Edinburgh Research Explorer The selective -2 inhibitor mavacoxib (Trocoxcil™) exerts anti-tumour effects in-vitro independent of cyclooxygenase-2 expression levels

Citation for published version: Hurst, EA, Pang, LY & Argyle, DJ 2019, 'The selective cyclooxygenase-2 inhibitor mavacoxib (Trocoxcil™) exerts anti-tumour effects in-vitro independent of cyclooxygenase-2 expression levels', Veterinary and Comparative Oncology. https://doi.org/10.1111/vco.12470

Digital Object Identifier (DOI): 10.1111/vco.12470

Link: Link to publication record in Edinburgh Research Explorer

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Published In: Veterinary and Comparative Oncology

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Download date: 05. Oct. 2021 This articleisprotected bycopyright.Allrightsreserved. apoptosisand induce inhibit migrationthe ofthesecells. Interestingly, we establish that osteosarcoma,and canine mammary and bladder cell carcinoma that itlines; can cancer Acceptedmavacoxib. inhibitor We demonstrate here that mavacoxib to of cytotoxic apanel is human we aim effects COX-2 the anti-tumourigenic toestablish of selective, long-acting the isepidemiologicaland there a clear link between thisand cancerNSAID risk.In study use to dueavailablean attractive(NSAIDs), readily anti-inflammatory option non-steroidal drugs is inanumber incancer elevated oftumourshumansand canines. ofboth COX-2 Targeting E2(PGE Abstract scholarship.Edinburgh Article bladdercanine transitionalcellcarcinoma lines. study cell of This wasfunded byaUniversity wouldThe author like to Dr thank Deborah KnappofPurdue University forkindly the gifting

article as doi: 10. differencesbetween version lead to this may been through the copyediting, typesetting, pagination andproofreading process, which This article hasbeen accepted for publication andundergone full peer review but has not The selective cyclooxygenase The Roslin Institute and Royal (Dick) School ofVeterinaryRoyal (Dick) InstituteandStudies, School The Roslin TheUniversity of tumour effects The inducible inflammatory enzyme cyclooxygenase-2 (COX-2) and its (COX-2)and The inducibleproduct inflammatory enzyme cyclooxygenase-2 Corresponding author Corresponding 1111/vco.12470 EmmaHurst A. Edinburgh, Bush,Edinburgh, Easter Midlothian, EH25 9RG in 2 ) are prominent) are tumouris and promoters, expression ofCOX-2 - vitro The anti-tumour effectsmavacoxib of independent of cyclooxygenase * : Emma ( Hurst A. Short running title: title: running Short Acknowledgements Acknowledgements - * 2 inhibitor mavacoxib ( , Lisa Y.Argyle PangandDavidJ.

and the Versionof Record. Please cite this [email protected] Trocoxcil - 2 expression levels ™ )

exerts anti )

- This articleisprotected bycopyright.Allrightsreserved. damaged cells to cancer toformgrow rapidly a an ofexpression pro protein p53,andbyincreasing theexpression of anti Acceptedderegulation ofapoptotic proteins viath involvedalso intheability toevade ofcancer cells apoptosis. This the due to occurs stimulation growth endothelial of vascular factor (VEGF) potentialinvasive of cancercellsvia mobility increased increasingprogression through cancer proliferation cell Pro pathways. prostaglandin targets axis many factorsthatplay important roles intumourigenic downstream COX therapeutics. types Manycancer (bothhuman andcanine)haveincreasedexpression of are withincreased2 levels more aggressive, incidence ofmetastasis and toresistance COXIn cancer, Article and inflammatoryfactors, produces prostaglandinsinvolved in signalling swelling pain and enzyme ofbyinflammatory growthas part and stimulated animmuneresponse cytokines , and from arachidonic Introduction Keywords: mavacoxib mayanti effective bean highlights the potentiallyof noveluse mavacoxib acancer as therapeutic, suggesting that mavacoxib theseeffects exert independently can ofCOX elevated - 2, including breast 2, including The cyclooxygenase(COX) (COX enzymes

canine; comparative oncology;COX canine; comparative staglandins, particularly PGE -

2 is overexpressed 2 isoverexpressed withadverse tumours consequences; withelevated C - apoptotic proteins 2 – 5 , lung 2,6 - , skin cancer agent independent oftumour COX 15 . The ability to avoid controlled cell. Theabilitytoavoid death allows e loss, damagee loss, ofthe tumour inhibition or suppressor 7 , colon 2 , are involved involved in , are enhancing tumour and growth - 2; COX ggressive tumour.ggressive 2,8,9 - - 1 andCOX apoptotic proteinsand reducing the

and bone cancer 11,14 15,16 - 17 2 independent 2 independent effects; mavacoxib , increasing themetastatic and . PGE

and via enhancing angiogenesis -

acid. COX 2) are key 2) are inthe synthesisof 2 - 2 expression. This This study 2 expression. signalling mechanismssignalling are

10 – 13 - . TheCOX 2 is an inducible inducible 2 isan - 2

expression. expression. - 2 / OX 1 -

.

This articleisprotected bycopyright.Allrightsreserved. to,ablecytotoxic to andis inhibit the migration particular ofthese cell lines.We begin aim to positive therapeutic effects progression disease in thesecancertypesand both species useofNSAIDs the shown has Accepted bladder,urinary whichexhibit cross species similarities. COX osteosarcoma,include mammary carcinoma andtransitional cell carcinoma of the (TCC) effec ofcaninepanel cancercell and lines cancer stem cells previous regimen.dosing Consequently, the benefit clinical potential mavacoxib of using ishigh. rate, a relatively largedi 34 withassociated canineosteoarthritis termmonths)where long 6 treatment (upto isrequir Article(Trocoxil of potential suchdrugs ascancerusing therapeutics intherapeutic combination witheither chemotherapeutics or demonstrates surgery the pre potentialinvasive of cells growth and proliferation have showntoinhibit coxibs NSAIDs been tumorigenesis and inflammatory conditions arthritis isuniversally suchas accepted. Exp afamilyare which selectively ofNSAIDs target COX . Mavacoxib unique is family asitexhibitsin the and coxibs ofNSAIDsclearance alow - clinical and clinical trials in which ts of mavacoxib on ofcaninea panel andhumants of studied cell lines. Thecancers cancer There islimitedThere research theinto potential anti Non

