keratinocyte chemoattractant (kc)/human growth-regulated oncogene (gro) chemokines and...

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Keratinocyte chemoattractant (KC)/human growth-regulated oncogene (GRO) chemokines and pro-inflammatory chemokine networks in mouse andhuman ovarian epithelial cancer cellsDeok-Soo Son, Angelika K. Parl, Valerie Montgomery Rice & Dineo KhabelePublished online: 01 Aug 2007.

To cite this article: Deok-Soo Son, Angelika K. Parl, Valerie Montgomery Rice & Dineo Khabele (2007) Keratinocytechemoattractant (KC)/human growth-regulated oncogene (GRO) chemokines and pro-inflammatory chemokine networks inmouse and human ovarian epithelial cancer cells, Cancer Biology & Therapy, 6:8, 1308-1318, DOI: 10.4161/cbt.6.8.4506

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e1 CancerBiology&Therapy 2007;Vol.6Issue8

Research Paper

Keratinocyte Chemoattractant (KC)/Human Growth-Regulated Oncogene (GRO) Chemokines and Pro-Inflammatory Chemokine Networks in Mouse and Human Ovarian Epithelial Cancer CellsDeok-Soo SonAngelika K. ParlValerie Montgomery RiceDineo Khabele*

Department of Obstetrics and Gynecology; Meharry Medical College; Nashville, Tennessee USA

*Correspondence to: Dineo Khabele; Division of Gynecologic Oncology; Department of Obstetrics and Gynecology; Meharry Medical College; 1005 DB Todd Blvd; Nashville, Tennessee 37208 USA; Tel.: 615.327.6284; Fax: 615. 327-6296; Email: [email protected]

Original manuscript submitted: 05/08/07Revised manuscript submitted: 05/25/07Manuscript accepted: 05/26/07

This manuscript has been published online, prior to printing for Cancer Biology & Therapy, Volume 6, Issue 8. Definitive page numbers have not been assigned. The current citation is: Cancer Biol Ther 2007; 6(8):http://www.landesbioscience.com/journals/cc/abstract.php?id=4506Once the issue is complete and page numbers have been assigned, the citation will change accordingly.

Key woRDS

inflammation, ovarian epithelial cancer,chemokine,pro-inflammatorycytokine

AbbReViAtionS

IL-1β interleukin-1KC keratinocytechemoattractantNFκB nuclearfactor-κBIκB inhibitorofNF-κBGRO growth-regulatedoncogeneLPS lipopolysaccharideTNF tumornecrosisfactor

AcKnowleDgeMentS

We thank Drs. Katherine Roby and PaulTerranova (University of Kansas MedicalCenter, Kansas City, KS) for the ID8 cells.This research was supported in part bythe Clinical Research Center of MeharryMedical College, RCMI Clinical ResearchInfrastructureInitiative,NIHP20RR011792(VMR),theRCMINIHG2RR03032(DK)andtheAmosMedicalFacultyDevelopmentProgram/Robert Wood Johnson Foundation(DK).

[CancerBiology&Therapy6:8,e1-e11,EPUBAheadofPrint:http://www.landesbioscience.com/journals/cbt/abstract.php?id=4506;August2007];©2007LandesBioscience

AbStRActChronic inflammation is an important underlying condition for ovarian tumor develop‑

ment, growth and progression. Since chemokine networks are activated by inflammation, patterns of chemokine gene expression were investigated in ovarian cancer cells. Chemokine specific microarrays were performed after mouse (ID8) and human (SKOV‑3) ovarian surface epithelial cancer cells were exposed to the inflammatory agent bacte‑rial endotoxin lipopolysaccharide (LPS, 10mg/ml) and pro‑inflammatory cytokines interleukin‑1β (IL‑1, 10ng/ml) and tumor necrosis factor‑a (TNF, 10ng/ml). In the mouse ID8 cells, LPS, IL‑1 and TNF led to robust upregulation of keratinocyte chemoattractant (KC) chemokines CXCL1/2, mouse homologues of human growth‑regulated oncogenes (GRO). Other chemokines, interferong inducible protein (IP)‑10 (CXCL10), CCL7 and CCL20 were moderately upregulated. ID8 cells constitutively expressed CXCL16 and CCL2, but only CCL2 expression was enhanced by LPS, IL‑1 and TNF. In the human SKOV‑3 cells, LPS had no effect on chemokines expression due to the absence of the LPS receptor, toll‑like receptor 4 (TLR4). However, IL‑1 and TNF induced GROa/β (CXCL1/2) in human SKOV‑3 cells in a similar manner as observed with mouse ID8 cells. In SKOV‑3 cells, IL‑8 (CXCL8) was highly expressed and other chemokines GROg (CXCL3) and CCL20 were moderately expressed in response to IL‑1 and TNF. The nuclear factor‑κB (NF‑κB) is a known mediator of cytokine and chemokines signaling. The NFκB inhibitor BAY 11‑7082 attenuated expression of inflammatory‑induced chemokines in the mouse and human ovarian cancer cells. Taken together, the results indicate that KC/GRO chemokines are the principal chemokines induced by LPS and pro‑inflammatory cytokines IL‑1 and TNF via NFκB signaling in ovarian surface epithelial cancer cells.

intRoDuctionIncreasing evidence has emerged to indicate a role for chronic inflammation in the

development, growth and progression of malignancies, including ovarian cancer.1-3Ovulationhasseveralsimilaritiestolocalinflammatoryreactions4,5andincessantovula-tionishypothesizedasariskfactorforepithelialovariancancer.3Tissuechemokinesaremediatorsofanumberofbiologicalprocesses,includinginflammation,angiogenesisandcellmigration-someoftheveryfeaturesobservedinmalignanttumors.6Itisknownthatchemokinenetworksregulateinflammationintheovary.7,8However,theprecisecontri-butionsofindividualchemokinesinovarianandtumorgrowthandprogressionarenotasclear.

