Repurposing Drugs in Oncology (ReDO)—Propranolol as an anti-cancer agent

Pan Pantziarka1,2, Gauthier Bouche1, Vidula Sukhatme3, Lydie Meheus1, Ilse Rooman1,4 and Vikas P Sukhatme3,5

1Anticancer Fund, Brussels, 1853 Strombeek-Bever, Belgium 2The George Pantziarka TP53 Trust, London, UK 3GlobalCures, Inc, Newton MA 02459, USA 4Oncology Research Centre, Vrije Universiteit Brussel, 1090 Brussels, Belgium 5Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA

Correspondence to: Pan Pantziarka. Email: [email protected]

Abstract

Propranolol (PRO) is a well-known and widely used non-selective beta-adrenergic receptor antagonist (beta-blocker), with a range of actions which are of interest in an oncological context. PRO displays effects on cellular proliferation and invasion, on the immune system, on the angiogenic cascade, and on tumour cell sensitivity to existing treatments. Both pre-clinical and clinical evidence of these effects, in multiple cancer types, is assessed and summarised and relevant mechanisms of action outlined. In particular there is evidence that PRO is effective at multiple points in the metastatic cascade, particularly in the context of the post-surgical wound response. Based on this

evidence the case is made for further clinical investigation of the anticancer effects of PRO, particularly in combination with other agents. Study Clinical A number of trials are on-going, in different treatment settings for various cancers.

Keywords: Propranolol, beta-blockers, drug repurposing, ReDO project

Published: 12/10/2016 Received: 06/06/2016 ecancer 2016, 10:680 DOI: 10.3332/ecancer.2016.680

Copyright: © the authors; licensee ecancermedicalscience. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1 is 38ng/ml(0.12µM)[9]. Athe extended a peakinthe range200–245ng/ml(0.770.96µM),while oral doseof160mgproduced single a singleoral doseof 40 mg,infastingconditions, concentrationfollowing around 10hoursforextendedreleasetablets.Meanpeakplasma doseor single oral dosing,withaplasma half-lifeofaround4hoursfollowing occur 1.5–3hoursfollowing Peak plasmaconcentrations eters (timetomaximumconcentration,half-lifeetc.)areunchanged other param- however meal, taken afteraprotein-containing when reported 50% of around increase with amean food intake, concomitant variability inter-patient there isconsiderable although due toextensivefirst-passhepaticclearance, lungs, liver,to tissues,particularly There iswidedistribution is intherange25–35% after oraldosing kidneys,andheart. Bioavailability and metabolites drug in faecesasunchanged or IVdoseofthedrugappears 1 –4%ofanoral renal, though tion. Excretionisprimarily rapid absorptioninthe PRO gastrointestinal tract and undergoes is and morethan90%undergoes plasmaprotein absorp highly lipophilic Pharmacokinetics bolic acidosisandcardiogenicshock.Itisnotrecommendedinpregnancyduringlactation. in patients sufferingPRO iscontra-indicated meta- artery disease, severe peripheral heart failure, asthma, uncontrolled from hypotension, treatments ofmanyyearsduration should betaperedratherthanstoppedabruptly. In general however, PRO has a good toxicologyprofileandcanbeusedfor long-term for patientssufferingof treatmentisnotadvised,particularly termination dose. Sudden – insuchcasesthedose heart disease ischaemic and psychosis.Ingeneralthe initiation of PRO may lead to initial, mildadverseeffects which resolveduringdosetitrationto a maintenance Rarely PRO vomiting, diarrhoea. is associated withheartfailure,block,hypotension,worseningof symptoms in psoriasis,asthma effectsside Common and Raynaud’sfatigue, coldextremities insomnia, include effects syndrome.Lesscommonside nausea, include Toxicity kg/day for1week,then2mg/kg/dayweekand3asamaintenancedose6months. the doseis120–240mg/day.angina is intherange80–240mg/day[4]. prophylaxis Migraine is1mg/ hemangioma The doseininfantile 160 mg–240mg, required toamaintenancedosethat in divideddosesorasonceadayuseofextendedreleasetablets.For is generally by indication. varies The PROdose mg/day,– 320 160 the range is in dose The anti-hypertensive as increasing mg and at 80 starting Dosage Current Usage evidenced byinvitro,vivoandarangeofclinicaldata. In haspotentanti-cancereffects,there isaccumulatingevidencethat addition to propranolol clinical useinthesevariedindications as akathisia associatedwith diseaseorpsychosis Alzheimer’s use of PRO in a syndrome associatedwithsevereburns[1], number of haemorrhage, sepsisandhypermetabolic conditions, including bytheEMAalso beenlicensed hemangioma. andFDA,forthetreatmentofinfantile the off-labeltrial datasupporting There isalsoclinical Inderal, mon tradenamesinclude Syprol, Ciplar.Angilol, Ain theUSA),has in Europe,Hemangeol oral formulation,(Hemangiol special tablet formulations,as The an oralsolutionandalsoas in bothstandardandextended-release drug is intravenous injection.Com- available Medicines. form andisontheWHOListofEssential in generic globally in the1960s,drugisnowavailable infarction. Firstdeveloped anxiety,myocardial and bleeding variceal migraine, against tremor andasaprophylaxis essential hyperthyroidism, arrhythmia, cardiac receptor antagonistusedinthetreatmentof angina, hypertension, beta-adrenergic (PRO) is acommonlyusednon-selective Propranolol Introduction [5]. [2], andaggressionassociatedwithbraininjuryordisease 2 www.ecancer.org [8]. . Bioavailability can beincreasedby [7]. Bioavailability ecancer [3]. 2016,10:680 [6]. -

Clinical Study rate ofLTRelevated one administered etodolac, orcelecoxib, (indomethacin, treated, butthatCOX-2inhibition comparedtonon-surgically controls to assess theimpactontumourcellretentioninlungs(LTR), and it was shownthat had an all surgicallytreatedanimals treated Rats pre-treatedwithCOX-inhibitors(SC560,indomethacin, etodolac,orcelecoxib)vehiclewerecomparedto non-surgically growth metastatic tumour and function immune Benish post-inoculation. These effects weredose-dependentandreversiblebyco-treatmentwiththenon-selectivebeta-blockernadolol. tumour cellsretainedinthelungs1-daypost-inoculation, andacorrespondingincreaseinpulmonarymetastaticlesionsthree-weeks were abletosuppresstheseeffects. Furthermore,treatmentwithMP wasassociatedwitha10-foldincreaseinthenumberof injected however, therewasaconcomitantdecreaseinNKactivityoverthesameperiod. The beta-adrenergicantagonistsnadololandPRO beta-adrenergic agonistmetaproterenol(MP)ledtoatransient increaseinNKcellnumberswhichreturnedtobaselinewithinonehour; and activity inF344ratsinoculatedwithhighly metastatic MADB106syngeneicmammaryadenocarcinomacells[24].Injection of the Shakhar andBen-Eliyahureportedontheinfluenceofbetaadrenergicagonistsantagonistsnaturalkiller (NK)cellnumber Breast leukaemia celllines growth factor (VEGF) in human vascular endothelial (MMP-2) andthepro-angiogenic factor matrix metalloproteinase-2 tissue remodelling and thatthe effectgrowth anddissemination ofthe the expression to inhibit shown by PRO[22].hasalsobeen be inhibited could by Lamkinet al An investigation in amurinemodelofleukaemia(ALL)foundthat chronic stresstumour acute lymphoblastic enhanced 200 µM. lines and Mirshafiey investigatedthe Hajighasemia cytotoxicity of PRO against Molt-4andJurkat human leukaemiaandthe U937 monocyte cell reversing resistanceinmulti-drugresistanthumanepidermoidKBcarcinomacelllines respectively lines cell P388/ADR in resistantP388/VCRand doxorubicin and the parentalP388cellline.Similarly, Tsuruo(P etalshowedthat10µMofPROsignificantly the cytotoxicityofvincristine <0.05)enhanced shown tohavemoderateeffectseffectsof additional in P388/ADRcells,butshowednoevidence in restoringsensitivitytodoxorubicin in that lookedattheactivityof cell line[18]. a rangeof PRO was drugs inreversingvitrodrugresistancetheP388/ADRmurineleukaemia One ofthefirst a report findings thatPROmaybeofsomebenefitincancertreatmentwasreportedbyRamuetal, who, in1984,published Leukaemia catecholamines incarcinogenesisandidentifyingbeta-adrenergicreceptorbindingsitesdifferent tissues[15–17]. Investigation of the possible anticancer properties of PRO began in the late 1970s, primarily with regards to elucidating the roles of Pre-clinical EvidenceinCancer- opathy, congestiveheartfailure,orrecentmyocardialinfarctionduetothenegativeinotropicandchronotropicactionofthesedrugs Caution isadvisedwhenPRO blocking drugs,particularlyIV is verapamil, inpatientswithseverecardiomy used withcalcium-channel plasma levelsofPRO are possible of druginteractions therefore arange and 2C19), 1A2 and system (CYP2D6, P450 in thecytochrome pathways multiple ofPROinvolves metabolism Hepatic tumours inchildren that theeffectsevidence in thetreatmentofrarebenign including that areachieved, concentrations on theplasma ofPROaredependent release tabletsproducedapeakintherange18–50ng/ml(0.070.19µM)[10]. PRO crosses thebloodbrainbarrier[11] . There issome [21]. of by PRO aboveconcentrations reduced of allthreecelllineswasdose-andtime-dependently that theviability They showed investigated the effectet alinvestigated on post-surgical receptors beta-adrenergic of blocking (COX-2) and cyclo-oxygenase-2 ofinhibiting [12]. [23]. [13]. . For example, concurrent cimetidine increased the area under the curve (AUC) and doubled peak the curve(AUC)anddoubled under the area increased cimetidine concurrent [4, 6].Forexample, In Vitro and . F344 rats underwent laparotomy and were injected with syngeneic MADB106 cells. MADB106 syngeneic with injected were and laparotomy [25]. F344ratsunderwent In Vivo 3 www.ecancer.org . Other investigators have also reported similar effectssimilar reported also have [19]. Otherinvestigators in [20]. ecancer 2016,10:680 [14]. -