ly shown that mavacoxib exhibitsly shownanti mavacoxib that  - steroidal anti ) is of interest because it isofinterest it because is currently ) to licensed pain treat and inflammation - stribution volumestribution longplasma anda half inflammatory drugs (NSAIDs) inhibit inflammatory inhibit (NSAIDs) theCOX drugs enzymes,coxibs and 14,21 16,25,26 2,4 , modulating apoptotic activity - 5,11,20,35,37

and by inhibiting angiogenesis

coxibs havecoxibs preventative beenused asa a agentoras – 48 . We todetermine d ifmavacoxib aim is - proliferative and pro 17,30 - cancer effects mavacoxib.Weof have - in 2. The use of NSAIDs to treat useofNSAIDstotreat 2. The – - 33 vitro . In veterinarymedicinemavacoxib 22 –

- 24 35,36 8,18 2 has an importantrol2 hasan 27 , reducing me the - – – life, resultinglife, reducedin a . Here, we investigate the 29 20 - . Positive from results

apoptotic effects a in by inhibitin erimentally, both g cancercell tastatic tastatic and irectly e in e in

ed This articleisprotected bycopyright.Allrightsreserved. (ThermoFisher Scientific) andwere maintained 37°Cina at humidified 5%CO supplemented 10% bovine with fetal serum 100 μg/mL and (FBS) penicillin/streptomycin (ThermoFisher Scientific) Accepted HT and the wereline maintainedin RPMI The w University). T24cell line bladder cell T24,5637lines andHT containing HEPESL and (PurdueStewart University) K9TCC,K9TCC (Peprotech, CaninetransitionalcellLondon, UK). carcinoma oftheurinary bladder cell lines human (Sigma insulin inRPMI medium Scientific)were grown 10 supplemented with 1640(ThermoFisher μg/mL Articlemammary LILY(gifted cells Raffaella, carcinoma from DeMaria University Dr of Turin) REM134carcinoma (REM) cells 5Amodifiedwas growninMcCoys medium Scie (ThermoFisher (ThermoFisher Scientific, USA). Massachusetts, The humancelllineosteosarcoma U2OS weremaintainedin mediumKhanna (NIH)) Dulbecco‟s modified Eagle‟s (DMEM) cultureCell methods and Materials but responsetypes, also ifthis comparable between is to establish if not only mavacoxib potential has anti as an The canine celllines,The canine osteosarcoma KTOSA5

- 1376 cell 1376 cell linemaintained were MEMin Advanced (minimum essential medium)

- AXA, K9TCCAXA, - Aldrich, Missouri, USA)Aldrich, Missouri, and10ng/mL human EGF recombinant - glutamine. cell Human carcinoma transitional oftheurinary supplemented 1% with glutamine. medium culture Allcell was ) were maintained) were inDMEM/F

- 1640 mediummodification) (ATCC (ThermoFisher Scientific) ere maintained inMcCoy‟s5A (modified) medium, cell 5637 - In and K9TCCIn and 50

were maintainedinDMEM. Canine inflammatory - 1376 cells were gift 1376 cells - Sh (gifted fromSh (gifted DeborahKnappandJane 35 species.

and CSKOS ed - cancer agent against thesecancer

- from (Oxford AnneKiltie 12 (ThermoFisher Scientific)

ntific). Canine mammary 49

(gifted from Chand 2 incubator. incubator.

This articleisprotected bycopyright.Allrightsreserved. media fixed once were waschanged aweek. Colonies byincubating with methanol 5 for wereincubated at Plates 37°C in humidified 5%CO andtreatedKTOSA5 with cells) REM, K9TCC, K9TCC Accepted assay Colony formation the using log(inhibitor) vs response variable usingcalculated GraphPad Prismfor 7.0c OSUSA) version Mac X(GraphPadsoftware,CA dose andaveraged normalizedtheaverage against signalofveh Cambridge, software UK) theViktor3 using (PerkinElmer,USA). Massachusetts, Data was was Luminescence hours. recordedby aspectrophotometer (WPA Biochrom, Biowave, Article mavacoxibdilutions of 96 in a was usedaccordingthe manufacturer‟s instructions. Briefly, per wereseeded 500 cells well viabilityCell assay media before immediately Vehiclecontrols use. were included allexperiments. in drugCytotoxic treat - response was curve produced. Cells wereseededassingle cellsatlowCells (250 /10 density cells cmCSKOS, for plate The CellTiter Mavacoxib (Trocoxcil - well 37 plate Cells in triplicate. for24hoursat were incubated -

Glo ment

(0 - ® AXA, K9TCC

Luminescent CellLuminescent Assay Viability (Promega, –

200 ™ , Zoetis, London,, Zoetis, UK)in wasdissolved dilutedin DMSO and  0, 25, 50or75 M) The for IC50 line cell each with treated mavacoxib was

were added were added and cel - In, K9TCC slope equation.  M -

Sh cells and100cellsSh cells cm /10 for plate 2 mavacoxib still insuspension. while

incubator for10 incubator l viability wasassayedafter72l viability icle controlicle treatedsamples anda

- o 14 culture days.Cell C, 5%CO Madison, WI,Madison, USA) 2 . Serial

This articleisprotected bycopyright.Allrightsreserved. initialwoundthe 0hours. widthat to„relativemigrationwas converted of distance‟ where cells, distance isapercentagethe of an average take Acceptedmicroscope withanAxioCAM coupled HRmcamera Ltd,Cambridge, (Carl Zeiss and UK) assaysmeasuredIn both widthof 6points the at gapwas the 40 the using Axiovert CFL mediumculture well. each wasaddedto Afterhours. 24hours incubationeach wasremoved insert mediumculture containing cellcultureOnce confluent, mediumwas removed from andreplaced well each withnew cell volume culture μlcell of70 medium. were Plates forincubated assayMigration Articlemonolayer ofcentre downthe pipettetip. eachplate cells witha 37°C inhumidified 5%CO media and wasreplaced were treatedwith plates assayScratch migrationCellular assay conditioncalculated. each The total number counted wasmanually of andtheaveragenumber colonies ofcoloniesfor Scientific). concentration For each tested, plates up wereset in triplicate or quadruplicate. minutes room at with and stained temperature . Cells were. Cells grownuntil80 n. Measurements made were Scratch width 0,4,8,24,28,32and48hours. at . Cells . Cells were seeded at3x10