Itiswell-establishedthatthenuclearfactorκB(NF-κB)playsakeyroleinregulatingautocrine and paracrine signaling between cytokines and chemokines.9 In the mouseovary, NFκB provides a similarly important link between cytokine and chemokinenetworks.8MorerecentlyithasbeenshownthatNFκBiscriticalforcancerdevelopmentandprogressionandcontributestotheupregulationofTNFinendothelialandinflam-matorycells.10,11

Keratinocyte chemoattractant (KC) chemokines [CXCL1/2, mouse homologues ofhuman growth-regulated oncogenes (GRO)] are the most highly induced chemokinesbypro-inflammatorycytokinesinterleukin-1(IL-1)andtumornecrosisfactor-a(TNF)in the mouse ovary.8The human chemokine GRO is significantly expressed at higherlevels intissueandserumfromwomenwithovariancancercomparedtothosewithoutovarian cancer.12 Other chemokines, CXCL8, CXCL12 and the monocyte chemoat-tractantprotein-1(MCP-1orCCL2)havebeenisolatedfromovariancancerascites.13-15Stromalcell-derivedfactor-1(SDF-1orCXCL12) ishighlyexpressedinovariancancercells and promotes ovarian cancer cell growth through CXCR4.16-19 CXCL12 and

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InductionofKC/GROChemokinesinOvarianSurfaceEpithelialCancerCells

vascular endothelial growth factor (VEGF) synergistically induceangiogenesisinhumanovariancancers.20MCP-1ishighlyexpressedinprimaryovariancancersversusbenigncysts21and is reportedtobe the predominant chemokine in tumor epithelium.22 Althoughthese reports demonstrate an association between chemokines andovariancancer,thereislimitedinformationregardingtheregulationofspecificchemokinesinresponsetopro-inflammatorystimuli.

Withthegoaloffurtherunderstandingthecomplexityofchemo-kine signaling inovariancancer, thepresent studywasdesigned toassessinflammation-drivenchemokinesinovariansurfaceepithelialcancer cells, by utilizing chemokine specific microarrays. In thisstudy the inflammatory agent, bacterial endotoxin lipopolysaccha-ride(LPS)andpro-inflammatorycytokinesIL-1andTNFledtotherobustinductionofKC/GROchemokinesthroughNFκBsignalinginmouseandhumanepithelialovariancancercelllines.

MAteRiAlS AnD MethoDSReagents. Recombinant cytokines were obtained as follows:

murine IL-1β andTNF from R&D Systems (Minneapolis, MN),human IL-1β from Cell Science (Canton, MA) and humanTNFfrom Biomol (Plymouth Meeting, PA). The following reagentswere purchased from Sigma (St. Louis, MO): penicillin G/strep-tomycin and lipopolysacharide (LPS). LipofectamineTM, TRIzol®,M-MLV,Taq DNA polymerase and all liquid culture media wereacquired fromInvitrogen (Grand Island,NY).Antisense and sense

oligonucleotides for chemokines were obtained from IntegratedDNATechnologies(Coralville,IA).TheOligoGEArray®microar-rays for mouse (OMM-022) and human chemokines (OHS-022)and the TrueLabeling-AMP™ Linear RNA Amplication Kitcame from SuperArray (Frederick, MD). The phospho-IκB(Ser 32/36) and IκB antibodies from Cell Signaling Technology(Beverly,MA)andβ-actinantibodyfromSantaCruzBiotechnology(SantaCruz,CA)wereacquired.TheNF-κBinhibitorBAY11-7082waspurchased fromBiomol (PlymouthMeeting,PA). IκBexpres-sionandpNF-κB-lucvectorsfromBDBiosciences(PaloAlto,CA)andtheLuciferase®ReporterAssaySystemfromPromega(Madison,WI)wereacquired.ChemiluminescentdetectionkitscamefromGEHealthcare(Piscataway,NJ).

Cell culture and treatment.Themouseovariansurfaceepithelialcancercell line(ID8)waskindlyprovidedbyDrs.KatherineRobyand PaulTerranova (University of Kansas Medical Center, KansasCity, KS) and the human ovarian cancer cell lines SKOV-3 andOVCAR-3andhumancolonadenocarcinomacelllineSW480werepurchased from the AmericanType Culture Collection (Manassas,VA).Cells (approximately5x104 cells/ml)were cultured at37˚Cinawater-saturatedatmosphereof95%airand5%CO2in24-or6-wellplateswithappropriatemedia:4%FBScontainingDulbecco’sModified Eagles Medium (DMEM) medium supplemented withpenicillin (100 U/ml)/streptomycin (100 U/ml) for ID8 cells,10%FBScontainingMcCoy’s5Amediumwithpenicillin/strepto-mycinforSKOV-3cells,20%FBScontainingRPMImediumwithpenicillin/streptomycinforOVCAR-3cellsand10%FBScontainingRPMImediumwithpenicillin/streptomycinforSW480cells.Afterovernight culture to allow cellular attachment to the plates, themedium was removed and fresh medium was added. TreatmentswiththevariousagentsaredescribedindetailinResults.