Clinical Study centration of 10 µM, with and without exogenous norepinephrine, significantly reduced proliferation (P < 0.001) and increased apoptosis experiments PROwasusedalongsidetwoselectivebeta3 adrenergicreceptorantagonists,SR59230A andL-748,337.PRO,atacon- Dal Monteetalinvestigatedtheroleofbeta3adrenergicreceptorsinmelanoma growthandvascularisation[34].Insomeoftheinvitro ated withincreasedtumourgrowthandthymicatrophythan isolation. associ- to bemore strongly was shown BALB/c mice.Over-crowding in sarcoma-bearing mice and C57BL/6 cohorts ofmelanoma-bearing thymic massandtumour(P < 0.05). Repeatedexperimentscomparedtheeffects versus isolationinadditional of over-crowding was significantlylowerthaneitherof the stressed groups.Organ weight wasalsoassessedanda negative correlationdetectedbetween tation. and cohort (non-stressed) the grouped than rate lower growth with theinitial dynamics, growth tumour reduced This cohortshowed animals. PRO,atadoseof30ppmfromdays-21to+21aftertumourimplan cohort wasadministered over-crowded another Additionally the effectgrouped and isolated over-crowded, the order most in increased rates were growth assessed. B16melanoma ontumourgrowth Meth mice and C57BL/6 Ain BALB/cmice). fibrosarcoma – and over-crowded and grouped – isolated, used were conditions Three housing of micewerehousedindifferentto simulatetheeffects conditions tumours (B16melanomain on growthofsyngeneic ofsocialcrowding and Saiki[33]. stress oncancergrowthintwomurinemodelof The impactof Groups cancers wasstudiedbyHasegawa psychosocial Melanoma plied increasedboththe number andareaof agonist isoproterenol osteolytic lesions comparedto beta-adrenergic controls andthat PRO, sup- mammary carcinomacellmigration. An invivomodel,usingthesamecelllineathymicmice,showedthat chronic stress or exogenous RANKLupregulated signalling that beta-adrenergic showed human MDA-MB-231VU that thisincreased and osteoblasts in expression by Campbell was investigated metastasis tothebone in breastcancer signalling of beta-adrenergic The role median survivalby19%(P =0.0005). (Palone to paclitaxel compared by 79% survival median increased paclitaxel mg/kg, with of 10 PRO with5-FUincreased and =0.0005) injected withMDA-MB-231humantriplenegativebreastcancercells[30].PRO, at adose in NMRInudemiceorthotopically combination Pasquier cell lines. of cancerandnon-cancer drugs inapanel chemotherapeutic of thesestandard effectsanti-angiogenic and the anti-proliferative increased 5-FU orpaclitaxel of PROand concentrations that low first ascertained Having [28–29]. in themetastaticprocessandreportedasimilaranti-metastaticactivityofPROmurinemodelsbreastcancer role ofnorepinephrine (Pstressed animals metastases in in increase the enhanced inhibited but completely animals the investigated also <0.001).Othershave stress was notassociatedwithsignificantchangesinprimarytumourgrowth.PROtreatmenthadnoeffect on metastaticgrowthincontrol the rateofmetastasis(Pnumber andsizeofmetastasescomparedtounstressedcontrols.Increased =0.04),bothintermsofincreased 66cl4mammarycarcinomacells.Stressedanimalsshowedreducedweightanda38-foldincreasein of days priorto syngeneic injection for five stress, orcontrolconditions catecholamine-mediated toinduce previously were subjectedtotwohoursperdayofrestraint,shown The effect of was investigatingina sympathetic nervous system murine breastcancermodelby signalling Sloan et al the PRO+etodolaccombination CpG-C increasedtheeffecttreatment withtheimmunostimulant showed thatcombination similar resultsandadditionally reproduced of cytotoxicity,NK cell reduced work bythesamegroup PRO. Subsequent and of etodolac by thecombination be reversed also whichcould andacutetreatmenthadsimilaroutcomes.Finally,in combination,andthatchronic with it wasshownthatsurgeryalsoassociated that LTRshowed and again onehourpriortoincision), and PRO,(bothadministered etodolac by eithertreatmentaloneand wasreduced hour priorto surgical incision)significantlyattenuatedtheincreasecomparedto the vehicletreatedgroup.Other experiments combined at 2mg/kg). they showed that PRO could inhibitthis antagonist effect, and resensitiseresistantcells,bothinvitro and ina xenograft model(PRO dosed and isoproterenolantagonisedthe effectanti-proliferative catecholamines epinephrine of trastuzumab bothinvitro and invivo. Additionally that the in patienttissuesamples,theyshowed signalling and beta2adrenergic trastuzumab-resistance between ing astrongassociation breast cancer,In HER2-amplified show- and PROonresistancetotrastuzumab [32]. After Liu etalinvestigatedtheroleofcatecholamines ad libitum (0.5g/L)viadrinkingwater, couldreversethisincrease(P <0.05). [26]. 4 www.ecancer.org studied the invivoeffectset alstudied ofthe et al ecancer [31]. Invitroexperiments [27]. BALB/c mice 2016,10:680 -

Clinical Study leading toa59%decreaseinvolumeafter5daysascompared tocontrolspheroids(P <0.001). (Puntreated spheroids <0.001). the growthoftumourspheroids, suppressed completely and vinblastine of propranolol The combination to after 5daysoftreatmentascompared in volume in a19–20%decrease resulting down the growthoftumourspheroids, cantly slowed to form spheroids of ~600 μm in diameter before treatment was initiated. When used alone, 10 μM propranolol and 1 nM vinblastine signifi but thattheeffecttreat angiosarcoma, BMST-RasIn a3Dinvitromodel with vinblastine. in combination wassynergistic cellswereallowed effectsor additive to haveantagonistic Furthermore, PROwasshown used to commonly or paclitaxel, with doxorubicin combined when effectsto theanti-proliferative sensitive were cells endothelial NRAS-transformed and that immortalised at alshowed Pasquier ofPRO[44]. revealed activecelldivision,suggestingtheneedtoemploy combinatorialtherapywithPRO. sham 984+/-92mg;N=15,P <0.0001).Despitethereductionintumoursize, sections frombothshamandPROconditions 20 the exceptionofmurinehemangioendotheliomacellline.Finally, inamurinemodelofangiosarcomaPROtreatment atadoseof cell lines(P <0.05),andshowed synergisticactionincellstreatedwithchemotherapy(cisplatin,busulfan,vincristine,orH2O2) proliferation in all lines compared to untreated controls (P < 0.05). At a higher concentration of 100 µM, PRO induced apoptosis in all canine angiosarcoma,murineangiosarcomaandhemangioendotheliomacelllines,itwasshownthat25µM ofPROinhibited angiosarcoma toinvestigatetheimpactofbeta-blockadewithPROoncellproliferation,migrationandapoptosis[43] . Usingapanelof of investigatorshaveexploredthepotentialbenefitPRO.Stilesetalusedinvitroandvivomodelshemangioendothelioma randomised controlled trial In lightofthepositiveclinicalexperiencePROintreatmentinfantilehemangioma,mostrecentlyconfirmed alargemulti-centre Angiosarcoma intervention inhibitedthepost-surgicalincreaseintumourgrowthrateandreducednumberofmetastaticnodules. RMG-II receptor-negative mice [40]. beta-adrenergic Treatment withPRO via micro-osmoticpumpstartingsevendayspriorto surgical but notin SKOV3ip1) and (HeyA8 models cancer ovarian of metastasesinatwomurine the multiplicity tumour growthrateand primary tumour,distant fromtheimplanted stress, fromawound thatsurgical showed investigation Subsequent with increased wasassociated tumour growth[39]. this increaseinVEGF abolished of 1µM, lines. Pre-treatmentwithPRO,ataconcentration of VEGFinbothcell the production agonist), andcortisolenhanced adrenergic beta- (a nonspecific isoproterenol epinephrine, tion usingtheSKOV3andEG ovarian carcinomacelllinesshowedthatnorepinephrine, incontrast to associated withincreasedangiogenesis, patients withlowerlevelsof social isolation[37]. Subsequent invitroinvestiga- Lutgendorf Ovarian treatments. increases insurvivalrates, although therewerenodifferences ineffect sizes betweenIL-12,IL-12+ PRO + etodolac andPRO + etodolac prior tosurgery.and treatedwithPRO+etodolacorvehicle ment groupswerefurthersubdivided significant All treatmentgroupsshowed vival rates(Ptreat- prior toamputation, 24-hours with IL-12orvehicle mice werepre-treated model carcinoma =0.0345).InaLewisLung differencesur- improved significantly PRO +etodolac combined whereas options, for anyofthesurgical in termsofsurvival with vehicle etodolac, orvehicle30-minutespriorto amputation and/orlaparotomy. Treatment witheitherdrugsinglyshowednostatisticallysignificant aCOX-inhibitor,PRO andetodolac, onsurvival Glasner of 1.7mg/day, tumourvolumesweresignificantlylower(P <0.001)thaninuntreatedcontrols. metastatic humanmelanomatumourstransplantedintoimmunodeficient(NodSCIDGamma)miceshowedthatPROatanaveragedose apoptosis invitroonapanelofmelanomacelllinesathighconcentration100µM[35].Invivoexperimentsusingbothprimaryand (P <0.01)ofB16F10cellscomparedtocontrols.Wrobel andLeGalalso showed thatPROhadsignificanteffects onproliferationand mg/kg every 2 days led to a significant reduction of tumour growth compared to controls (tumour weight of 357+/-58 mg; N = 17 vs investigated the effectset alinvestigated of pre-operative on theimpact and models, mouse two syngeneic in survival on intervention ofsurgical et alnotedthatovariancancerpatientswithgreaterlevelsofsocialisolationanddistresshadserumVEGF, [41], and evidence of beta-adrenergic receptor expression in a range of vascular tumours [38]. Later workshowedthat in a murineSKOV3 model PRO was ableto reduce isoproterenol-induced . B16 melanoma-bearing C57BL/6J mice were administered PRO, etodolac, PRO+ PRO, etodolac, administered mice were C57BL/6J [36]. B16melanoma-bearing 5 www.ecancer.org ecancer 2016,10:680 [42], a number -