2 0

incubator overnight.incubator Ascra –

125

 M

mavacoxib

- 90% confluent 10 in cmculture Thecell plates. 5

Giemsa at1:20dilution stain (ThermoFisher cells / well ofthe / cells culture cell insertina 0 – and

125 incubated at 37°C, incubated at 5% CO tch made confluentwas the in 

from its well. fromml well. One its ofcell M

mavacoxib incubated and at 24 hours at24 hours 37°C, 5%CO

2

for 24 2 . This articleisprotected bycopyright.Allrightsreserved. calculate the concentration of samples. the standards (a linear relationship wasdetermined) andtheequation oftheline wasused to Acceptedwith Viktor3softw measuredsamples at450nm wasspectrophotometer the using (WPA Biochrom) Biowave, ELISAoutand the carried following manufacturer‟s protocol. the Optical of density the After incubati 24hours withwhich wasreplaced culture fresh cell media cellsreached50% whenthe confluency. cell culture media. numbers seeded were inequal usingrelevant Cells culturemedia the cell Abingdon, UK)determine to wasused PGE Article control treatedvehicle samples. caspase activity present. signal Datawasaveragedand normalized theaverage of against Viktor3 the softwareusing (PerkinElmer). tois proportional t Luminescence treatment. Luminescence wasrecordedbyaspectrophotometer(WPA Biowave,Biochrom) – 96 according tomanufacturer‟s the instruction. Briefly, cellswereseededat per in 500 cells well Apoptosis assay

200 - well plateswell andincubatedfor24hours at37 2

Apoptotic activitywas Apoptotic measured Caspase the using  EL The Canine fromProstaglandin E2ELISAKit BlueGene (AMSBiotechnology, M) M) ISA were added toappropriatewells. Apoptoticwere added activitymeasured was48hourspost

are (PerkinElmer).are curvewasproduced bythe Astandard plotting on cellculture the media wasremovedfrom flask,centrifuged each

the the amount ofPGE

o C, 5%CO - Glo 2. 2

exported from intoexported thecells the Serial dilutionsmavac of Serial ®

3 - 7 Assay(Promega) he amount of oxib (0 - This articleisprotected bycopyright.Allrightsreserved. from were HRP Accepted isoformof beta the ofactin residues) (Akt1 phosphorylatedat Ser473, and Akt2 andAkt3whenphosphorylatedcorresponding at phosphorylatedatThr202/Tyr204)dually USA (C Pharmacia Biotech). bysubsequent chemiluminescencedetection enhanced (Amersham Article primary tohybridised anappropriate and antibody HRP C SDS polyacrylamid amountsUSA). ofprotein Equal usingQuickblotting Start™Bradford 1xDyeReagent (Bio Bradford assaywas used to buffer 0.1MDTT, urea, X (7M 0.05%Triton analysis.for western blot were and Cells pelletsharvested cell werelysed withurea lysis blotanalysis Western ™ -

terminus of COX nitrocellulose membranenitrocellulose (Amersham Biotech, Pharmacia UK)and Buckinghamshire, ) Dako, Agilent TechnAgilent Dako, ; The followingprimary were antibodies utilizedtoprotein assess expression:COX

ERK1/2 Cells were withCells treated

all from - conjugated conjugated swine anti

(p44/42 (p44/42 MAP Cell Signalling Te Cell Signalling e gel electrophoresis (SDSPAGE)e gel -

2 ofhumansc origin,

ologies ologies , quantify of protein concentrations samples all western prior to ab6276 K (137F5)) 0, 25,50 - (30 rabbit IgG, rabbit antirabbit IgG, ( CA, USA). ) from chnology  g)

or 75 , were resolved based based molecularwere resolved ontheir weight by , Akt

phosphorylated Abcam - 1745 -

 (

100,mM 25 20mMHepes pH7.5).A NaCl, (Akt1, Akt2andAkt3) Massachusetts, USA M ) from

( mavacoxib prior toharvesting for24hours Cambridge, UK 51

Santa Cruz Biotechnology Santa Cruz - - mouse and IgG rabbit anti , transfer conjugated secondaryconjugated antibody for - ERK1/2 - Rad Laboratories, California,Rad Laboratories, red toAmershamred

™ ) (phospho ) . Secondary antibodies. Secondary ; E

, phosphorylated and β CL ™ , Amersham - actin - p44/42 MARK,

(N - ( ™ goat IgG Texas,

- Hybond terminal - Akt - 2 - This articleisprotected bycopyright.Allrightsreserved. (Supplementary table 1). wasalsoarange There in time doubling from (T24cells) to 10.6 hours(K9TCC 7.0 Accepted CSKOSlines andKTOSA5inflammatorymammary andthe LILY).carcinomaline cell withprotrusions associated mesenchymal (for cells example, canine osteosarcoma the cell K9TCC,carcinomalines cell K9TCC mammaryexample, the line REM cell carcinoma humanbladdertrans and the morphology frommorphology cobblestone classic associated with epithelial cells (for morphology anddoublingtime We (Supplementarytable 1). showedaspectrum of lineshuman cancercell Characterization Article Results oflessthan0.05. value OSMac X(GraphPadsoftwa forcorrelation nonparametric analysis wasperformed Prism7.0c version GraphPad for using Whitney test were performed statistical softwareMinitab® USA). (PA, Spearman‟s using Statistical analysis Data were expressed as expressed Data were mean andhumanA panelofcanine chara cell lines were cancer

of COX

- 2 expressionandPGE re, CAUSA).werere, with deemed Differences significant ap  - AXA,K9TCC