Microarray.TotalRNAwasextractedutilizingTRIzol®reagentaccordingtothemanufacturer’sinstructions.ThecDNAtemplatewasreversetranscribedfromtotalRNAusingtheTrueLabeling-AMP™LinearRNAAmplicationKit.Invitro-transcriptionandbiotin-16-labeling of the cRNA target were produced from the cDNA andsubjectedtoOligoGEArray®microarrays.Hybridizationanddetec-tion were performed as follows: prehybridization for 2 h at 60˚C,hybridization overnight at 60˚C, washing and exposure on X-rayfilm.The signal intensities were quantified by densitometry usingQuantityOne(Biorad,Hercules,CA).Absolutevalueswereobtainedafterdeterminingtheaveragedensityofeachspot, thebackgroundwith minimum value and the interquartile ranges. Target signalsabove 150 (lowest detectable) were chosen for further analysis.Finally,thevaluesfromthetreatedcellswerecomparedtothevaluesfromthevehiclecontrolcells.

RT‑PCR.TotalRNAwasisolatedusingTRIzol®reagent.TheRTreactionconditions,usingrandomprimerswithreversetranscriptaseM-MLV, were at 42˚C for 60 min followed by 94˚C for 10 min.Specificprimersformouseandhumanchemokinesweredesignedas(Table1).L19wasusedasaloadingcontrolofmousechemokines.8PCRwasperformedunderthefollowingconditions:denaturationat94˚Cfor1min,annealingat58˚Cfor1minandextensionat74˚Cfor 1 min with 25 cycles. Amplified PCR products were analyzedby electrophoresis in 2% agarose gels containing 1 µg ethidiumbromide/ml.Thefluorescent imageswerephotographedunderUVlight.

Western blot. Cell lysates were prepared, fractionated onSDS-polyacrylamidegelsandtransferredtonitrocellulosemembranesaccordingtoestablishedprocedures.Blockingofnonspecificproteins

Figure 1. Identification of chemokines induced by LPS in ID8 cells. (A) LPS‑induced chemokines by microarray. ID8 cells were incubated with vehicle (Control) or LPS (10 mg/ml) for 1 h. After isolating total RNAs, biotin‑16‑labeling of the cRNA target was produced from the cDNA template transcribed reversely from total RNA. Upper and lower arrow heads indicate CXCL1 and CXCL2, respectively. (B) Time course of LPS‑induced chemokines observed by microarray were confirmed by RT‑PCR. ID8 cells were incu‑bated with LPS (10 mg/ml) for 0, 0.5, 1, 2, 3 and 6 h. After isolating total RNAs, RT‑PCR was performed using specific primers of each chemokine with 25 cycles. L19 was used as an internal housekeeping gene. M = molecular marker in base pair. Experiments were performed in duplicate and a repre‑sentative result is shown.

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wasperformedbyincubationofthemembraneswith5%nonfatdrymilk in Tris buffered saline Tween-20 (TBST containing 10 mMTris,150mMphosphatebufferedsaline,0.05%Tween20,pH8.0)for 2 h at room temperature. Blots were incubated with primaryantibodies at 1:1,000 dilution in blocking solution overnight at4˚C.The membranes were washed three times withTBST for 10minandfollowedbyincubationfor1hwithhorseradishperoxidaseconjugatedsecondaryantibodyaccordingtoprimaryantibodyusedat 1:2,500 in 5% milk/TBST. The membranes were then rinsedthree times withTBST for 10 min and the bands were visualizedbyenhancedchemiluminescence.Aftermembrane stripping for10minwithmethanolcontaining3%H2O2,β-actinwasdetectedasaninternalloadingcontrolforthecelllysates.

Transient transfection and luciferase assays. SKOV-3 ovariancancercellsatapproximately50%confluencyin24-wellplateswerewashedoncewithfreshmediawithoutadditivesandweretransientlytransfected with IκB expression or pNF-κB-luc vectors for 3 h at37˚C using Lipofectamine solution. Transfected cells were treatedas outlined in Results and incubated for 6 h. After rinsing cellswithice-coldPBSandaddinglysisbuffer(Promega,Madison,WI),cell lysates were used for determination of luciferase activity usinga microplate luminometer. Luciferase activity, expressed as relativelightunits,wasnormalizedtomeasuredproteinlevels.

Statistics.Datawere analyzedby thepaired student’s t-test andone-wayanalysisofvariance (ANOVA)asappropriate. If statisticalsignificance (p≤0.05)wasdeterminedbyANOVA, thedatawerefurther analyzedbyTukey’spairwise comparisons todetect specificdifferencesbetweentreatments.