Clinical Study metastatic spread in 50%of mice, compared to none inthe controls. PRO treatment, via osmoticpumpdelivering10mg/kg/day, blocked mice (P < 0.01) andincreasedtumourmass five-fold (7.5 mg ± 5 vs. 41 mg ± 13, P = 0.03). chronic stress Additionally was associatedwith rate ofpancreatic tumourgrowthby11%levels, wasassociatedwithanincreased tissue catecholamine ± 3 perdaycomparedtocontrol to mimicstressorcontrolrespectively.cell conditions torestraintorhome Mice weresubject and in bodyweight by changes Stress,verified Kim-Fuchs MIA PaCa-2andBxPC-3celllinesatthesameconcentration in of 100µM[51],andreduceinvasiveness at concentrations cancer cellline pancreatic in thePC-2human apoptosis was abletoinduce Guo Pancreatic cancer PC3cells Brohée pared tocontrols,andPROtreatmentreducedmetastatictumourgrowthbelowcontrols(P =0.009). effect on with asignificantincrease(Pprimary wasassociated tumour growth, norepinephrine = 0.014) inmetastatictumourgrowthcom- via micro-osmoticpumps,andtherateofprimarymetastatictumourgrowthwasassessed.Whileneithertreatment hadsignificant administered PRO oracombination, with PC3cancercellsweretreated norepinephrine, Furthermore, athymicBALB/cmiceinjected this increase inhibited levels, andthatPROatthesamedosesignificantly on proliferation in vitrobuthadno influence cells carcinoma Workprostate at aconcentrationof10µM,increasedthemigratoryactivityPC3human that norepinephrine, byMasuretalshowed Prostate produced lowertumourvolumeatday14comparedtocontrols,(P =0.0246)andwasassociatedwithlongermediansurvival(P =0.0135). alone, P =0.0009forSN-38alone). UsingSK-N-AScellsinxenograftmodels,treatmentwithPROat2mg/kg/dayinjectedsubcutaneously the active metaboliteofirinotecan,withsignificantlyreducedviabilitycomparedtotreatmenteitheragentalone(P = 0.008forPRO reducing proliferation and viability, in all lines at IC50 values in the range 114 μM to 218 μM. It was also shown to be synergistic with SN-38, Wolter significantly lower5mg/kgand10groups(P <0.05). microvessel density(MVD)waslowerinthetreatedgroupsthancontrols(P <0.01)andVEGF, MMP-2,andMMP-9proteinlevelswere group. Tumoursignificant. not statistically than control,afigure lower 18.3% in the10mg/kgwere weights that showed analyses Additional (P36.6% were 5 mg/kggroups in the2mg/kgand tumour weights on day9and (P34.4% and <0.002) than thecontrol lower <0.005) were treatedwithdifferentto untreatedcontrols. dosesofPRO–2,5or10mg/kgforninedaysandcompared Animals weresacrificed Abased ontheBE(2)-Ccellline,wasusedbyXuetal neuroblastoma, modelofpaediatric xenograft complete remissioninoneanimal(often),whichremainedtumour-freeuntilstudycompletion(day60). (Palone vincristine treatment with the mosteffectiveseemed vincristine with Carvedilol <0.0001). in sustained resulted and combination analysis ofsurvivaltimesshowedthatmicetreatedwithPROandvincristinehadafourfoldincreaseinmediancomparedto treatment (Pby vincristine induced the rateoftumourregression that PRO,atadoseof50mg/kgi.p.,increased showed <0.05).Finally, suggesting the effect was related to microtubule disruption.In vivo results, using a TH-MYCN transgenicmousemodelof neuroblastoma, (Psignificant showed paclitaxel, and vinblastine with obtained were results Similar vincristine. alkaloid the vinca with synergy <0.001) effectsimpact ontheanti-cancer not significantly did these threeagents PRO. While and nebivolol they of chemotherapeutics, ofarange agents werecarvedilol, roblastoma celllinesinvitro[45]. and anti-angiogenic Of the sevendrugstestedmostpotentanti-proliferative Pasquier Neuroblastoma showed that PRO inhibited MMP-2,MMP-9andVEGF thatPRO inhibited et alshowed in pancreatic cancercelllines[50]. Zhang etalalsoreportedthatPRO . PRO inhibited cell growth, et altestedPROagainstapanelof15neuroblastomacelllinesrepresentingrangegeneticprofiles[47].inhibitedgrowth, showed that in vitro PRO concentrations of 100 µM potentiated the anti-proliferative effectof 100µMpotentiatedtheanti-proliferative et alshowedthatinvitroPROconcentrations of rapamycinonhumanprostate tested a panel of seven beta-adrenergic antagonists, alone and in combination with vincristine,onBE(2)-CandSHEPantagonists, aloneandincombination et altestedapanelofsevenbeta-adrenergic neu- used Panc-1 human pancreatic cancer cells to establish orthotopic tumoursinBALB/c-Foxn1nunudeathymic mice[53]. cancer cellstoestablish et alusedPanc-1humanpancreatic [49]. [52]. 6 www.ecancer.org [46]. tumours Animals withestablished ecancer 2016,10:680 [48].

Clinical Study of radiolabelled prior toadministration at adoseof10mg/kg,minutes ofsmalltumours[64].PROwasadministered, imaging improve The effectby Bomber was investigated ofPROontumourperfusion However, whiletreatmentincreasedtumour growthlatency, therewasnodifference ingrowthratesoncetumourswereestablished. manner.a dose-dependent the effectdecrease inIgGplasmavalues,confirming There was alsoadose-dependent seenontumoursize. cells. ofLPC-1 injection subcutaneous mice following controls, in to untreated tumour growth compared delayed All treatedmiceshowed BALB/c mice [63]. Mice weretreated with dosesof 0.6. 6 and 60mg/kg/day, viaosmoticpumps, and comparedwithuntreated administered tumour growthofLPC-1plasmacytomatumoursinfemale of PROdelayed invivo,thatadministration Grzanna andco-workersshowed, Other SCC9, SCC17a,SCC25,andFaDucelllines,thatitsynergisedwithcisplatinradiotherapyintreatingSCC17a cells Wolterthis increase. of 1µM,blocked and PRO,ataconcentration cell proliferation of the viability that PROreduced etalalsoshowed cells and similar observations weremadeforinSCC9andSCC25cells. isoproterenol These effects were associatedwithanincreasein in SCC9,SCC15,andSCC25 IL-6 production relevant levels,induced andcortisol,atphysiologically They showedthatnorepinephrine and cortisolonoralsquamouscellcarcinomalines(OSCC)[61]. of norepinephrine assessed theinfluence Bernabé andcolleagues been confirmedinhumanbrainmicrovascularendothelialcells cell invasiveness. Treatmentin MMP-2,MMP-9andVEGF.this increase withPROinhibited by PROofMMP-9secretionhasalso Inhibition increased VEGF and MMP-9) and MMP-2 and (specifically of metalloproteinases levels upregulated of HONE-1cellswithnorepinephrine Yang Head andNeck blocked, invivo,usingPROatadoseof2mg/kg Lin µM, respectively).PROinducedG1-phasearrestandapoptosisinaffected celllines. lines cells Chin the IC50ofPROas65.4µM. of HT-29receptor agonistsandantagonistsonthe proliferation a numberofbetaadrenergic cells [56]andreported colonadenocarcinoma in migratorycellnumbers.Similarly,increase did notinterferewiththenorepinephrine-induced atenolol Coelho antagonist adrenoreceptor beta1 Incontrasttreatmentwiththeselective this increase. µM) abolished (1 µM,10µMand100 concentrations increase inthenumberofmigratingcellsabovebasallevels,treatment withPROatthesame caused adosedependent norepinephrine Masur Colorectal P <0.0058). controls, in 52 days versus (59 days controls to untreated compared survival increased rates and growth tumour reduced kg mouse/day), P <0.0058). Treatmentat 30–60mg/ water aiming of 0.5g/ldrinking water usingaconcentration orally viadrinking withPRO,(given reduced comparedtounstressedcontrolmice(52daysversus66days, cell invasion.Mediansurvivalinstressedmicewassignificantly 6606PDAand cancer in tumourangiogenesis an increase and withimmunosuppression to beassociated tumours[54].Stresswasshown Partecke frame oftheexperiment. the effect of stress on primarytumourgrowth(41mg± 13 vs. 21 mg± 5), although therewasnochangeinthemetastaticratetime- et al that showed chronic restraintstress promoted tumourgrowthinxenograftmodelsof colorectal cancer, and that this effect could be et alalsoinvestigatedtheeffectcancer of colorectal PRO, inapanel receptor antagonists,including of selective beta2-adrenergic investigated the effectset alinvestigated (HONE-1, HNE-1,andCNE-1)[59].Treatmentcell lines carcinoma on nasopharyngeal ofnorepinephrine et al investigated the effect and PRO of on the norepinephrine in vitro migratory activity of SW 480 coloncarcinomacells[55]. Where et al also investigatedtheimpactof chronic stress on pancreaticcancer growth usingC57BL/6micebearingorthotopicsyngeneic [57]. (IC50 119.5,HT29 cells and of SW620,Colo205, the viability inhibited that PROsignificantly They showed 69.1 86.38, and [58]. [60]. 7 www.ecancer.org in a small 1986 study focused on enhancing uptake of Ga-67 to of Ga-67 uptake on enhancing study focused 1986 et alinasmall explored the impactof et alexplored ecancer 2016,10:680 [62].

Clinical Study prostate cancers(HR=0.52;95%CI0.33–0.83). CI =0.49–0.93),and = 0.68;95% (HR CI =0.30–0.98),colon = 0.54;95% – 0.96),stomach(HR CI =0.13 = 0.35;95% (HR oesophagus 0.67–0.85; Pa decreasedriskinheadandneckcancers(HR=0.58;95%CI0.35–0.95), showed <0.001),andsitespecificanalysis non-PRO usegroup(n=12 a largecohortstudyofover24 [75]. Chang etalpublished CI =0.09–0.65;Pratio (HR)=0.25;(95% with ahazard (HCC), carcinoma risk hepatocellular with adecreased was associated <0.004) odds ratio(OR)of0.86(95%CI=0.77–0.96)[74].InpatientswithhepatitisCassociatedcirrhosisNkontchou etalreportedthatPROuse had aprotectiveeffectthat treatmentwithbeta-blockers and found drugs of anti-hypertensive men treatedwitharange withanadjusted patients.For example Perronet dence inhypertensiveandnon-hypertensive of al prostate cancerinCanadian reported ontheincidence effectThe earliesthumandatatosuggestapositive cancer inci- studies comparing came fromepidemiological on cancer ofpropranolol Human Data associated withadecreasedtheexpressionofHIFtargetgenes,includingVEGF. in apoptosis and increase in viability decrease reported adose-dependent of theretinaandcentralnervoussystem. Albiñana hemangioblastoma of arangetumourtypes,including by thedevelopment gene.It is characterised tumour suppressor Hippel–Lindau syndrome, is araregeneticdisordercausedbygerm-linemutationinthevon (VHL) disease, orvonHippel-Lindau von Hippel-Lindau mg/kg showedreducedtumourvolumeincreasecomparedto controls, with reducedSUVmaxonPET/CT and Ki76stainingof tumour cells. treated withPROatadoseof10 and cells 8505C using model of apoptosis.Invivodatafromaxenograftmouse levels ated withincreased lines, with IC50 values of 200 μM and 280 μM, respectively. Growth inhibition was further analysed in 8505C line was shown to be associ- Wei HSP70 andiNOSlevels. survival (P withimproved that wasassociated in IL-10, reductions with statisticallysignificant levels wereassociated <0.05).Bothdosage doses wereassociatedwithreductionstumourgrowthratesandvolumescomparedtountreatedcontrols,itwasonlythe higherdosegroup both tumours [71].While carcinoma Ehrlich solid 10 mg/kg,inmicebearing used twodosesofPRO,5mg/kgand and colleagues Abdi respectively. effectdependent of 141,100,and75µMafter24-,48-,72-hPROexposure, with IC50values and apoptosis, oncellproliferation Kozanoglu showed asimilarnicotine-driveautocrinecatecholaminefeedbackloopinpancreaticadenocarcinomamodel PRO also viability comparedto reduced cells not treated with nicotine. The same authorssubsequentlyextendedthis line of research and (Palone to nicotine cells compared of viable in thenumber reductions significant showed to nicotine <0.0001).Interestingly,to exposure and increased proliferation.CellstreatedwithPRO epinephrine at a concentration of 1 µM for 10-minutes priorto acute or chronic exposure proliferation (NSCLC), cancer lung cell In non-small Al-Wadeithe effectsinvestigated colleagues and cell cancer on to nicotine exposure ofchronic regulation ofVEGF, COX-2,andEGFRexpression. down- and NF-κB of level decreased a with associated was PRO apoptosis. and radiosensitivity in increase significant a displayed lines same gastriccarcinomacelllineswasalsoinvestigated[67]. When pre-treatedfor 24 hourswithPRO at a concentrationof 50 µMbothcell cell cyclearrestandapoptosisinbothlines. of 200µMinduced In vitroconcentrations The effectof the ofPROontheradiosensitivity The effect of PRO was alsoinvestigatedintwo human gastriccarcinomacelllines(SGC-7901andBGC-823)by Liao andcolleagues ment (P <0.05)comparedtoeithernorepinephrineorPROalone in thetumourcompart- rate ofmicrospheres the embolisation supply tohepatictumoursinrabbitsanddoubled the relativeblood enhanced and PRO Burton andGray of reported that ing therelativeuptakeintumourcompartment.Subsequently norepinephrine a combination citrate. gallium tissues, therebyincreas Ga-67 uptake innon-tumour tumour perfusionanddecreased that PROdoubled Analysis showed et theuseof al PRO explored in apanelof human thyroidcancercelllines[72]. PRO wasshownto inhibit growthof8505CandK1cell et al investigated theinvitroeffect of PRO on theU266humanmultiplemyelomacellline[70]. They showedadoseandtime . Results showed that nicotine-treated lung adenocarcinoma cells, nor- [68]. (NCI-H322, NCI-H441andNCI-H1299),released lungadenocarcinoma Results showedthat nicotine-treated 119) overa12yearperiod[76].OveralltheriskofcancerwaslowerinPROgroup(HR =0.75;95%CI explored the effectet alexplored cells fromVHL ofPROonhemangioblastoma patientsand 238 patients,withaPROgroup(>6monthsuse,n=12 [65]. 8 www.ecancer.org [73]. This effectand was in hypoxicconditions wasenhanced [69]. ecancer 119) comparedtoa 2016,10:680 [66]. -