SD. Statistical analysis ofstudent‟s Statisticalanalysis SD. t 2

production inapanel ofcanine and - In and K9TCCIn and cterised bycellular - Sh) andelongated Sh) - test or Mann test or itional itional cell - In cells) In cells)

- - This articleisprotected bycopyright.Allrightsreserved. ofshown isrepresentative lines). allcell However, sens mavacoxib celllines indicating that all tested in mavacoxib (Figure 2Ai cytotoxic is treatment. cellviability wasreduced inadose that We showed cell lines withincreasingdosesofmavacoxib and assessed cell via Accepted death cell Mavacoxib celllinesis cytotoxic to humanandcaninecancer andinducesreplicative statistically significant (p=0.00 areproduction positively (r=0.7478, correlated Figure 1B)and correlationis that this nonparametric natureofthe Here, data. weconfirmed COX that PGE and mammaryTo celllines carcinoma (Table1). determinecorrelation ifthere is a between Article 1).(Table We TCC bladder showthat lines morePGE cell produce isPGEreaction and isdisplayedline this data Table in 1.Oneof main the ofthe products COX COX K9TCC,and in K9TCC expression was detected inREM(caninemammary carcinoma), 5637(human osteosarcoma) mammary and carcinoma) HT expression was detected inKTOSA5 osteosarcoma), (canine LILY(canine inflammatory (canine osteosarcoma) andT24(humanCOX TCC)lines, lowlevel cell whereas bladder 1A).(Figure COX 2 -

2 expression was quantified from wasquantified analysis2 expression blot relativeto western production andCOXproduction The expression of COX To determine ifmavacoxibcytotoxic tocancercells we treatedapanel is of cancer

2

and weconfirmed amountPGE the of - 2 expression was negligible in2 expression U2O - AXA, K9TCC - 2 expression weu 2 expression - 1376 (human TCC) cell l bladder 71). - 2 protein in 2 protein in celllineeach wasdetermined blotting bywestern

- In and K9TCCIn and tilized the Spearman‟s test statistical due tothe - S (human osteosarcoma),S (human CSKOS 2 itivity betweento mavacoxib varied Sh (canine bladderTCCSh cell lines).

produced by each cell by byeach cell line ELISA produced - dependent manner by - ines ines and high COX 2 expression andPGE bility 72hoursafter 2 than the osteosarcoma the than  - actin foreachactin cell - 2 enzyme - - 2 v. Data 2 - 2 This articleisprotected bycopyright.Allrightsreserved. effects andthatthis withthe can occur ofCOX use usually associated withenhancing cell proliferation can andsurvival, induce anti studiesInterestingly, other havenoted that Accepted canine Akt pathwayin celllinesosteosarcoma (CSKOS viability. cell and U2OS) reduce to increasedalso mavacoxibtreatment. with sug Thisresult expression dosesofmavacoxib,with increasing inK9TCC and p cells U2OSwas totalInREM Aktin both cells. cells p InCSKOSvariable. cells,p oftheseresponse pathways increasingdosesofmavacoxib to apoptosis and cellproliferation via reduce ERK andsurvival the pathways andAkt progression. cell cycle factorsnumerous transcription downstream areinvolved in that promotinggrowth cell and of bot bytheactionofPI3K/Akt pathways PGE Article 2B. (Figure shown).representative data forERK,and probed phosphorylat be agentcytotoxic against these cellt factors these (r= IC50 values and COX tomavacoxib,sensitivity statistical Spearman‟s test Todetermine acorrelation1376) (Table2). is between ifthere COX celllines,the valuesrangingfrom withIC50 34.5 affected by mavacoxibaffected bywith wetreated cells doses ofmavacoxibincreasing for24 hours h ERKAkt and To elucidatethedownstream pathways signalling - 0.2222, mavacoxib p =0.4839),suggesting that mayaneffective be is required is required - 2 expression. expression. wasnoThere 2 statistically significant correlation between Several studies havedemonstratedthat COX - Akt was downregulatedwithi for ypes, including withlow those COX ed

activat - ERK (p COX 2

via EP2/EP4receptorsvia the activation ofthe the ERK which is pathway, ion - - 2 is known to isknown activate 2 the MAPK/ERK and ERK), Akt and phosphorylated ERK), Aktand , which boththen goonto phosphorylate -  ERK andp -

M (K9TCC 2 inhibitors 2 inhibitors was used correlation toassess between gests thatmavacoxib may the inhibit involved involved in cell survival thatmay ncreasing does ncreasing mavacoxib,of as - in the celllines herein the tested Akt were increased in Akt wereincreased in - 24,57,58 AXA) to 157.7 AXA) to 52 – - - 54 2 inhibitors induce 2 inhibitors 2 expression and 2 expression . Further i . - - ERK was expression Dual phosphorylation 2 expression. 2 expression. - Akt (p nvestigation  - M (HT proliferative 55,56 -

Akt) .

- T was he

This articleisprotected bycopyright.Allrightsreserved. remainmore to sensitive mavacoxib caspase induced 2, howeverKTOSA5and U2OSexpress also lowlevels cells of COX LILY (Figure4D)andT24cells4F). and T24cells ofexpress low levels COX however onlythehighestmavacoxibproduced doseof statistically a significa Accepted cellsisdetectedapoptotic inKTOSA5, U2OS,REMandK9TCC (Figure4A cells when treated 100μM at Adose (Figure 4). orabove demonstrate thatmavacoxib induce can caspase damagedallowing cells to grow cancer rapidly to form tumour. anaggressive we Here, lines Mavacoxib can inducecaspase withviability tre Article COXby the forming detected ability panelacross the (Figure 3C). Theinduction ofreplicativecell death canine andhumanbladderTCCbetween 3A (Figure significantly colony reduced formationadose in forming as an assay ability ofreplicative cell d types. mechanisms responsible in for thereduction cellviability othercaused bymavacoxib in cell into these and