ReSultSKC chemokines (mouse homologues of GRO) were the main

chemokines induced by the pro‑inflammatory stimuli LPS, TNF and IL‑1 in mouse ID8 cells. A microarray containing genes thatencode mouse chemokines was employed to assess chemokineexpressioninducedbyLPS,TNFandIL-1inID8cells.Thenomen-clature approved by IUIS/WHO Subcommittee on ChemokineNomenclaturewasusedinthepresentstudy.23Basedonthenumberof amino acids between the first cysteine motif, chemokines havebeenclassifiedintofoursubfamilies:C,CC,CXCandCX3C.24

ID8cellswerefirsttreatedwithLPS,becauseLPSinducesinflam-matoryeventsintheovary8andbecauseLPSisrelatedtometastaticgrowthoftumorsinamurinemodel.25Theresultsshowthatafter1h,LPSinducedthehighestlevelsofexpressionofKCchemokines(CXCL1/2,mousehomologuesofhumanGRO).Moderateexpres-sionofinterferonginducibleprotein(IP)-10(CXCL10),CCL7andCCL20 (Fig. 1 andTable 2) were observed. CXCL16 and CCL2were the constitutive chemokines detectable in ID8 cells. LPS didnotaffectCXCL16expressionwhereasitenhancedCCL2(Fig.1andTable2).TofurtherdelineatetheresponseofchemokinestoLPS,atime-courseofchemokinesinducedbyLPSwasinvestigatedbyusingRT-PCR.LPSincreasedchemokinesinID8cellstomaximumlevelswithin1-2hafterexposureandthenthe levelsdecreasedgradually(Fig.1B).ID8cellshaveconstitutiveexpressionoftheLPSreceptor,TLR4,whichremainedunchangedbyLPStreatment(Fig.1B).

ID8cellswerethentreatedwiththeIL-1andTNFat1handat6htodeterminetheeffectsofcytokine-inductionofchemokinesinID8cells.AsobservedwithLPStreatment(Fig.1),IL-1andTNFprimarilyinducedKCchemokines(CXCL1/2),withmoderateinduc-tionofCXCL10,CCL7andCCL20(Fig.2andTable2).SimilarlytoLPStreatment,theconstitutivelyactiveCCL2wasenhancedbyIL-1andTNF(Fig.2andTable2).Additionaltime-pointswereevaluatedbyRT-PCR.IL-1andTNFinducedKCchemokinesandCCL20to

Table 1. PrimersandanticipatedproductsizeusedintheRT‑PCRanalysis.

chemokines Accession PcR Primers Product Size (bp) number Mouse mCXCL1 NM_008176 5’‑ATG ATC CCA GCC ACC CGC TCG CTT‑3’ (sense) 5’‑CCG TTA CTT GGG GAC ACC TTT TAG CAT C‑3’ (antisense) 294 mCXCL2 NM_009140 5’‑ATG GCC CCT CCC ACC TGC C‑3’ (sense) 5’‑GGG CTT CAG GGT CAA GGC A‑3’ (antisense) 250 mCXCL10 NM_021274 5’‑ATG AAC CCA AGT GCT GCC GTC ATT‑3’ (sense) 5’‑AGG AGC CCT TTT AGA CCT TT‑3’ (antisense) 294 mCCL2 NM_011333 5’‑ATG CAG GTC CCT GTC ATG CTT‑3’ (sense) 5’‑GTT CAC TGT CAC ACT GGT CAC‑3’ (antisense) 444 mCCL7 NM_013654 5’‑ATG AGG ATC TCT GCC ACG CTT‑3’ (sense) 5’‑TCA AGG CTT TGG AGT TGG G‑3’ (antisense) 294 mCCL20 NM_016960 5’‑ATG GCC TGC GGT GGC AAG CGT‑3’ (sense) 5’‑CAT CTT CTT GAC TCT TAG GC‑3’ (antisense) 291 mTLR4 NM_021297 5’‑GCA GTG GGT CAA GGA ACA G‑3’ (sense) 5’‑TCA CCC AGT CCT CAT TCT G‑3’ (antisense) 296Human hCXCL1 NM_001511 5’‑ATG GCC CGC GCT GCT CTC TC‑3’ (sense) 5’‑GTT GGA TTT GTC ACT GTT CAG C‑3’ (antisense) 321 hCXCL2 NM_002089 5’‑CGC CCA AAC CGA AGT CAT A‑3’ (sense) 5’‑TCA GTT GGA TTT GCC ATT T‑3’ (antisense) 118 hCXCL3 NM_002090 5’‑ATG GCC CAC GCC ACG CTC TC‑3’ (sense) 5’‑GTT GGT GCT CCC CTT GTT CAG‑3’ (antisense) 321 hCXCL8 NM_000584 5’‑AAG AGC CAG GAA GAA ACC ACC‑3’ (sense) 5’‑ATT GCA TCT GGC AAC CCT ACA‑3’ (antisense) 466 hCCL15 NM_004167 5’‑ATG AAG GTC TCC GTG GCT GC‑3’ (sense) 5’‑TAT TGA GTA GGG CTT CAG GA‑3’ (antisense) 339 hCCL20 NM_004591 5’‑ATG TGC TGT ACC AAG AGT TTG C‑3’ (sense) 5’‑TAC TGA GGA GAC GCA CAA T‑3’ (antisense) 271 hTLR4 NM_138554 5’‑CCT TCA GAT AAG CAG GGC ATG‑3’ (sense) 5’‑TGG ATG AAG TGC TGG GAC AC‑3’ (antisense) 395 β‑actin NM_001101 5’‑ACC TTC AAC ACC CCA GCC ATG TAC G‑3’ (sense) 5’‑ATG CCC AGG AAG GAA GGC TGG AAG‑3’ (antisense) 4

mCXCL: mouse CXCL chemokine, mCCL: mouse CCL chemokine, mTLR-4: mouse toll-like receptor 4, hCXCL: human CXCL chemokine, hCCL: human CCL chemokine, hTLR4: human toll-like receptor 4.

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Table 2. EffectsofLPS,IL‑1andTNFonchemokinesinID8cellsasdeterminedbymicroarray (Unit:expressionsignalonmicroarray).