Clinical Study (40 mgBID),weekly vinblastine (i.v. 6 mg/m Subsequently thesamegrouphavereportedonaseriesofsevencasesadvancedangiosarcomatreatedwith combination ofPRO but ultimatelydiedofprogressivedisease. 100 mgwithsomeresponse and oralthalidomide radiotherapy palliative treatment andwastreatedwithlocal of themetronomic initiation treatment ceased. months afterwhich days forsixadditional mg) andPRO(20mgBID),onalternate 20 monthsafter The patientrelapsed after two cycles of therapy. After one yearof treatment the patient remained onamaintenancetreatment(50 of oral cyclophosphamide and PRO(40mgtwiceaday)[81]. celecoxib (etoposide andcyclophosphamide), chemotherapy The patientshowedacompleteresponse of metronomicorallow-dose treated withacombination metastatic angiosarcoma onacaseofrelapsing reported and colleagues Banavali Angiosarcoma CI =0.32-0.80)andnoimpactofall-causemortality(HR1.02;95%0.751.37) cancer recurrence(HR= 0.67; 95%CI = 0.39-1.13),asignificantreductionintheriskof breast cancermortality(HR= 0.50; 95% Ause andbreastcancerbyChilders systematicreviewandmeta-analysisofbeta-blocker CI =0.06–0.60). There wasnodifference ineitheroutcomebetweenatenololusersandmatchednon-users. probability of lower for breast cancer-specificmortalitywasalsosignificantly PRO users compared to matched nonusers(HR= 0.19; 95% 0.24, 95% CI = 0.07 – 0.85) or N2/N3/M1 (OR = 0.20; 95%C = 0.04 – 0.88) diagnosiscompared to matched non-users. The cumulative with a risk ofpresenting with alower in theratio1:2.PROusewasassociated treatment (n=4738) beta-blocker not receiving T4 (OR= were matchedwithwomen (n =525)intheyearpriortobreastcancerdiagnosis [79]. Irish womentreatedwithPRO(n=70)oratenolol protective effectsof breastcancerpatientsshowed by Barronetalinalargerpopulation Results published with PROuse associated Breast Cancer address immortaltimebias no attemptismadeto when studies isthereforerequired of observational interpretation time bias.Careful to immortal not prone to studies use andsurvivalwhenrestrictingtheanalysis for anassociationbetweenbeta-blocker evidence meaningful studies andfoundnoclinically (HR =1.17;95%CI1.04–1.32;P =0.01).However, Weberpals (HR =0.81;95%CI0.67–0.97;Pcancer typeshowedbothabenefitformelanoma cancer risk forovarian =0.02)andanincreased tal orprostatecancer.effectno significant there was of beta-blockers use Forpre-diagnosis mortality,all-cause on by butstratification Peffecttype therewasnobeneficial on stratificationbycancer =0.03).However for breast,colorec use ofbeta-blockers ofpost-diagnosis cancer-specific mortality, beta-blocker usewasassociatedwitha reduced risk of cancer-specific mortality(HR= 0.89; 95% CI = 0.79 – 0.99; forbreastcancer(HR=0.82;95%CI0.68–0.99;Pin riskofdeathwasonlysignificant fied bycancertypethereduction =0.03).Intermsof CI =0.81–0.98;Pmortality (HR=0.89;95% in theriskofall-cause reduction signification with astatistically associated =0.02).Whenstrati- was use ofabeta-blocker patients. Post-diagnosis 76 538 results byMay2015,involving reported that had studies 24 relevant identified Zhong dose of40mg TID, withclearsignsofdiseaseregressionandnoevidence ofmetastaticdisease. radiotherapy.and index ofthedrug) peutic to takePROata has continued the patient radiotherapy and of paclitaxel cessation Following the thera- to increase designed of paclitaxel (a formulation poliglumex of paclitaxel 10-weeks concurrently withPRO,included administered Staining for Ki-67 showedthat the PRO monotherapy wasassociatedwitha34%reductioninproliferationrate. Subsequent treatments, 40 mgBID was initiatedleadingto clinical improvementwithina week andasubsequentincreaseinthe dose to 40 mgthreetimesaday. metastatic multifocal stage witha2-yearsurvivalrateof T2 0%. cutaneous angiosarcoma, adiagnosis Treatment withPRO at a dose of Chow response andthreeverygoodpartialresponses.MedianPFS was11 months(range5–24)andOSwas1610–30). secutive daysincyclesof30[44]. The treatmentwaswelltoleratedandshoweda100%responserate,including onecomplete 12 months followed by maintenance of PRO (40 mg BID), oral etoposide (50 mg/day) and oral cyclophosphamide (50 mg/day) for 20 also published a case report in angiosarcoma treated withPRO[82]. a casereportinangiosarcoma et alalsopublished non- disseminated The patientpresentedwithawidely performed a systematic review and meta-analysis of observational studies of beta-blocker use andcancer mortality[77]. They studies ofbeta-blocker of observational et alperformedasystematicreviewandmeta-analysis [78]. 2 toa maximum of10mg)andmethotrexate (35 mg/m 9 www.ecancer.org evaluated a possible immortal timebiasintheseobservational a possible et alevaluated [80]. found a non-significant reduction in breast reduction et alfoundanon-significant 2 toa maximum 50mg)forupto ecancer 2016,10:680 con- -

Clinical Study 10 www.ecancer.org Ewing’srhabdomyosarcoma, four extensioncohortsof 9 patients(neuroblastoma, dose ofthecombinationisestablished recommended and PROwillbeperformed.Oncethe of vinorelbine analysis of thefirstcycle.Pharmacokinetic PRO 1.5mg/kg/dayBID)aftercompletion (PROVIN) NCT02897986 – This planned PhaseI trial (10, dose escalation 20 and30mg/m² of onlyplusdaily thrice weeklyoralvinorelbine vironment. Secondaryoutcomemeasuresincludeone-year PFSandOS. of toxicity, areincidence Primary end-points and tumourmicroen response of impactsonimmune in VEGF levels, measurement change label studyaretreatedwithPRO BID (dose notspecified)for four monthsintheabsenceof toxicity.disease progressionor unacceptable – NCT02013492 Atumours. Patientsinthisopen- or metastaticsolid recurrent with non-resectable PRO inpatients study oforal feasibility Treatment commences48–72hourspriortosurgicalresectionandcontinues forsixcyclesofchemotherapypost-surgery. cancer.apy inovarian study,as afeasibility This isdesignated treatment. of patientscompleting of theproportion withaprimaryend-point – NCT01504126 Achemother or taxane platinum standard and (n =25)trialofPRO,20mgBID,withsurgery arm, open-label single small Feasibility/Phase I As of31 Clinical Trials distress, measuredintermsofthenumberandrateintrusivethoughts,associatedwithacancerdiagnosis effectsThere hasalsobeensomeinterestinthepsychological of emotional the level in reducing ofPROincancerpatients,forexample in heartrate,wassignificantlymorepronouncedcomparedwithatenolol(P<0.05). solidtumourpatients[89] . While bothdrugssignificantlyreducedREE(P10 weight-losing <0.05), PROtreatment,accountingforadecline in theothertreatmentgroupsorcontrols. A(50 mg/day)in PRO (80mg/day)andatenolol study bythesamegroupcompared subsequent after 5daysoftreatment(Pto base-line in REEofaround10%compared decrease changes there werenosignificant <0.02),whereas or placebo morphine indomethacin, COX inhibitor mg BID),thenon-selective PRO (80 taking in late-stagecancer patients [87]. Hyltander et al cancer explored influencesonrestingenergyexpenditure(REE) in weight-losing patients and mortality of morbidity cause a significant cachexia, cancer-related of PROinaddressing some interestintherole been There hasalso was 9.6monthsversus17.2inthesorafeniband+VT-122 armsrespectively. VT-122OS in HCC[86].Median with sorafenib trial incombination controlled wasalsousedinasmall(n=20),multi-centre,randomised + VT-122 armexperiencedreducedpainandneuropathyincreasedweightgaincomparedtotheGemNabpatients. OS was 10.5monthsversus15.9fortheGemNaband+VT-122 armsrespectively. patients intheGemNab Additionally with VT-122with GemNab.PFSwas7.2and11.8of GemNab,andthencontinuously to commencement foroneweekprior months,and VT-122cancer or metastaticpancreatic advanced with locally patients in with andwithout (GemNab) nab-paclitxel and trial ofgemcitabine centre open-label (n =37)single on asmall reported also The samegroup respectively at ASCO, with 41patientsincluded,showedamedianOS of 9.2monthsversus17.6months,andaresponserate 35% versus57% arm. Survival at six months was40%and63%,OS at one yearwas12%versus22%respectively. An update in2015,alsoreported 400mgBID). of PRO20mgBIDandetodolac The median TTP was5.2monthsinthemTMZ arm and8.8monthsinthemTMZ + VT-122 at glioblastoma recurrent ASCO 2014[83]. AmTMZ orwithVT-122to either randomised were of 32patients series (the combination Bhattacharyya Other or soontocommencerecruitment)on-goingareincluded. st May 2016, a number of clinical trials are investigating the anti-cancer uses of PRO. Only trials which are currently open (recruiting open are currently uses ofPRO.Onlytrialswhich the anti-cancer are investigating trials May2016,anumberofclinical [84]. et al reported ontheuseof (mTMZ), the combinationof metronomic temozolomide the COX-2inhibitoretodolacand PRO in . Patients in the GemNab + VT-122the GemNab [85]. Patientsin were treated arm (n=20) . Patients in the PRO group showed a showed the PROgroup [88]. Patientsin [90]. ecancer 2016,10:680 - -