To substantiate the previous we data, determinedeffect the ofmavacoxib oncolony Cancer cells abilityCancer havethe byderegulatingapoptotic to evade apoptosis pathways, - C). Theabilitymavacoxib ofinducereplicativecell to death is comparable - 2 inhibitor mavacoxib previous demonstrating supports data incell a reduction other proliferationother pathways be to will required de atment ofthisdrug. - dependent incanine cancercell apoptosis andhuman

cell lines, adose cell with eath. We mavacoxibeath. that treatment showed - - dependent apoptosis inall cell lines te dependent manner celllinestested inall - dependent increase in number the of - dependent apop - dependent in decrease colony termine the downstream tosis. Mavacoxib was - 2 and these cell 2 andthese lines nt response in - C and4E), sted sted - This articleisprotected bycopyright.Allrightsreserved. Acceptedmigration in cell to resistant mavacoxib 5Biv). results These indicate (Figure mavacoxib that inhibits cell beforegap width asignificant response con wasdetected, mammary line LILY cell of carcinoma higher doses required mavacoxib initial alarger and compared canine bladder the TCC to cell line 5Bv).Theinflammatory(K9TCC) (Figure requires(T24) ahigher concentration ofmavacoxib yield a to signifi respectively) respond in a comparable manner, howeverthe TCChuman cell bladder line andhumancanine osteosarcoma (KTOSA5 cell lines (Figure 5Bi) andU2OS(Figure 5Bii) a representativeimage assay the conducted in of K9TCC shownin cells concentration a significant ofdrugyield statistically to isresult shownforline, cell each with migrationthe ofhumanand canine cancer cell lines Article incubatedcells with mavacoxibfor24 Here, hours. wedemonstrate that mavacoxib reduce (K9TCC T24cell lines (Figure and 5 Bv REM a lines migrationCellular is inhibited by mavacoxib in acell butapoptosis, other apoptoticpathways may beinvolved. cell these lines may resistant to mavacoxib beinherently to able reduce viability inLILY cell atlower andT24cells concentrations, suggesting that in nd LILY cell lines5Bind LILYcell (Figure - The ability ofThe ability mavacoxib inhibit to the migration cancerof canine cell human and

vitro was investigated scratchwas investigated (Figure5).Either a standard assay (KTOSA5,U2OS, - line dependent manner. - iv)) orthe Ibidi -

vi)) was tomeasurevi)) used the migration of cancer in ® - vitro Culture - - induced caspase line dependent manner

sistent withthiscell beingmoreline (Figure 5B) (Figure - Insert migrationInsert assay cant result cant result (Figure 5Bvi)

. Figure 5A.The Figure

The lowest The lowest - dependent dependent

s This articleisprotected bycopyright.Allrightsreserved. NSAIDs toVarious been shown changesinERKphosphorylation between have induce 4and incongruously demonstrated that and apoptosis results celldeath.induces in cancer studies However, several havealso AcceptedCOX inothers decreasing (CSKOS hasbeen that it shown the andU2OS).Generally, of inhibition with phosphorylation increasing with treatment and insome lines(REM K9TCC) cell and response in thephosphorylationstatus treatment ofbothERKandAktafter w investigatedand Aktwere after treatment cell mavacoxib. with We avariable demonstrate celldeath. Tofurtherinduce understandmechanisms the theactivation this, of behind ERK 60,61 adaptive immuneandalter response metastasis and encouraging invasion, andlymphangiogenesis, angiogenesis inhibiting the Articletumour effects, proliferation including promoting andsurvival, enhancing promoting EMT, microenvironmentinflammatory resultingcells, surge ina activate the factorssame ininflammatory keytranscription tumour cells, andstromal cells including COX activated in tumour which produce cells, activationinflammation andoncogenic converge,inflammatory transcriptionfactorsare patients.canine Thelinkbetweeninflammation, COX Discussion .

- This study demonstratesThis study mavacoxib abletoreducecancerthat is cellsurvival and highlightsnovelThis study a usefor mavacoxib 2 decreases phosphorylation 2 decreases

, this , this increase inphosphorylation was - 2, that recruit andactivateinflammatory2, that These cells. immune cytokines inhibition of of inflammatorymediators giving cancerrelated rise to a COX 59,60 of ing responses chemotherapeutic tohormonesand agents - . Thiss 2 increases the2 increases phosphorylationofthesemolecules and both ERK and Akt which in turand Aktwhichin both ERK inflammatory mediators, cytokines chemokines, and mouldering inflammatory environment hasmany also associated withalso apoptosis

- as a potentialas a anti 2 and cancer cancer is wellestablished;2 andwhen n inhibitsproliferation, - cancer agentin ith mavacoxib,ith 24,57,58,62 . This articleisprotected bycopyright.Allrightsreserved. mesenchymal transition(EMT), which epithelial allows primary tumour togain invasive cells witha those primary tumour.COX Accepted stagemethods detectearly apoptosis that to may able becaspase investigation the into mechanisms mayinhibition bethe ofamitochondrialresult explanation withcaspaselines, cell deathlike do caspaseinhibitors that notprevent death cell through apoptosis autophagy, necrosisand mechanisms. Caspase suggesting at concentrations, lower mavacoxib may caspase induce (Figurehere 4 Article to order investigate what theorcauses active suppressive response. a type and cell dependent. of response cellline ERKeach the to /Aktphosphorylationmay effects ofNSAIDsbetime the influence extent to which cells waymay inthis toNSAIDs respond was activationpathway occurring. The basal level of ERK the andAktmay pathways also but 24 carcinoma ofpancreatic hour) with suldinac cells notinduce did inphosphorylation changes ofincubation 24 hours ctivation ofctivation pathwaysincubated these with various different NSAIDsfor various times in The survival rate ofpatientsThe survival rate w caspaseMavacoxib can induce - hour incubat 35,37,65 ). This occurs at higher occursat drugconcentrations ). This than thereductionviability, incell 64 . Previous have NSAIDs studies similar resultswith noted cancer in cell . We previously have shownthatinc - independent and independent and BAX ion did,suggesting that long - independent cellindependent deathhasbeenpreviously highlig characterised, 24,62,63 Future studiesFuture shouldincludecelllines low with andhighbasal . Yip - driving Schneider alet discuss (2003) that shortterm (1 incubation - 2 has been implicated2 hasbeen inthe of process epithelial to ith metastaticith cancer greatlyreduced incomparison is to - dependent anumber apoptosisin oftested cell lines -

