treatedcells;C+:cellstreatedwithmethylmethanesulfonateon* Signiﬁcantlydifferentfromthecontrol(p<0.05;byKruskal-WallistestfollowedbyGames-Howellpost-hocanalysis).**Signiﬁcantlydifferentfromthecontrol(p0.001;by

Kruskal-WallistestfollowedbyGames-Howellpost-hocanalysis).

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Fig.7.Kaurenoicacideffectongenetranscriptioningastriccancercelllines.A)MYC;B)CCDN1;C)BCL2;D)CASP3;E)ATM;F)CHK2;G)TP53.Thedrugtreatmentsandgene transcription analyses were performed in ACP02, ACP03, AGP01 and PG100 cell lines. C-: control, which were non-treated cells; C+: cells treated with methylmethanesulfonateon*Signiﬁcantlydifferentfromthecontrol(p<0.05;byKruskal-WallistestfollowedbyGames-Howellpost-hocanalysis).**Signiﬁcantly differentfromthecontrol(p0.001;byKruskal-WallistestfollowedbyGames-Howellpost-hocanalysis).

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inGCcelllines,reinforcingthatkaurenoicacidalsoactsinmitosis inhibition.

AlthoughreductionofCASP3wasdetectedinGCcellinrelation

tonon-cancerous cells and withthe kaurenoic acidtreatment,

higherconcentrationofthiscompoundinducedhigherATM,CHK2

and TP53in GC cell lines.These genes present several rolesin

humancells,includingcellcyclearrestandinductionofapoptosis

in response toDNAdamage as observed bycell cycleanalysis.

Considering that the DNA damage also increased with the

kaurenoicacidconcentrationtreatment,ourresultssuggestthat

kaurenoicacidmayalsoactasanapoptosisinducerinresponseto

DNAdamage.Furtherinvestigationsarestillnecessary,however,

kaurenoicacidmaypresentantineoplasticactivityasthediterpene

paclitaxel.

4.Conclusion

OurresultsshowedthatkaurenoicacidobtainedfromCopaifera

inducesDNAdamageandincreasesthemicronucleifrequencyina

dose-dependentmannerinGCcells,withasigniﬁcantgenotoxicity

observed above the concentration of 5

m

g/mL. Moreover, this

compound seems to be able to induce cell cycle arrest and

apoptosisinGCcells.

Conﬂictofinterest

Theauthorsdeclarethatthereisnoconﬂictofinterest.

Acknowledgements

We gratefully acknowledge the ﬁnancial support of the

Conselho NacionaldeDesenvolvimentoCientíﬁcoeTecnológico

(CNPq)andFundaçãodeAmparoàPesquisadoEstadodeSãoPaulo

(FAPESP),forgrantsandfellowshipaward.

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