Clinical Study 11 sion foreachof20,000genesatbaselineandsurgicalresection. the dayof surgery andthenwillbetitratedoff PRO over twodaysinthepost-operative period. expres- The primaryoutcomeistumourgene cancer. to andincluding PRO (40mgBIDdays1–3,804-8)prior 7 daysofpre-operative receive will The treatmentgroup – ACTRN12615000889550 A Phase II randomised study of perioperative PRO vs breast placebo ongeneexpression innewlydiagnosed comparison to a historical control group. Secondary outcomes include the disease control rate at 6 months and overall survival at 12 months. a maximum of 120 mg/day, in two divided doses. The primary outcome is to demonstrate the non-inferiority of event free survival (EFS) in (PCCV) followedbyfivecyclesPCCVresultinginatotalof 13cycles(364daysoftreatment). The dailydoseofPROis0.5mg/kg/day, to phosphamide, fortnightly i.v. vinblastine, oral etoposide (PCCVE) and of PRO, celecoxib, oral cyclophosphamide, fortnightly i.v. vinblastine gressive highriskneuroblastoma(METRO-NB2012). Treatment consistsofeightalternating28-day-cyclesPRO,celecoxib,oralcyclo- NCT02641314 – A Phase II randomised trial of metronomic chemotherapy and PRO in children and adolescents with recurrent or pro- that aprevioustrialofVT-122 inNSCLC-relatedcachexiahasyettoreportresults(trialcompletedDecember2012). of cachexia. at 6-months. survival outcome isfailure-free The primary outcome. Note is thesecondary rate at6-months benefit The clinical – NCT01265576 Atrial ofVT-122placebo-controlled PhaseIIrandomised, (HCC) patientsatrisk carcinoma in hepatocellular withsorafenib PSA doublingtime,PSA progressionandtimetosymptomprogression. twice aday.mg ofetodolac 340 this trialis22mgPROand in PSAis change Primaryoutcome outcomes include at12weeks.Secondary as VT-122,cific doseof22mgPROand340etodolac,isdesignated of thetrial,Vicus bythesponsor Therapeutics. The doseusedin – NCT01857817 Atrial ofPROandetodolacin prostate cancer. PhaseIIcontrolled randomised, placebo at aspe- The drugcombination, currently suspendedduetofinancialreasons]. to amaximum of160mg/day.will receivePROatadoseassessedbycardiologist, The primaryoutcomeisfive-yearPFS.[Recruitment – NCT01988831 Arecurrence risk ofdisease Patients athigh primary melanoma. trial inhigh-risk controlled PhaseIIrandomised,placebo outcomes relatetosafety, toxicityandadherence. for three weekspriorto administered surgical resection. The primaryoutcomeisareductionintheproliferativeindex(Ki-67),secondary – NCT02596867 Aof opportunity’‘window PhaseII open-label breast cancer. trialinnewlydiagnosed PRO, at a doseof 1.5 mg/kgBID, is comes includeadverseeventratesandchangesintumourproliferation(Ki-67staining). Diffuse(as assessedusing in angiogenesis changes Optical Tomography)Secondary out- stress levels. in psychological and changes chemotherapy,during are PROcompliance outcomes to 40mgthen80BID.Primary dose ofPROis20mgBID,butup-titrated ing – NCT01847001 Achemotherapy.neo-adjuvant breast cancerpatientsundergoing study innewlydiagnosed PhaseII open-label The start- Phase II The primaryclinicalend-pointisthefive-yearrecurrencerate. ers (numberandcytotoxicactivityof NK cells,levelsof NK T-cells, lymphocytes, monocytesandgranulocytes;levelsof cortisol andVEGF). days post-surgery. biomark- immune andangiogenesis-related primary outcomemeasures including The trialhasanumberofbiochemical Womenuntil three starting twodayspriortosurgeryandcontinuing receive 40mgofPROand800etodolac inthetreatmentarmwill – NCT00502684 Abreast cancersurgery.PRO andetodolacinwomenundergoing trial ofperi-operative placebo-controlled randomised, Randomised –nophaselisted or metastaticangiosarcoma(PROPAN) – NCT02732678 Aadvanced in patientswithlocally with metronomicfixedoralcyclophosphamide trial ofPROincombination dose-finding isotypes) willalsobeevaluated. receptor expressionandbeta-tubulin tumours) willbeadded.Potentialbiomarkers(tumourbeta-adrenergic sarcoma, andmiscellaneous www.ecancer.org ecancer 2016,10:680

Clinical Study 12 www.ecancer.org by IL-6. rate induced proliferation the increased to inhibit rate. PROwasalsoshown in proliferation the increase ies inhibited A decrease IL-6 usingantibod neutralising or isoproterenol, in responsetonorepinephrine fromOSCCcellslines to showingincreasedIL-6production Bernabé line this inhibition[102]andotherauthorshavereportedadecreaseinproliferation inbreastcancercellline,forexampletheMCF7 adrenergic agonists isoprenaline and salbutamol reduced breast tumour growth in animal models, and that PRO treatment reversed In contrast,therehavealsobeencontradictoryresults,for exampleforbreastcancerwherePérezPiñeroetalhavereportedthatbeta breast (MCF7),colon(HT-29), andhepatocellular(HepG2) cancer cellsthanatenolol showed that colleagues PRO and ICI118,551 weremorepotentinreducingthe proliferation, migration,andinvasionof non-stimulated ICI118-551and and (beta1) Işeri atenolol 2), antagonists (beta receptor adrenergic beta selective the and PRO between comparison a In catecholamines orisoproterenolinanumberofcancercelltypes with associated in cancercellproliferation the increase that PROisabletoinhibit rate ofgrowth[97].It has sincebeenshown a reduced Iwata, blockade, withPROandotheragents,wasassociated with Kariya andFujimotoshowedthat in aciliatedprotozoanbeta-adrenergic DNAand triggered synthesis[96]. in thesameera,forexample due toPROwasalsoreported of proliferation The converse,areduction the rateofmitosis increased that isoproterenol confirmed mitotic rate[95].Barkasubsequently to anincreased most likelydue glands, of salivary growth excessive displayed isoproterenol agonist the beta-adrenergic treated with that ratschronically reported and Cantin leux extend backmorethanfifty years. In1961Selye,Veilproliferation on cellular signalling of beta-adrenergic Investigations oftheinfluence - Proliferation the metastaticprocess[94]. effects of PRO, pathway andwhichmaybeparticularlyimportant inthecontext of many of them associatedwiththebeta2-adrenoreceptor repurposing and thereexists some evidencethattheseparticulardrugsmayalsohavepotentialin and carvedilol blocker drugs,particularlypindolol 100-fold) for the beta3-adrenoreceptor (approximately PRO receptorantagonist,with is beta-adrenergic a non-selective similar bindingaffinity for beta1- andbeta2-a much lower affinity of Action Mechanisms and sizeofretinalhemangioblastomasat12-months. retinal hemangioblastomas. and juxta-papillary thegrowthofpapillary kg, incontrolling in thenumber is areduction The primaryendpoint EudraCT – 2014-003671-30 This open labelPhaseIII trial inpatientswithVHL syndrome willassessthe efficacy of PRO, at a dose of 2 mg/ Secondary endpointsareimmune-related. mg PO BID forthesecondpostoperativeweek. end-pointistherateoflocalanddistantrecurrenceat three years. The primaryclinical days, 80mgBIDonthedayofsurgery,vention period,PRO20mgBIDfor5pre-operative 40 mgBIDforthefirst postoperative week,20 resection. Patients in thetreatmentarmreceiveetodolac800mgBID etodolac incolorectalcancerpatientsundergoing for the entireinter- – NCT00888797 PRO and trial ofperi-operative and isaPhaseIII randomised, placebo-controlled This isasistertrialtoNCT00502684 Phase III metastases. undergoing surgicalexcisionoftheprimarytumour. The primaryoutcomeisareductionoftwo-yearraterecurrenceanddistant ACTRN12612000852853 – A PhaseIIrandomisedcontrolledtrialofperi-operativePROandetodolacincolorectalcancerpatients [101]. showed that OSCC proliferation in response to increased beta2 adrenergic signalling was mediatedbyIL-6[61]. In signalling addition in responsetoincreasedbeta2adrenergic et alshowedthatOSCCproliferation [92–93]. There area number of distinct putativemechanismsof action that have beeninvestigatedinrelationto the anticancer [91]. In usedbeta- this respectPRO has asimilarselectivitytosomeotherclinically [56, 98–100]. [101]. ecancer 2016,10:680 -

Clinical Study showed increased invasiveness, in response to co-culture with norepinephrine via a beta2-adrenergic signalling pathway signalling via abeta2-adrenergic norepinephrine with to co-culture in response invasiveness, increased showed Shan Tissuetransition (EMT)whichisimportantinmetastasis[112] is oneofthekeystepsinprocessepithelial-mesenchymal remodelling . 231-HM breast cancer cell line cell breast cancer 231-HM Pon 13 www.ecancer.org cancer [107]andofMMP-9inmedulloblastoma carcinoma also beenreportedinnasopharyngeal could reducethe rate of tumour growthandinfiltrationinvivo. A of similar inhibitionof the upregulation MMP-2 and MMP-9 by PRO has increase inovariancancerinvasiveness expressed duringthenorepinephrine-induced process [104–105] a centralroleinthetissueremodelling ases (MMPs)playing and themetastaticcascade,withmatrixmetalloprotein matrixisakeyfactorintumourprogression of theextra-cellular Degradation cell lines Strell Migration andInvasion growth comparedtountreatedcontrols in circulatingIL-6 to levels inresponse PRO has beenreportedin a murine melanomamodel,concomitantwithalowerlevelof metastatic congenital lymphangioma between cAMP andCa ICI-118551.antagonist with thepositivefeedback to beassociated was shown cell line of theMDA-MB-231-HM The increasedinvasion receptor by PROandtheselectivebeta2adrenergic ERK, whichwascompetitivelyinhibited and adecreaseinphosphorylated signalling [120–121] The processof metastasis dependsalsoonthecreationof a ‘metastaticniche’,inadditionto the propertiesof the primarycancercells response toisoproterenolandanincreaseincellmigration,whereastreatmentwithPROreducedthelevelofmigration andmaypromoteametastaticphenotype cell adhesion small GTPase that increases cell-celladhesion. There is evidence to suggest that loss of Rap1B at the plasma membranedecreases cell- is a is active.Rap1B signalling that adrenergic and heretoothereisevidence adhesion, Also importantinthemetastaticprocessiscell-cell inhibited thisincrease increased the rate adrenergic signallingformation of in humanandmurinebreastcancercelllinesvitro, invadopodia and that PRO to beactiveinthesecretionofMMPs[115]structures formedonthesurfaceofcancercellsbelieved . Creedetalhaveshownthatbeta2 matrix andtheEMTof theextra-cellular to thedegradation Related which areactin processistheformationoftumour cellinvadopodia, 10 µM,inhibitedtheincreasein TGF-β1 andpartiallydecreasedthemigrationinvasivenessofnorepinephrine-treatedcells. lines of EMTand initiation signalling catecholamine increased between al showedasimilarassociation inHT-29 and cancer cell A549 colorectal elling was mediated by VEGFC production, which was induced by COX-2/PGE byVEGFC production, whichwasinduced was mediated elling by isoproterenol or inhibited by PRO treatment of BALB/c mice injectedwithMDA-MB-231breastcancercells. The remod- stress-induced leading to elevated levelsof lymphatic draining[123]. These effectsand couldbeenhanced signalling were sensitiveto beta-adrenergic an increaseinlymphaticvesseldensityanddilation of stress the tumourlymphaticvasculature, including induced aremodelling that chronic in recentyears[122].Leetalshowed recognised increasingly The roleofthelymphaticsysteminmetastasishasbecome reversed byPROortheRANKL inhibitordenosumab. that thiseffectand signalling, to catecholamine inresponse lesions of osteolytic the number increased of RANKL, which lation be could effectsof bone viaadrenergic signalling cancer cellcolonization on thebonemarrowstromalcompartment[31]. This effect was viaupregu described a beta2 adrenergic receptor activated feed-forward loop driving the invasiveness of the highly metastatic MDA-MB- of thehighly the invasiveness driving loop feed-forward activated receptor adrenergic abeta2 et aldescribed [114]of concentration . a at PRO, EMTthat induced and norepinephrine process that the showed mediated they TGF-β1 Additionally et al have shownthat PRO increaseinmigratoryactivity is of able to a abrogate therangeof norepinephrine-induced breast cancer et al that showed changes typicalof the EMT,human gastriccancercelllinesBGC-823andSGC-7901underwentmorphological and [28–29]. . Campbell at al showed that in a mouse model restraint-induced stress or exogenous isoproterenol promoted MDA-231breast isoproterenol stress orexogenous at alshowedthatinamousemodelrestraint-induced . Campbell [116]. 2+ butindependentoftheeffect onpERK. [124]. [111] . Ain cAMPincrease dependent a dose caused agonists receptor adrenergic of beta2 range Ca and [103]. [108] andinfantilehemangioma , pancreatic cancer [50,53],gastricadenocarcinoma [59], pancreatic [117–118]in adhesion cell-cell reduced breast cancercellsshowed . MDA-MB-231 reported that MMP-2andMMP-9werehighly . Soodetalreported 2 [109–110]. . PRO has also been explored as atreatmentoption for . PRO hasalsobeenexplored [39]. Furthermore theyshowedthatPROtreatment [66], melanoma ecancer [119]. 2016,10:680 [113]. Zhang [106], prostate et 2+ - -