mavacoxib independent apoptosissuggested asapossible independent - independent apoptoticindependent pathway - term thantransient activation rather of this - induced celldeathinclude should reased reased Bcl - - independent. 2 levels COXafter -

independent cell independent cell death 62 . Therefore, the

35 . Further - 2 hting hting - This articleisprotected bycopyright.Allrightsreserved. forevidence involvement the COX ofthe “phoenixthe pathway” rising regeneration Acceptedapoptos stimulates cellproliferation migration. and There is increasing evidence that cellsundergoing withapipettecarcinoma tip result cells line,LILY cell whereby a the metastasis and lymph invasion node cancer type fromderived inflammatory acanine mammary carcinoma, fastgrowing,highlymalignant a mavacoxib other the than canine mammary c treatment, alongwithof expression EMT transcriptionfactors andmesenchymal markers. Articledetermine the expression ofE mavacoxibwhether itsanti exerts andhuman canine cell lines cancer carcinoma (IL demonstrated bewhich can by reversed COX the cancer cells. Inhuman cancer, EMT theT24celllineexhibits bladder astrong phenotype mesenchymal toepithelial transition ( 66 and metastatic pote . Severalstudies have established potential for COXutilizing - 6) upregulation of COX Interestingly, the LILYcellInterestingly, the migrated fasterandmoreline was inherently resistant to is signal theirpresence to the surroundingto tissues elicit tissue repair and 69

in which . In this study, mavacoxib. Inthis wereport that inhibi can 73

in lung cancercells in lung that . This phenomenon of tissue damage. Thisphenomenontissue ofinitiating tissue repair coinehas been ntial by converting toamesenchymal with mobility phenotype enhanced

COX - 2 hasbeen showntobeoverexpressed - 2/PGE ct confluent ofscratchinga ofinflammatory layer mammary - 74 cadherin ineac cadherin

a nd - migration effects byreversing EMT,futurestudies should

- 2 and 2 inhibitor 2 inhibitor

a 70 68

MET), therebymigration inhibiting and invasion of

seminal cancer has studyinbladder – migration and invasion stimulated interleukin are via , with a , witha similar andnecksquamous findinginhead cell 72 s that that thi

. Here, aninflammatorywe suspect response bythe in an apoptosis induced inflammatory response that - 2/PGE arcinoma cell linearcinoma The cell tested. LILYcellline was h celllinebeforemavacoxib andafter s

action is 2

pathway inpathway cancer stem cell (CSC) 67

cell line dependent . Che al. et (2017) have - t migration ofapanel 2 inhibitors toinduce with increased risk of demonstrated . Toestablish d

- 6

This articleisprotected bycopyright.Allrightsreserved. oranhydrase polyamine pathways. COX lacks Acceptedand COX elucidated. An proliferative,angiogenic apoptotic, andmetastatic pathways antitheir Interestingly, derivatives thethat ofNSAIDslack to ability inhibit COX the enzymes retain spermidine/spermine of polyamines,regulators of tumour cell environments carbonic anhydraseisoforms, importantwhich are incancer survival cell enzymesinhypoxic discuss(2015) the inhibitionofinclude the carbonicanhydraseand polyamines. Articleanti therapeutics anumberofpropert ademonstrated NSAIDs have tumour inaCOX effects withlowlines COX anti along ofline, withexpression the COX investigation isrequired toassessapop ofrepopulation - - angiogenic effects cancer effects. Here,we mavacoxib showthat exer can Many NSAIDsandCOX

- - tumour activity 2 - - 2 inhibitory 2 inhibitory action , including independent effectsmavacoxib by a of producing mavacoxib derivative that 79 interesting future research avenue futureresearch interesting ; and residual tumoursresidual tochemotherapy inresponse ability ofsulphonamide COX -

2 expression, advocating2 expression, that mavacoxibmay some exert anti ofits - indomethacin acetyltransferase (SSAT)cancer cell inhuman colon lines

anti 78 - . 2 independent manner. AsidefromtheirCOX 81 Potential Potential - – proliferative pro and 83

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se NSAID, h determin totic activitytotic after induction LILY damage inthe cell -

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whether mavacoxibwhether inhibits thecar 2 selective inhibitors2 selective to inhibit several apoptotic activityapoptotic would betoconfirm COX the ies that make them anti attractive t anti 81

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- dent and This articleisprotected bycopyright.Allrightsreserved. modalitydual treatment andmetronomic chemotherapy, increased significantly when compareduntreated to dogs more towardsbut is selective COX Accepted (a preferential COX notedcomplete andpartial remission treatment oftransitional cell carcinoma bladder dogs, with theurinary in cas of single as a mode treatment hasbeen effective established as an anti COX modalitydual ormetronomic chemotherapy treatment Piroxicam, anon options. of potential a cancer treatmentNSAIDs asasingle asboth treatment forof dogs, andaspart cancer types approaches. For example, modality dual treatments currently are offered forseveral Articleh unlikely that COX cardio toxicity side havestudies notindicated asignificant benefit to patient,andincombinationwith the the use ofthese fordrugs prevention cancer therapy and remains controversial.clinical Many demonstratedall positive anti drugscancerthese foruseas therapeu incidence and progression ofcancer epidemiological evidence an demonstrating correlationbetweenNSAID inverse useandthe owever, they may adjuvantswhen conjunctionused in beeffective - effects withassociated NSAIDs

inhibitor used in human usedin medicineinhibitor In the fieldIn the Currently, thereareanumber of NSAIDs , including colorectal and prostate ) , there is limited enthusiasm use for their of veterinary oncology,numerous trials areattemptingto elucidate the - 2 inhibitors inparticular willbe2 inhibitors utilized as amonotherapy for cancer - - neoplastic neoplastic effects clinical during trials 2 inhibitor; this class of this class drugs 2 inhibitor; affects both COX

in stageandmetastaticboth early disease - 2) significantly increased time survival s as ,

there havethere beennumerous attempting trials validate to ( 95 tic which can which can range from GI moderate . In canine prostatic . Incanineprostatic carcinoma, piroxicam both and ,

s has been Piroxicam oneofthemostwidely studied. .

cancer approved forclinical us approved , ,

92 – 94 91 . . Given these controversies it is

- cancer agentthe in with therapeutic other 76,84 e.

and piroxicam have –

90 toxicity tosevere W .