The nomenclature is that approved by the IUIS/WHO Subcommittee on Chemokine Nomenclature. ID8 ovarian cancer cells were cultured in the presence of vehicle (control), LPS (10 mg/ml), IL-1β (10 ng/ml) or TNF (10 ng/ml) for 1 h and 6 h. Expression signals were normalized to background intensity. Target signal (lowest detectable) = 150. Underline indicates the presence of detectable mRNA. ? = Unknown. Where common name is not available, systemic name is alternated.

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maximumlevelswithin1handthenthelevelsdecreasedgradually(Fig. 2B). Both cytokines enhanced constitutive CCL2 expression,whichwasmaintainedat6haftertreatment(Fig.2BandTable2).IL-1inducedCXCL10andCCL20tomaximumlevelswithin1handthenthelevelsdecreasedgradually(Fig.2B).Ontheotherhand,TNFmaintainedtheinducedconcentrationsofCXCL10at6haftertreatment(Fig.2BandTable2).

GRO chemokines were the principal chemokines induced by pro‑inflammatory cytokines IL‑1 and TNF, whereas LPS had no effect on chemokine expression in human SKOV‑3 ovarian cancer cells. Inorder todetermine if the effectsofLPSand cytokinesonchemokines expression was similar in human surface epithelialovariancancercells,ahumanoligonucleotidechemokinearraywasemployed.The results show thatLPShadno effecton chemokineexpression,mostlikelybecauseoftheabsenceofTLR4inSKOV-3cells(Fig.3B).Ontheotherhand,IL-1andTNFincreasedGROa/β(CXCL1/2)inasimilarfashiontowhatwasobservedinthemouseID8cells (Fig.3andTable3). Interestingly,GROa (CXCL1)was

constitutivelyexpressedinSKOV-3cellsandwasabun-dantlyenhancedbybothIL-1andTNF.IL-8(CXCL8)wasalsohighlyexpressedinSKOV-3cells,butwasnotobserved in ID8 cells since there is no known mousehomologforIL-8.TherewasmoderateupregulationofGROg (CXCL3) andCCL20.Constitutive expressionofCCL15 thatdidnot changewith exposure to IL-1andTNFwasobserved.

NFκB was found to be a key regulator of the induction of specific chemokines in ovarian cancer cells. It isknownthatNF-κBregulates the inflamma-tory response. Therefore, the involvement of NF-κBsignaling on the induction of the particular chemo-kinesthatwereupregulatedbyLPS,IL-1andTNFinovariancancercellswasinvestigated.Theexpressionofphosphorylation of IκB (p-IκB), a NF-κB regulatoryprotein was evaluated. LPS treatment induced p-IκBexpressioninID8cells,within15minofexposure,toamaximum level of expression at 30min.Expressionlevels gradually decreased, but remained present at120 min (Fig. 4A). At the same time, total IκBwas reduced, consistent with protein degradation. Toverify the role of NF-κB in LPS-induced chemo-kine expression, ID8 cells were treated with BAY11-7082, a NF-KB inhibitor. Pretreatment with BAY11-7082blockedcytokine-inducedIκBandattenuatedthe expression of the LPS-induced chemokine genes(Fig.4BandC).

As expected, IL-1 and TNF increased p-IκB inSKOV-3cells.Theinductionofp-IκBoccurredwithin5minofexposuretobothcytokines.TotalIκBproteinwasreducedat5–60minafterIL-1andat5-120minafterTNF(Fig.5A).Therewasnoalterationinlevelsofp-IκBafterLPS,againlikelyduetothelackofTLR4inSKOV-3cells(Fig.5A).

Further evaluation of the NFκB signal transduc-tion pathway was assessed by investigating the effectsofLPS,IL-1andTNFonNFκB(-GGGAATTTCC-)promoteractivity.IL-1andTNFincreasedtheluciferaseactivity of pNF-κB-Luc in SKOV-3 cells (Fig. 5B).OverexpressionofIκBinhibitedbasalandpro-inflamma-torycytokine-inducedluciferaseactivityofpNF-κB-Luc

(Fig.4B).Asexpected,LPShadnoeffectonthe luciferaseactivityof pNF-κB-Luc (Fig. 5B). Pretreatment of BAY 11-7082, NFκBinhibitor, inhibited pro-inflammatory cytokine-induced luciferaseactivity of pNF-κB-Luc in a dose-dependent manner (Fig. 5C).In addition, BAY 11-7082 blocked the induction of p-IκB byIL-1 andTNF and restored the reduction of total IκB (Fig. 5D).BlockageofNFκBsignalingwithBAY11-7082attenuatedexpres-sionofchemokinegenesthathadbeenupregulatedbyIL-1andTNF(Fig.5E).