Clinical Study while PROsignificantlyreducedexpression(P <0.05)[66]. The COX-2/prostaglandinE2(PGE In addition to VEGF, MMP-2 and MMP-9, Liao been showninvitroandvivoamurinepancreaticcancer model adrenergic signalling on HIF-1α, VEGF and angiogenesis. Similar results, using a beta2-adrenergic receptor antagonist (ICI118 551), have the effecttube formation.FurthermorePROpre-treatmentabrogated and capillary in VEGF increase expression epinephrine-induced of nor- the p70S6K pathway,reduced HIF-1α in additionto VEGF,of Silencing and that process [132]. it plays akeyroleintheangiogenic Park potentially reducingtumourangiogenesis. MMP-9 the expression andreduced endothelial rate PRO of cellstubologenesis, down-regulated human brainmicrovascularendothelial was effective [131], biopsy samples[60]. investigated PRO ininfantilehemangiomas It activity inhumanglioblastoma was reportedthat effectsthe anti-angiogenic in a role play may also Other mechanisms ofPRO.Forexample, Annabi increased survivalandreducedangiogenesisinamousemodelofneuroblastoma hemangiomas geal carcinoma[59], melanoma effectAn anti-angiogenic nasopharyn including lines of cancercell in arange shown of VEGFhasalsobeen ofPRO,viadown-regulation and theconsequentincreaseinangiogenesistumourgrowth. growth andtumourangiogenesis rates oftumour with increased and thatthiswasassociated cancer model ovarian in amurine signalling beta-adrenergic increased induced tumour angiogenesis stress enhanced behavioural of thecAMP-PKAactivation beta-adrenergic MMP-9. Inparticular and by which mechanism as amajor identified was pathway signalling stress chronic behaviouralwasassociatedwithincreasedtumourgrowthandvascularisationenhancedexpression of VEGF, MMP-2 In vivoworkusingamurinemodelofovariancancershowedthat tumour growthviaincreasedangiogenesis. ioural stressandenhanced 14 www.ecancer.org of 1µM,blockedthisincrease and SKOV3),thatPRO, at aconcentration (EC lines cell cancer of VEGFintwoovarian the expression increased agonists that betaadrenergic showed colleagues and Lutgendorf ergic signallingbynorepinephrineinducedincreasedlevelsofVEGFexpressioninbrownadiposetissue in thelate1990s,whenitwasshownthatbetaadren was firstelucidated and angiogenesis signalling between adrenergic The relationship Angiogenesis tigated thepro-apoptoticeffect ofPROonneuroblastomacelllinesandshowedthattreatmentincreasedexpressionp53p73[47]. anti-apoptotic ΔNp63a and induction of the pro-apoptotic TAp73b in both SCC9 and SCC17a cell lines. Some of the same authors also inves- to down-streamactivityofp63andp73,bothp53-familyproteins.FollowingPROtreatmenttherewasevidencedownregulationthe (HNSCC) cell lineswith differing p53 status The pro-apoptoticactivityofPROwasalsoinvestigatedbyWolter andcolleaguesintheirworkheadnecksquamouscellcarcinoma tosis wasassociatedwithadecreaseinlevelsofNF-κB,VEGF, COX-2,MMP-2andMMP-9expression[66]. Liao prolol. ChinandcolleaguesalsoshowedthatincolorectalcancercelllinesPROwasassociatedwithcyclearrestapoptosis[57]. the increasedlevelofapoptosisinducedbyselectivebeta2antagonistbutaxamineandreducedduetobeta1blockermeto- tosis Zhang Apoptosis reported that in vitro PRO concentrations of 200 µM induced cell cycle arrest and apoptosis in gastric carcinoma cell lines. of 200µMinducedcellcyclearrestandapoptosisingastriccarcinomalines. et alreportedthatinvitroPRO concentrations Apop- [51]. The pro-apoptoticactionofPROwasfoundtobeviablockadethebeta2-adrenergicreceptorratherthanbeta1,asshownby showed that hypoxia-inducible factor 1α (HIF-1α) expression is also upregulated by norepinephrine, via the cAMP/PKA/Akt/ the via norepinephrine, by upregulated also is expression (HIF-1α) 1α factor hypoxia-inducible that showed et al et al showed that PRO limited the expansion of the PC-2 pancreatic cancer cell line and that this was due to an increased rate of apop- [129–130] . Pasquier [128], pancreatic cancer[50], leukaemia [23], head andnecksquamouscellcarcinoma[62]infantile [40]. PRO was shownto Perioperative inhibit thesurgicalstress-inducedincreaseinVEGF expression showed that the combination of beta-blockers, including PRO, with vincristine was associated with was associated PRO, withvincristine including ofbeta-blockers, that thecombination et alshowed [62]. PROtreatmentwas shown to cause apoptosis irrespective of p53 status and was related et al showed that isoproterenol increased levels of COX-2, in gastric cancer cell lines, . In subsequent work some of the same authors showed that surgical stress work someofthesameauthorsshowedthatsurgical [127]. In subsequent [133]. [38]. This findingsuggestedaputativelinkbetweenbehav [45]. 2 ) pathwayisalsoknowntobeinvolvedin , following initial reports that PRO reports initial et al,following [125–126] ecancer . 2016,10:680 - - -

Clinical Study 15 www.ecancer.org (P <0.05)andinhibitthereductionofphagocyticactivityinducedbyphysicalrestraint-induced stress(P <0.01)[146] . Teshima receptor antagonists. for example[144–145] of cancer [143]. While thereareanumberof papers whichreviewtheeffect system, stress ontheimmune of physical andpsychological important inthe logical stress context and increasingly and immuneresponse,akeyconcernwithinthe field of psychoneuroimmunology cells. and, insomecases,directlyoncancer microenvironment psycho- is thelinkbetween relationship An importantaspectofthiscomplex mediated by theeffectsare primarily on different ofsympatheticnervous system(SNS)signalling of immunecells,thetumour populations A mechanisms of number of action havealsobeenoutlinedasimportantintheanti-cancereffectsimmune-related of PRO. These effects Immunological NF-κB/COX-2/PGE the to related be may radiosensitivity increased for mechanism The [62]. We mayalsonotetheconcurrentuseofPROandchemoradiotherapyinonecasereportsangiosarcoma the effectthat itenhanced celllinesandshowed PRO onHNSCC of synergywithcisplatin evidence to displaying ofradiation,inaddition the effecttion of50µMfor24hours,increased on cellviabilityinvitro[67]. Similarly,of radiotherapy Wolter etalassessedtheimpactof with PRO,ataconcentra- BGC-823 andSGC-7901 (HGC)celllines gastric adenocarcinoma that pre-treatmentofthehuman et alshowed radiation. Liao PRO and between the relationship work investigating some preclinical been therehasalso to chemosensitisation In addition and drugspecific,forexamplePROseemedtohavelittleimpactonthecisplatinsensitivityofNSCLCcelllines resistant P388murineleukaemia in differentresistant phenotype to revertthedrug the potential that PROhad showed in vitrowork Early doxorubicin- including lines, cell Treatment Sensitisation induced IκBαdegradationandenhancedNF-kBtranscriptionalactivityinatime-dependentmanner[136]. and colleaguesshowedthatgeneticdeletionofb2-adrenergicreceptorsimpairedangiogenesisinamousemodel,isoproterenol cancer-associated angiogenesis[134–135]. These effects were partly due to activation of the activation of NF-κB pathway inhibitor vemurafenib thyroid cancercellstothetargetedBRAF-V600E [142]andsensitising cancerinanvivomodel activity incolorectal tion ofsunitinib human prostatecancerPC3cells[49], reverting resistanceto trastuzumab inHER2breastcancer[32],thestress-relatedreduc- inhibiting the effectsensitivity todrugtreatmentenhancing cancer cell of PROactingtoimprove There arestillotherexamples on ofrapamycin group publishedresultswhichshowedPROsynergisedwithvincristineinamurinemodelofneuroblastoma for paclitaxel alone,or 47 for control, P = 0.0005) and5-FU (56 daysvs 47 for 5-FU or 44 for control, P = 0.0005). Subsequentlythe same week for5weeks). improvedmediansurvivaltimesbothfor paclitaxel (125daysvs treatmentsproducedsignificantly 70 The combination of paclitaxelandPRO. combination The sameprotocolwasusedwith5-FU,andthedoseofdrug5-FUalone(30mg/kg,3daysa (10 mg/kg,5daysaweekforweeks)orthe (20 mg/kg,3daysaweekforweeks),PROalone alone paclitaxel trol (saline-treatment), mice wereused,con- drug. Fourtreatmentgroupsoftumour-bearing (MDA-MB-231) xenograftmodelwasusedwitheachchemotherapy genic effectsbreast cancer (10 µM)ofPRO.Invivoamurineorthotopictriplenegative drugs bylowconcentrations ofthechemotherapy on dose, cell lineand depending chemotherapy drug. The synergistic effects were shownto be dueto an enhancementof the anti-angio effectsantagonistic and sub-additive additive, of synergistic, a range showed lines non-cancer and cancer human proliferation cell on Pasquier cancer celllines resistance insome radiation may conferincreased have reported[36,52,67]. Evidence existstosuggestthatelevatedCOX-2expression et alshowedthatinC3H/Hand activity ofmacrophages the phagocytic AKR micePRO,atadoseof5mg/kg, was abletoincrease et al investigated the synergism of PRO and 5-FU, with paclitaxel both in vitroand[30]. vivo In vitro analysis of a numberof [139–141] [72]. , the primary focus in this paper is on direct evidence of therolePRO,andwhererelevant,otherbetaadrenergic , theprimaryfocusinthispaperisondirectevidence . [18] and humanmulti-drugresistant (MDR) CEMleukaemia 2 pathway inhibition that anumberofinvestigators pathwayinhibition [137] for example. But results were cell line [45]. [138]. ecancer 2016,10:680 [66]. Ciccarelli [82]. -