96 ith ampleith However . Aspart of - es ofboth ingle agents 1 and COX 1 and - selective human , the the ,

- 2 This articleisprotected bycopyright.Allrightsreserved. activity, rendering them more sensitive to the actionsofCOX If even inhibitors. occurs, this chemotherapyTherefore, may for clone select a of with increased tumour cells COX Accepted thetumour, repopulate process shownthat COX a hasbeen toinvolve the ofCOXaction which raisestheinterestingpossibility chemotherapy may that sensitize the tumour the to developingof foreach time drugresistance separately drug over than concurrently, rather thereceiving same simult agents was single agents there a significantincreasein survivaloverall time dogs to compared notedthatwhen Knapp etal. (2016) vinblastine treatment wasfollowedbypiroxicamas two of COX insurvivalfactor time study this in combined alone,compared surgery to buttherewas difference nosignificant whenchemotherapy was Article surgery that plus adjuvant compared treatmentregimens other to demonstrated differencefirocoxib no significant inpr lymphoma combinations ofchemotherapeutics radiotherapy utilizing or with piroxicam and in transitional cellcarcinoma bladder, nasalcarcinomas oftheurinary multicentric and in combination withchemotherapy mammaryinflammatory pa used carcinomas as when NSAIDsare alone concludes that including NSAIDs significantly intreatment improves especially in survival, carcinoma treatments piroxicam without ofcancer range types ina timesurvival andincidences ofcompleteor partialremission compared same the to - 2 should be included as a predictive beincludedasa 2 should factorfor cancer type this

with or without or with NSAIDs 98

and primary lungcarcinoma

inhibitors inhibitors 98 chemo . After chemotherapy treatment aneously. It aneously. It is suggested that this could consequencebe the therapy therapy 100 and 104 . However, conflicting evidenc . COX 101 the authorsuggest the – significantly significantly improve 103 99 . Arecent review mammaryon canine carcinoma . Astudy inmamma - 2 expression was significant2 expression foundtobea ogression free andoverallsurvival 97

s including invasive urothelial ,

that immunohistochemical score resistant cellsresistant goonto can d

overall survivaloverall time ry carcinoma e still remains. Studies 104 lliative treatmentor - . Interestingly, 2 pathway 2 pathway demonstrated - 2 75 . This articleisprotected bycopyright.Allrightsreserved. which wouldallow research concerted effort to develop targeteddrug systems delivery therapies forcancer mavacoxib are utilized inthis study higher than plasma there is concentrations, currently a Accepted benefitsthat the ofreduceddos mavacosafety profileof ofstate arou doses initial mg/kgof 20and day at day14,maintaining plasma at a steady concentrations dosingusual regimenmavacoxib for comprises ofadministrationatmonthly intervals after oflife regarding treatment optionsinveterinarycancer care. relevclinically improvementsubjective of inquality life Articledogs remain onpiroxicam tumour treatmentevenif remission occur,duetotheir didnot compared treatmentoptions other to questionnaires) pain) above, treatmentcurative of aprimaryquality when life, veterinarycancer concern treating patients. Inmany cases ofconsisting chemotherapy andNSAIDs sequentially. tumours COX withlow were allnotedtobe improved treatedwithNSAIDs inpatients (reported throug Currently theofmavacoxib use in the is clinic unorthodox topicsOne ofacrossmanythestudiesmentioned recurring aboveisthat the the drugthe potentially resultin several nd 0.52

ant findingant andshouldinto betaken are consideration whendecisions made factors ,

even in where noimprovementcases NSAIDs showed time insurvival

is not andis not an option palliative studiescare mentionedis optedfor.the In increased concentrations oftherapeutic agents to the target tumour site –

1.11 μg/μl(1.35 used assess to of (includingappetite,activitylevels quality life and - xib is comparable xib is to other veterinary NSAIDs 2 expression initiallymay from benefit atreatme ing should be exploited. Althoughing shouldbeexploited. concentrations of the g in 95,99,101,102

side –

2.88 μM) 95 - . Given importanceofquality the oflife, this is effects whichare problematic for thepatient. The . I n one study 105,106

. S tudies have demonstratedtudies have that the ,

most owners requested that their most ownersrequested

as aresultof 107,108 nt program , andsuggests

the half long h owner - This articleisprotected bycopyright.Allrightsreserved. Accepted The authors that declare no there are ofinterest.conflicts ofinterestConflict an anti Articlevivo understand the COX expressing ofCOX a on cells low level tumour across We and types species. highlightthat mavacoxibanti exert can exploited. diminishedcan be byimproved systems,anti drugdelivery their in therapeutic and agents release them controlled ina manner diseased totarget cellswith undesirablesidewhile minimising creased specificity

and preclinical elucidatestudies willberequired to fully potentialmavacoxib the of as In - cancer agentconjunction in clinical benefitswith its as conclusion, mavacoxib weshowthat aneffective is agentcytotoxic on of arange

- 109 2 dependent and COX2 dependent – 111 . Ifthe challenges toxicityfor mavacoxib ofdosingand drugslike

- effects. using These include nanoparticles toencapsulate - 2, however further mechanistic 2, howeverfurthermechanistic studies arere - 2 independent actionsmavacoxib. of Further2 independent

an anti - cancer potential maybe - inflammatory drug. - tumour effects quired to in

This articleisprotected bycopyright.Allrightsreserved. 14. 13. Accepted12. 11. 10. 9. 8. Article7. 6. 5. 4. 3. 2. 1. References

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2. 9.

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invasive invasive 304. - 2) in 2) in - 2 in - 2 in

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- - This articleisprotected bycopyright.Allrightsreserved. 82. 81. Accepted80. 79. 78. 7 76. 75. 74. Article73. 72. 71. 70. 69. 68. 67. 7.

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- This articleisprotected bycopyright.Allrightsreserved. 97. 96. Accepted95. 94. 93. 92. 91. 90. 89. Article88. 87. 86. 85. 84. 83.