Specific κB Sites in the Promoter Regions of the Chemokines Appear to be Critical for the Response to Pro‑Inflammatory Stimuli. To further evaluate the role of NFκB in the regula-tion of chemokines, κB sites (5'-GGGRNNYYCC-3'; R, purine;Y,pyrimidine;N,anynucleotide)intheenhancerregionsofspecificchemokinesinducedbypro-inflammatorystimuliwerecollectedbyperformingasearchoftheEntrezNucleotidedatabaseandPubMed.Using the obtained sequences of chemokine promoters, in silicopredictionofκBbindingsiteswasperformedbasedontheliterature

Figure 2. Identification of chemokines induced by IL‑1 and TNF in ID8 cells. (A) IL‑1‑ and TNF‑induced chemokines by microarray. ID8 cells were incubated with vehicle (Control) or IL‑1 (10 ng/ml) or TNF (10 ng/ml) for 1 and 6 h. After isolating total RNAs, biotin‑16‑ labeling of the cRNA target was produced from the cDNA template transcribed reversely from total RNA. Upper and lower arrow heads indicate CXCL1 and CXCL2, respec‑tively. (B) Time course of IL‑1‑ and TNF‑induced chemokines observed by microarray were confirmed by RT‑PCR. ID8 cells were incubated with IL‑1 (10 ng/ml) or TNF (10 ng/ml) for 0, 0.5, 1, 2, 3 and 6 h. After isolating total RNAs, RT‑PCR was performed using specific primers of each chemokine with 25 cycles. L19 was used as an internal housekeeping gene. M = molecular marker in base pair. Experiments were performed in duplicate and a representative result is shown.

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Table 3. EffectsofLPS,IL‑1andTNFonchemokinesinSKOV‑3cellsasdeterminedbymicroarray. (Unit:expressionsignalonmicroarray)

The nomenclature is that approved by the IUIS/WHO Subcommittee on Chemokine Nomenclature. SKOV-3 cells were cultured in the presence of vehicle (control), LPS (10 mg/ml), IL-1β (10 ng/ml) or TNF (10 ng/ml) for 1 h. Expression signals were normalized to background. Target signal (lowest detectable) = 150. Underline indicates the presence of detectable mRNA. ? = Unknown. Where common name is not available, systemic name is alternated.

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searchandtheuseofMatchTM(publicversion1.0),asearchprogramfor transcription factor binding sites provided by Weill MedicalCollegeofCornellUniversity (NewYork,NY). In themouse,KCchemokine CXCL1 (mCXCL1) contains three κB sites; whereastheKCchemokineCXCL2harborsoneproximalκBsite(Table4).In thehuman,homologousGROchemokines (hCXCL1,CXCL2,hCXCL3)andIL-8(hCXCL8)shareaconservedproximalκBsite.TheproximalsitesappeartobeessentialforgeneactivationandthesecytokineswererobustlyactivatedbyinflammatorystimuliwithLPS,IL-1andTNF.

The chemokines hCXCL3, mCXCL10 and CCL20 have aproximal κB site that is essential for their activation and weremoderately induced by inflammation. Although regulation ofmCCL7 is unknown, it holds a distal κB site the gene and wasmoderatelyupregulatedbyLPS, IL-1 andTNF in ID8cells (Figs.

1B, 2B and 4C). Interestingly, the mouse mCCL2wasconstitutivelyexpressedgenethatwasinducedbyinflammation(Figs.1and2).AlthoughthemCCL2gene has a nonfunctional proximal κB site, it hascontainstwodistalκBsitesthatarecriticalforactiva-tion.

DiScuSSionOvarian cancer is emerging as an inflamma-

tion-driven condition that involves activationof complex cytokine and chemokine networks.1-3Althoughthereareseveralreportsthatdocumenttheexpression of chemokines such as GRO,12 IL-8,14CCL2,21andCXCL1215inovariancancer,verylittleisknownaboutthepatternsofchemokineexpressioninovarianepithelialcancercells,particularlyindirectresponsetoinflammatorystimuli.Inthisreport,geneexpression patterns of inflammation-driven chemo-kines in mouse ID8 and human SKOV-3 ovariancancercellswereevaluatedandcategorizedas1)highlyexpressed, 2) moderately expressed and 3) constitu-tivelyexpressedchemokines.

The main findings in the present study were therobust and rapid upregulation of GRO/KC chemo-kines (CXCL1 and 2) in the mouse and humanovariancancercellsinresponsetoinflammation(Figs.3and4,andTables2and3).Arecentstudyshowsthatinflammation mediated by cytokines such as TNFin the microenvironment of the ovary, leads to theincreased production of chemokines and cytokinesthat promote peritoneal spread of ovarian cancer ina xenograftmodel.26Recent studies suggestbacterialinfectionsandanLPS-likeresponseprovideapossiblelink between inflammation and carcinogenesis, viaTLR4 signal transduction in ovarian cancer cells.27Ovarian cancer cells and tissues require TLR4 andits downstreamadaptorprotein for signaling. In thisstudy, LPS activated a chemokine response in ID8cells, but did not activate a chemokine response inSKOV-3 cells, due to the lack ofTLR4 in SKOV-3cells.ItispossiblethattheinflammatoryresponsemaybeenhancedinovariancancersthathaveconstitutiveTLR4expression.

KC chemokine has been shown to promote cellproliferation,28 cell invasion,29 tumor formation30 and angio-genesis.31GROchemokine is vital for cell survival and themalig-nant transformationofhumanovarianepithelialcells.12Aprimarybiological functionofKCchemokine isregulating leukocytetrafficincluding migration.32-34 The increased expression of KC/GROwithin ovarian cancer cells may be associated with the influxof leukocytes into the tumor microenvironment. The KC/GROchemokinescanimpactdynamiceventsthatoccurinthegrowthanddevelopmentovariancancer,includingincreasedperitonealdissemi-nationofcancercellsandthus,maycontributetothelinkbetweeninflammatoryreactionsandtumorgrowthandprogression.