Clinical Study 16 www.ecancer.org of greater inthedirection Th2 balance Th2 polarisation effecton thedifferentiationcatecholamines oflymphocyte-derived function of and Tthat theyshiftedthe (Th) cells,suggesting helper Th1/ Lymphocyteswith potentialdownstreamimpactsonimmunity. arealsoknowntosecretecatecholamines, the Huang et al investigated [153]. Treated animalsshowedlowerratesoftumourgrowth(P <0.01)andincreasedlevelofCTL activity(P <0.05). et al showed that PRO and a HSP-70-rich tumour lysate vaccine synergised to increase IFN-γ production in a murine model of fibrosarcoma production (IFN-γ) gamma interferon of down-regulation via response immune the impact to known also are Catecholamines complexity offollowingcellpopulationsinvivooverextended timeperiodsmakesinterpretationoftheseresultsdifficult [151]. stressful ‘fightorflight’ episodes. However, it has beenarguedthat and the complexityof multiple immunecompartments,NKcelllineages insult during the hostforpathogenic occurs inordertoprepare that thepriming suggesting for thesefindings, explanation an evolutionary and 14hoursafterstress markers, bothimmediately of activation NK cytotoxicactivityandtheexpression numbers, NK cell splenic mice), bothincreased aggressive contrast, TarrIn no effect onotherlymphocyte sub-setsinthelungs. priorto 20 mg/kg, restraint wasshownto administered reverse thereductioninNKcellnumberscomparedto untreated controls but had stress restraint-induced (P < 0.01), as were allotherlymphocytesubsetsassessed(CD8+,CD4+,B cell andNKT cells). PRO, at a dose of by were alsoreduced numbers NK cell of restraint.Lung four hoursofcessation within to base-line returned by restraint,butnumbers (P <0.001)whichreturnedtobase-line valueswithinonehour[149]. Lymphocyte(P numbers in bloodandlungsweredepressed <0.001) Kanemi that PRO,atadoseof30ppm,slowedtheratetumourgrowthover-crowdedgrouptobelowunstressed controls. induced similarstress-relatedthymicatrophy[33]. mouse modelsandalsoshowedthatover-crowding additional it wasshown Additionally tion inthymocytes in cytolyticactivityof and reduction NK cells.Subsequentresearchbythesamelaboratoryreplicatedthesefindingsin to unstressedcontrols(Pmetastases compared <0.05)[148]. in thymusweight,reduc- with areduction This wasshowntobeassociated Wu CD11b+F4/80+ macrophagesasbeingimplicatedinthemetastaticprocesssame mousemodel Sloan CTLgeneration. and TNF-α on norepinephrine of effects the reversed completely µM, 1 of tion concentra a at PRO, of use vivo Ex TNF-α. exogenous of addition the by confirmed result a macrophages, activated F4/80+ and cells T BALB/c mice[147]. Results showed that norepinephrine inhibited CTL generation via a reduction of TNF-α expression in CD4+ and CD8+ Kalinichenko acted theseeffects [36]. shown to be associatedwithreducedFasL and CD11a expressionpost-surgery, and that the combinedetodolacPRO treatment counter- cytotoxicity.and per-cell in NK cell numbers reduction induced the surgically reversed impacts onNKcellswerelater These deleterious (1.5 mg/kg) was moreeffective than eithersingletreatment and completelyinhibitedthe effect of surgery. Notably the combined treatment the LTRreduced mg/kg, alsosignificantly ratecomparedtountreatedcontrols. treatment ofetodolac(12.5mg/kg)andPRO The combined PRO, atadoseof1.5mg/kgand4.5 Pre-surgical etodolac, andcelecoxib). (indomethacin, treatment withCOX-2inhibitors pre-surgical (Pnificantly (LTR)retention tumour cell the rateoflung with increased <0.05)associated [25]. in LTRThis increase wasattenuatedby Benish etal showed that Subsequently with MADB106cellsinF344rats surgical intervention(laparotomy)priorto was sig- inoculation cells andthenumberoflungmetastases. NK-sensitive MADB106breastcancer receptor antagonist,reducedthenumberof lung-retained beta-adrenergic like PROanon-selective 0.1 –0.5mg/kg)ornadolol. in the range of PRO(atdoses by prioradministration inhibited that nadolol, showed experiments Additional (Pby metaproterenol at fivehours.BloodNKactivitywasdepressed values bythreehoursandreturningtobase-line <0.03),butthiswas significance, the time courseof NK numbersshowedthat the initialincreasesubsidedwithinonehourandbeforefallingbelowbase-line killer (NK) cell numberswithin10 minutes of administration inF344rats (P < 0.0001) [24]. It should benotedthat although it did not reach transient increaseinnatural induced adose-dependent agonist metaproterenol that the beta-adrenergic showed Shakhar andBen-Eliyahu showed that stress induced by social isolation in Balb/c mice injected with colon 26-L5 carcinoma cells increased the rateofliver withcolon26-L5carcinomacellsincreased in Balb/cmiceinjected bysocialisolation thatstressinduced et alshowed showed that stressed induced by physical restraint in C57BL/6 mice resulted in a significant increase in epinephrine levels epinephrine in increase in asignificant mice resulted C57BL/6 restraint in by physical induced that stressed et alshowed et al investigated the effect oncytotoxic of norepinephrine T lymphocytes (CTL) using a MOPC-315 plasmocytomamodelin showed that repeated social disruption stress (induced by repeatedly introducing aggressive mice intocagesofnon- aggressive introducing by repeatedly stress (induced disruption social that repeated et alshowed [150]. Administration of PRO (10 mg/kg)reducedthe‘priming’ of these NKcellsat 14 hours. The authors propose . Panina-Bordignon [154–155] had earlier suggested that beta2-adrenergic suggested et alhadearlier et alalsoidentifiedstress-sensitive [27]. ecancer 2016,10:680 [152]. Khalili -

Clinical Study of aspirinmayberelatedtoanti-plateleteffects viairreversibleinhibitionofCOX-1[172–173],andthereisalsosomeevidencethat tumour immunityby‘cloaking’ tumourcellsfromNK cellsandaroleinestablishingthemetastaticniche[171]. The anticancereffects Platelets playacomplexroleintumourprogressionandmetastasis viathereleaseofpro-angiogenicfactors,aroleinsubvertinganti- and HeyA8ovariancancercells. Skov3-ip1 ovariantumoursalsoshowedelevatedlevelsofPGE from micebearing from patients[170]. Tumourlevels ofPGE higher significantly patientsshowed samplesfromdepressed vivo analysisshowedthatPROalsoreducedtheimmunosuppressiveeffect of T-reg cellscomparedtocontrols. in the increase attenuated T-regeffector synergistic no additive there was though numbers Ex to PROalone. compared oftheparecoxib secretion ofPGE PGE impact ontumourgrowth. tive to PRO at a dose of 10 mg/kg, and that this was associatedwith a reduction inHK-2expression.PRO may therefore havea metabolic sensitive to PRO at a concentrationof 50 µM. In the vivo resultsusing 4T1 mousebreastcancerlineshowedthat tumours werealsosensi- transporter-1 (GLUT-1)glucose thatwhile showed analysis unaffected wasrelatively (HK-2) was by PRO,hexokinase-2 inallcelllines Kang and hasbeenshowntoreducethecellviabilityofEGFR-dependentcancerlines to ligands availability receptor restricting thereby surface toendosomes, EGFR fromthecell activity function may be to interfere withthe binding or endocytic processdirectly tyrosine rather thandirectlytargetingreceptor-ligand kinase EGFR [163–164] factor receptors,including growth multiple endocytic traffickingin theprocessbywhichtumoursgainself-sufficiency isimplicated of ingrowthsignalsbydelaystheinactivation in manycancertypesandisamajordrugtarget[162].Disrupted plays acentralrole growth factorreceptor(EGFR)signalling Epidermal have beendescribedintheliterature,someofwhichmaybeunrelatedtobeta-adrenergicreceptorantagonistactivity ofPRO. which of action mechanisms of otherpossible a number about, there arealso outlined been that have mechanisms tothemain In addition Other 17 www.ecancer.org [160], the datashowedthat the COX-2/PGE of CD11b+Gr1+ withanaccumulation state associated marrow in thebone MDSCs prognostic markerinsomecancers[157–158] Myeloid derivedsuppressorcells (MDSC) areimplicatedinthedysfunctionalimmuneresponseto cancer andareconsideredanegative immunity [156]. inhibits productionof IL-12, therebypromoting signalling Th2 differentiationthe andinhibiting associated withanti-tumour Th1 development sought to investigate the relationship between beta adrenergic signalling and PGE signalling beta adrenergic between the relationship et alsoughttoinvestigate tosis was associated with an increase in epinephrine, norepinephrine, PGE norepinephrine, was associatedwith an increaseinepinephrine, operative day. Patients inthe combination groupreceivedbothtreatments at the same doseandschedule.Resultsshowedthat surgery from dayofsurgeryuntilthirdpost-operativeday. group received40mgperday,The parecoxib IV, from dayofsurgerytosecondpost- Patients wereassignedto control, PRO, parecoxib andPRO + parecoxib groups.PatientsinthePRO group received20mg TID starting ied the impact of PRO and theCOX-2 inhibitor parecoxibon T-regradical mastectomy numbers inbreastcancerpatientsundergoing T-regulatory (T-reg)of immune cellsassociatedwithtumour-associateddysfunction.Zhouet cellsareanotherpopulation al stud- (P <0.001)andproportion(P =0.018). of MDSCs,bothintermscellnumbers the accumulation and thatPRO(10mg/kg)partiallyreversed implicated, were also catecholamines 2 [169]. shown,thesediverseeffectsbeen As hasalready of actionPRO.Nagaraja in someofthemechanisms arealsoimplicated is a key inflammatory molecule with multiple effectsmolecule withmultiple isakeyinflammatory incancer,effects including onimmunity,and apop- proliferation angiogenesis, investigated the impact of PRO on glucose uptake in breast cancer cell lines (4T1, MDA-MB-231 and MCF-7) [168].Invitro and (4T1, MDA-MB-231 lines in breastcancercell uptake the impactofPROonglucose et alinvestigated [165]. (PAP)Inhibition of phosphatidic acidphosphohydrolase has beenshownto cause a reversible trafficking of (empty) inactive 2 from tumour cells. Furthermore PRO was also shown to inhibit the catecholamine-induced increase inPGE fromtumourcells.FurthermorePRO was alsoshownto inhibit thecatecholamine-induced 2 axis acentralrole in thisaccumulation.In addition therewasevidencethat stress-related . Jin et al usedBALB/cmicetoshowthat chronic stressinducedanimmunosuppressive . It has been proposed by Shaughnessy proposed . Ithasbeen 2 levels and T-reg numbers. Treatment withPRO or PRO + parecoxib 2 . the increased Analysis showedthatnorepinephrine . In line with previous reports from other groups reports fromother previous with [159]. Inline [165]. 2 in more detail using primary ovarian cancer cells primary ovarian using inmoredetail . PRO is a known inhibitor of PAPinhibitor [166]. PROisaknown [167], that a strategy for inhibiting EGFR et althatastrategyforinhibiting 2 and PGF2α, tumour samples tumour PGF2α, and ecancer 2016,10:680 2 inSkov3 [161].