Cell Pharmacol [Internet]. 2009;1(1):29 SJ,ZhangRayburn ER,Ezell R.Anti Resulcancer: AC,DM,Vidal LE,Moreira Howard InitiativeWomenHealth 1.CancerRes.2003;63:6096 s ‟ and Anti Cancer Nonsteroidal from ProspectiveInflammatoryWomen Drugs : Results the sHealth ‟ RE, CancerHarris Anti RD,Chlebowski RT,Jackson al.Breast andNonsteroidal et Biomarkers Prev. 2000;9(December):1287 Adenomatous Polyp EffectsTrial ofPiroxicam E2 onProstaglandin inRectal Levels Mucosa of inRectal Levels Mucosa of Patients : AdenomatousPolyp ARandomized IIb Phase R, Calaluce DL, Earnest EffectsHeddens D,etal. ofPiroxicam E2 onProstaglandin Inst. Cancer 2010;102(24):1835 withcancer Acelecoxib: randomized, double Elmets Pentland JL, AP,et CA, Viner Chemoprevention of al. nonmela Pharmacol. CancerChemother 2008;62(4):717 biomarker evaluation. activity metastaticin pretreated breast cancer:Resultsofaphase II study with Fabi A,MetroG,Papaldo ofcelecoxib P,etal. Impact o Oncology GroupStudy. 2017;145(2):291 Gynecol Oncol. Predictive anNRG ValueofPotential SerumOncology/Gynecologic VEGF Levels: of Celecoxib for Tr SillRader J, al. M,Beumer Randomized J,et AStratified Double 2007;369(9573):1603 evidence fromconsistent randomisedLancet. andobservationalstudies. Flossmann E, Rothwell o PM. Eff ect cancer.IntJMolSci.2013;14(9):17972of colorectal steroidal anti C,F,MonteleoneStolfi DeSimoneV, Pallone G.Mechanismsof ofaction non classeffectnot a Pharmacol. ofcoxibs.Biochem 2008;76(2):179 mast Aphase Idose tumours): cell phosphate (Pallatoceranib L,Chon E, Kubicek LN,VailDM.Safetyevaluation Mccartan of combination http://doi.wiley.com/10.1111/j.1476 prostatic carcinoma. 2004;2(1):13 VetComp [Internet]. Oncol cyclooxygenase SorenmoKU, Goldschmidt FS, MH,Shofer 8. Transitional Cell Carcinoma Bladder. JVetIntern theUrinary Med. 1994;8(4):273 of Knapp DW,Richardson RC, Piroxicam ChanTCK,etal. Therapy http://bmcmedicine.biomedcentral.com/articles/10.1186/1741 and meta onprostateinflammatory drugs c Liu Y,Chen J from: http://gut.bmj.com/cgi/doi/10.1136/gut.2009.203000 and survival from colorectal cancer. Gut [Internet].2010;59(12):1670 Din FVN,E, Farrington Theodoratou SM, Mechanistic,Pharmalogic,Agents: and Issues. 2002;94(4). Clinical SJ,PatronoThun MJ,Henley C.Nonsteroidal Anti http://www.mcpharmacol.com/index.php/Journals/ar

- analysis. BMCanalysis. 2014;12(1):55. Med[Internet]. from: Available - ts from Cancer REDUCEClin the Res.2015;21(4):756 study. inflammatory (NSAIDs) drugs inthechemoprevention and - Q, Xie L, et al. EffectQ, Xie L,etal. and non of other aspirin - 2 expression the and effect ofcyclooxygenase inhib eating Patients with Cervical Cervical eating Patients with Intraepithelial Neoplasia: The Patients : ARandomizedTrial IIb Phase 1.Cancer Epidemiol – 13. dia®) and piroxicam intumour

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Walton Cowderoy MB, E,Lascelles D, 2014; mavacoxibsafety of and inthetreatmentcarprofen ofcanineos Payne AmVet Sci.2013;8(2):89 JAnim Kim T in osteoarthritic dogs.J Ther.2010;34:1 Vet Pharmacol Cox SR, Liao S,Payne and Cyclooxygenase Innibitors. InVivo (Brooklyn). 2012;26:375 MammaryGland neoplasmwith AdvancedClinical Staging GE, Lavalle Campos AC,Cassalide, Bertagnolli GD. CB Canine Malignant Am Assoc.2002;220(12):1813 VetMed doxorubicin aloneand piroxicamfor ordoxorubicin multicentric lymphomaJ dogs. in AJ,Mutsaers GlickmanNW,DB,DeNicola al.Evaluation et of treatmentwith 2015;56(3):335 forfirocoxib treatment the ofcaninenasalcarcinoma. Ultrasound. VetRadiol Ca carcinoma JAm theurinary bladder. of Vet 2011;238(8):1004 MedAssoc. gemcitabine incombination withpiroxicam treatment withtransitional indogs cell Marconato L, ZiniE,LindnerD, 2016;167(7 chemotherapy : AreviewfromRev ofstudies (Toulouse). 2000todate. Med Vet Karayannopoulou Lafioniatis M, S. advances oncanine Recent mammary cancer thalidomide. VetCompOncol. 2018;16(3):399 primary by lungcarcinoma treated metronomic cyclo G,FinotelloR,SabattiniS,etPolton Survival al. advancedanalysis ofdogswith urothelialinvasive carcinoma. 2016;2(2):241 BlCancer. enhances theactivityinhibitor Knapp DW,Ruple 93. nanocarriers , the future nanocarriers ,thefuture of EurJ chemotherapy. Ph Pérez from:Available http://dx.doi.org/10.1016/j.nano.2014.04.012 Nanomedicinetargeting. N SM, M.Drug Beyond JA,Kavallaris Sagnella delivery : Mccarroll tumour active http://dx.doi.org/10.1016/j.molmed.2015.01.001 Trendscombination therapy. MolMed[Internet]. 2015;21(4):223 Xu X,W, ZhangX, Ho osteoarthritis : randomised a clinical comparator trial.Rec. Vet 2014;1 ncedda S, Sabattini S, ncedda S,Sabattini Bettini G,etal.Combination ofradiation therapy and

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