IL-8 (CXCL8) was also abundantly expressed after exposureto IL-1 andTNF in SKOV-3 cells (Fig. 3A and B andTable 3).There is no mouse homolog for IL-8, but IL-8 is strongly impli-cated in ovarian cancer growth and progression.35 IL-8 blocks the

Figure 3. Identification of chemokines induced by LPS, IL‑1 and TNF in SKOV‑3 cells. (A) LPS‑, IL‑1‑ and TNF‑induced chemokines by microarray. SKOV‑3 cells were incubated with vehicle (Control) or LPS (10 mg/ml) or IL‑1 (10 ng/ml) or TNF (10 ng/ml) for 1 h. After isolating total RNAs, biotin‑16‑labeling of the cRNA target was produced from the cDNA template transcribed reversely from total RNA. Upper, middle and lower arrow heads indicate strong spots for CXCL1, CXCL2 and CXCL8, respectively. (B) LPS‑, IL‑1‑ and TNF‑induced chemo‑kines observed by microarray were confirmed by RT‑PCR. SKOV‑3 cells were incubated with LPS (10 mg/ml) or IL‑1 (10 ng/ml) or TNF (10 ng/ml) for 1 h. After isolating total RNAs, RT‑PCR was performed using specific primers of each chemokine with 35 cycles. β‑actin was used as an internal housekeeping gene. M = molecular marker in base pair; SW = SW480 human colon adenocarcinoma; SK = SKOV‑3 human ovarian carcinoma cells; OV = OVCAR‑3 human ovarian carcinoma cells. Experiments were performed in duplicate and a representative result is shown.

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TRAIL-inducedapoptosisinovariancarci-nomaOVCAR3cells36 and IL-8 increasescellular invasivenessthroughmatrixmetal-loproteinase7.37

The chemokine expression pattern inresponse to inflammation was rapid inovariancancercells (Figs.1Band3B)andwassimilartothatofimmediate-earlygeneexpression in response to inflammation inthe normal mouse ovary.8 Knowing thatNFκB is important in regulating inflam-matory pathway and that chemokines aretarget genes of NFκB signal transduc-tion,38 the roleofNFκB in the inductionof specific chemokines in ovarian cancerwas investigated. The results demon-strate that NFκB signaling is essentialto the rapid induction of chemokines inmouse and human ovarian cancer cells(Figs. 4 and 5,Table 4). LPS upregulatedKC (CXCL1/2) via transcriptional activa-tionofNFκBsignalinginTLR4expressingID8 cells (Figs. 1 and 4 and Table 2).On the other hand, SKOV-3 cells do notexpressTLR4,resultinginthelackofLPSresponsiveness(Figs.3and5).Theimpor-tance of κB sites in induced chemokinepromotersstrengthensthelinkforinvolve-mentofNFκBsignaling inovariancancercells since LPS, IL-1 and TNF inducedp-IκB (Figs. 4 and 5) Overexpression ofIκB and treatment with the NFκB inhib-itorBAY11-7082blockedNFκBpromoteractivity (Fig. 5). The fact that the NFκBinhibitor led to a decrease in inflamma-tion-induced chemokine mRNAs (Figs. 4and5)confirmstheroleofNFκBinregu-

lating chemokine expression. Further evidencethatNFκBsignalingisessentialtotheresponseofthehighlyexpressedchemokinesistheiden-tification of a conserved proximal κB site inKC/GROchemokinesandIL-8(Table4).

Table 4. TheκBsitesinpromotersofchemokinesinducedbypro‑inflammatory cytokinesinID8andSKOV‑3cells.

Figure 4. Involvement of NFκB signaling in regulat‑ing LPS‑induced chemokines in ID8 cells. (A) Effects of LPS on IκB activation. ID8 cells were incubated with LPS (10 mg/ml) for 0, 5, 15, 30, 60 and 120 min. (B) Effects of BAY 11‑7082, NFκB inhibitor, on LPS‑activated NFκB signaling. ID8 cells were pretreat‑ed with BAY 11‑7082 for 30 min and then incubated with LPS (10 mg/ml) for 30 min. After isolating total proteins, Western blots were performed using anti‑bodies for phospho‑IκB, IκB and β‑actin. As a loading control, β‑actin was used. (C) Effects of BAY 11‑7082 on LPS‑induced chemokines. ID8 cells were pretreated with BAY 11‑7082 (50 mM) for 30 min and then incu‑bated with LPS (10 mg/ml) for 2 h. After isolating total RNAs, RT‑PCR was performed using specific primers for each chemokine with 25 cycles. The L19 was used as a loading control. First lane = molecular marker in base pair. Experiments were performed in duplicate and a representative result is shown.

mCXCL: mouse CXCL chemokine, mCCL: mouse CCL chemokine, hCXCL: human CXCL chemokine, hCCL: human CCL chemokine. ?, Unknown.

In silico prediction of κB binding site was performed based on combination of literature and MatchTM (public version 1.0), a search program for transcription factor binding sites provided by Weill Medical College of Cornell University (New York, NY).