Clinical Study 18 www.ecancer.org of COX-2/PGE down-regulation and of tissue remodelling inhibition and lymphangiogenesis, of reduction intherateofangiogenesis invasion, down-regulation including value able is ofconsider agents for anti-metastatic the search therefore mortality and of cancer-related cause the primary remains Metastatic disease Anti-metastatic Agent confirm theefficacy ofthesecombinations withPRO. [44, 81,82],alongwithresponsestopropranololobservedinothervasculartumours[41,183–184].Clinicaltrialsareurgentlyrequired needs inthisdisease,thecasereportsshowingsomebenefittopatientsusingPROcombinationwithotheragents arenoteworthy treated withsurgicalresection,chemotherapy(doxorubicinorpaclitaxelmostcommonly)andradiotherapy[182].Giventhehighunmet reported five-yearoverallsurvivalratesintherange30%-40%[181]. Although nostandardtreatmentexists,themajorityofpatientsare Angiosarcoma isarareandaggressivesofttissuesarcomaarisinginvascularendothelialcells,diseasewith poorprognosisand Angiosarcoma to assessplasmaconcentrationsofpropranololespeciallywhenadirecteffect ontumourgrowthisdesirable. the effectnoteworthy.are particularly intervention surgical ontherateofdistant metastasesfollowing Inallfuturetrials,itwillbeimportant tial inthe same or other settings/tumours.For PRO the evidenceinangiosarcoma,anti-metastaticeffects in breastandovariancancers suggest that it be prioritised for further to clinical research.Needless say, of the repurposing other beta-blockersmay also carry great poten- andnebivolol example carvedilol in differentvalue of therapeutic effects,that otherdrugsinthesameclassashaveanti-cancer settings. Whilethereisevidence clinical for The evidenceoutlinedabove,andsummarisedin Our Take environment. PRO, signalling. at leptin inadipocytesviabeta-adrenergica dose of 0.5 g/l indrinkingwater, inhibited the protective effect of the enriched neurotrophic factor of growth rate(BDNF) hypothalamic brain-derived was associatedwithdown-regulationandincreasedproductionof in acontrolenvironment living growth compared rate ofB16melanoma with reduced cise), wasassociated and physicalexer- stimulation, socialstimulation levels ofinanimate (increased environment in anenriched of eustress,relatedtoliving positive stress (eustress) andnegativestress (distress) withdifferingcorrelates [179]. Cao et physiologicalal showed that a murinemodel nity. However,differentiatespsychology – clinical negative necessarily stress are forms ofsocial that notall be noted itshould between stress arising from social interactionshasbeenshownto have negativeeffects immu- on proliferation,invasion,metastasisandanticancer Finally,physiology.states and psychological between is attheintersection signalling beta-adrenergic mentioned previously As hasbeen (0.99±0.19) incomparisonwithbothatenolol(1.41±0.70;P =0.004)andbaseline(1.59±0.94;P =0.002)[178]. PRO (40mg TID) to atenolol (100 mg/day) confirmed that the number of circulating platelet aggregates decreased significantly with PRO tion more than selective non-lipophilic beta-blockers. A small randomised cross-over trial in moderate essential hypertension compared CI =-0.85–-0.24,P <0.0001)[177].Inparticularnon-selectivelipophilicbeta-blockers(includingPRO)decreasedplateletaggrega- lished in 2014 showed that they decreased platelet aggregation by 13% (95% CI = 8 - 17%, standardised mean difference=-0.54, 95% ity other anti-platelet agents, such as low molecular weight heparins and dipyridamole may also have anti-cancer or anti-metastatic activ- metastatic cancersmaythereforebeastrategytoreducethe rateofmetastaticspread. in breast and other cancers viadirect effects signalling on beta-adrenergic [174–176]. Beta-adrenergicreceptorantagonistsarealsoknowntohaveeffects onplateletaggregationandameta-analysispub- [185]. previously,As outlined may havesomeimpactonthemetastaticprocess, mechanisms ofaction which PROhasmultiple 2 expression. Data from a number of in vivo models has shown that PRO may reduce the rateofmetastasis that PROmayreduce has shown ofinvivomodels Data fromanumber expression. [45], PRO has boththe widest rangeof use, therefore pragmaticreasons evidence andextensive clinical Table 1, suggests that PRO has a number of distinct anti-cancereffects which may be [27, 36, 48]. The addition of PRO to standard of care for non- [180]. in tumour This reduction ecancer 2016,10:680 -

Clinical Study . Of note the combination of PRO with a COX-2/PGE of PROwith rate ofmetastases[40].Ofnotethecombination a reduced with PRO isassociated that peri-operative reported have in vivostudies and numerous metastatic process in thepost-surgical implicated is also signalling beta-adrenergic outlined, previously 19 www.ecancer.org benefit ofPROinothercancers. the potential in assessing to suggestthatthere maybesomevalue dence of catecholamine The ubiquity While wehaveoutlinedareaswherethereis a particularlycompellingcaseto be madefor further clinical study, there is also sufficient evi- Other Cancers interventions includechoiceof Options for anaesthesia such peri-operative growing levelof interest intargetingsomeaspectsofthispost-surgicalresponsesoastoreducetheriskmetastaticspread[192–193] signalling that lead to immunity andanincreaseinpro-angiogenic suppression of cell-mediated . with distantmetastases[186–187] response’healing The ‘wound events initiates acascadeofinflammatory duetothesurgical incision may beassociated intervention suggest thatsurgical models of patientoutcomesandfromanimal analyses Data frombothretrospective Perioperative Intervention osteosarcoma, headandneckcancers,upperGI NSCLC andovariancancer. breast cancer,distant metastases areafrequentoccurrence,including thecaseinthosecancerswhichpost-surgical This isparticularly inhibitor,investigation. setting andwarrantsadditional such asketorolacoretodolac,hasthepotentialtoshow synergisminaperi-operative pathway suchasketorolac[196–198] Table 1.Summaryofevidencebycancertype. Prostate Pancreatic Angiosarcoma Ovarian Oral SCC NSCLC Neuroblastoma Nasopharygeal Multiple myeloma Melanoma Leukaemia HCC Glioblastoma Gastric Colorectal Breast Cancer Type and diclofenac[199–200] In Vitro [50–52] [61, 62] [45, 47] [34, 35] [21, 60] [66, 67] [55, 57] [48] [38] [68] [59] [70] [43] , and theH2RA cimetidine [201–202] [53, 54] [39, 40] [46, 47] [33, 36] [24–31] In Vivo [22] [58] [43] [194–195] ACTRN12612000852853 ACTRN12615000889550 Case Report/Trial NCT02596867, NCT01847001, NCT01504126 NCT01857817 NCT00888797 NCT02732678 NCT02897986 NCT02641314 NCT01988831 NCT01265576 NCT00502684 [44, 81,82] [83, 84] , the useof drugs whichtargettheCOX-2/PGE [85] [86] [79] [188–191] . As should beclearfrom the results . However, there isnowa ecancer 2016,10:680 2 2 .

Clinical Study The peri-operativeuseofPROincombinationwithketorolacoretodolacisalsointerestthefollowingcancers: is discussedinthissetting. to erties ofPRO with otheragents exploit duringsurgicalintervention, andanumberof may beparticularlyvaluable possible combinations 20 www.ecancer.org COX-2/PGE repurposed as an anticancer agent. repurposing In particular the potential for synergistic interactions withotherdrugshasbeenoutlined,including effects associated withPRO. for This datahasbeensummarised and presentedtomakethecasethatPROisaverystrongcandidate anti-cancer relevant clinically that therearemultiple studies toindicate and human of datafrominvitro,animal volume There isasignificant Conclusion The dataarestrongestforclinicaltrialsofPRO,incombinationwithotheragents,thefollowingcancertypes: is tobehopedthatpositivereportsfromthesetrialswillforthcominginthefuture. trial activity,of clinical The currentlevel it and evidence of clinical drug, testifiestothestronglevel for arepurposed high is relatively which Next Steps effects inadditiontophysicaleffects onthehostenvironment. an anti-metastaticeffect,to theresultssupporting in termsofpsychological beneficial may be that betablockade to believe thereisreason ment stress manage meditation [206],cognitivebehavioural mindfulness investigated, including have beenclinically stress reductiontechniques stress andcancer,the intersectionbetweenpsychosocial data. models andepidemiological animal asevidencedinnumerous A varietyof are relatedto aspects ofiscentralto the hostenvironmentratherthandirectly on primarycancergrowth[205].signalling Beta adrenergic effectsis thattheapoptotic the evidence where as diclofenac, effectsthe anti-cancer that therefore and achievable, arenotphysiologically levels. In many respectsthisissimilarto achievable the concentrations whichmaynot case withsomeNSAIDs, reflect physiologically such Weeffect notethatthedataforapro-apoptotic ofPROonprimarytumourgrowthcomesprimarilyfrominvitrostudieswhichusehigh Psychological Stress posed drugssuchasketorolacoretodolac. repur- with otheragents,including in combination particularly cancer andneuroblastoma, in pancreatic for furtherinvestigation evidence ultimately,suggested toamuchlargernumberofcancertypesthanhashithertobeen and theapparentexpressionofreceptorsinmultipletumourtypeswouldsuggestthat adrenergic theeffectssignalling of PRO may extend, • • • • • • • • • • • • [207] andevencommunalsinging Pancreatic cancer Non-small CellLungCancer Ovarian cancer Breast cancer Oesophageal cancer Head andneckcancers Osteosarcoma Neuroblastoma Pancreatic cancer Ovarian cancer Breast cancer Angiosarcoma 2 inhibitors andarangeofat and standarddosing. both metronomic chemotherapeutics The anti-metastaticprop- [208]. Therefore, whilethedataforaneffect on primarytumourgrowthmaybelimitedincontrast [203–204] . Inthemeantimethereisagoodlevelof ecancer 2016,10:680 -

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