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The moderately induced chemo-kines were identified in response toinflammation: CXCL10, CCL7 andCCL20inID8cellsandCXCL3andCCL20 inSKOV-3 cells (Figs. 2 and3 and Tables 2 and 3). The mousehomologue of human CXCL3 is stillunknown. Interestingly CXCL10and CCL7 were induced after LPS,IL-1 or TNF in ID8 cells whereasSKOV-3cellsdidnotrespond(Tables2 and 3). The promoter region ofCXCL10 between mouse and humanis similar to conserving the inter-feron-stimulated response element(GGAAAGTGAAACCTA) and twoNFκB binding elements.39 MouseCXCL10promotercontainsAP-1site(-85TGAGTCA-78)thatislackinginthe human CXCL10.39 It is knownthat inflammation can activate AP-1.As previously described, TNF caninducec-JUNinmouseovariangranu-losa cells40 and AP-1 requires c-JUNactivation, a transcription factordownstreamofNFκB.11Interestingly,TNFmaintainedtheconcentrationsofCXCL10until6hincontrasttoLPSandIL-1(Fig.2),indicatingadifferen-tialmechanisminCXCL10induction.Thus,theregulationofCXCL10isanareaforfutureinvestigation.AlthoughCCL7promotercontainsonedistalκBsite(Table4),theregulationofCCL7was previously not known. Here forthefirsttimeitisshownthatCCL7ismoderatelyupregulatedbyLPS,TNFandIL-1inID8cells,likelyviaNFκB.CCL20expressionappearstobeunderthe direct control of NFκB (Table 4)through cooperation between essen-tialproximalandsupportivedistalκBsites.41

Constitutively active chemokineswere differently expressed betweenmouse ID8 and human SKOV-3ovarian cancer cells. CXCL16 andCCL2wereconstitutivelyexpressedinID8 cells. GRO/CXCL1 and CCL15weredetectableinSKOV-3cells(Tables2and3).AlthoughaproximalκBsiteappearstobecriticalfortheregulationofotherinduciblechemokines,CCL2has two functionaldistalκBsitesandonenonfunctionalproximalsite(Table4)andstillwasupregulatedinresponseto LPS, IL-1 and TNF in ID8 cells.There was no significant change inthe constitutive expression of CCL15inSKOV-3cells (Fig.4andTable3).

Figure 5. Involvement of NFκB signaling in regulating LPS‑, IL‑1‑ and TNF‑induced chemokines in SKOV‑3 cells. (A) Effects of LPS, IL‑1 and TNF on IκB activation. SKOV‑3 cells were incubated with LPS (10 mg/ml) or IL‑1 (10 ng/ml) or TNF (10 ng/ml) for 0, 5, 15, 30, 60 and 120 min. (B) Effects of IκB expression vector on IL‑1‑ and TNF‑induced NFκB promoter activity. SKOV‑3 cells were cotransfected for 3 h with the pNF‑κB‑luc vector (500 ng/ml) and either empty (100 ng/ml) or IκB (100 ng/ml) expression vectors. Where indicated cells were incubated with or without LPS (10 mg/ml) or IL‑1 (10 ng/ml) or TNF (10 ng/ml) for 6 h. (C) Effects of BAY 11‑7082 on IL‑1‑ and TNF‑induced NFκB promoter activity. SKOV‑3 cells were transfected for 3 h with the pNF‑κB‑luc vector (500 ng/ml). Where indicated cells were pretreated with vehicle (Control) or BAY 11‑7082 (10 and 25 mM) for 30 min and then were incubated with or without LPS (10 mg/ml) or IL‑1 (10 ng/ml) or TNF (10 ng/ml) for 6 h. The luciferase activity was normalized to total protein concentrations and expressed as a fold change by comparison to the control. A gray bar indicates significant increases (P ≤ 0.05) when compared with control as calculated by the paired Student’s t‑test. A dark bar indicates significant decrease (P ≤ 0.05) when compared with IL‑1 or TNF treatment as calculated by the paired Student’s t‑test. LUC = luciferase. Experiments were performed in triplicate and all data are shown as mean ± SE. (D) Effects of BAY 11‑7082 on LPS‑, IL‑1‑ and TNF‑activated NFκB signaling. SKOV‑3 cells were pretreated with BAY 11‑7082 (50 mM) for 30 min and then incubated with LPS (10 mg/ml) or IL‑1 (10 ng/ml) or TNF (10 ng/ml) for 5 min. After isolating total proteins, Western blots were performed using antibodies for phospho‑IκB, IκB and β‑actin. As a loading control, β‑actin was used. (E) Effects of BAY 11‑7082 on IL‑1‑ and TNF‑induced chemokines. SKOV‑3 cells were pretreated with BAY 11‑7082 for 30 min and then incubated with IL‑1 (10 ng/ml) or TNF (10 ng/ml) for 1 h. After isolating total RNAs, RT‑PCR was performed using specific primers for each chemokine with 35 cycles. First lane = molecular marker in base pair. Experiments were performed in duplicate and a representative result is shown.

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However, GRO/CXCL1 had basal levels of expression in SKOV-3cells and was abundantly enhanced by IL-1 andTNF (Fig. 3 andTable3).DifferentfromthemouseKCchemokineCXCL1,whichhas3κBsites,thehumanGRO/CXCL1promoterhasoneproximalκBsite.InadditiontotheκBsite,theGRO/CXCL1promoterhasupstreamregionsthatcontainC/EBP,42HMG(I)YandSp130,43thatlikelycontributetothebasalexpressionofGRO/CXCL1.

In summary, the results indicate that inflammatory stimuliprimarily induce KC/GRO chemokines in ovarian cancer cells viaNFκB signaling pathway. Thus, KC/GRO chemokines contributeto a pro-inflammatory microenvironment that is conducive totumorcellproliferationandinvasionandforthesereasonsKC/GROchemokinesarepotentialtargetsfortherapeuticintervention.

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