Study of new therapeutic strategies to combat breast cancer using drug combinations

biomolecules A icle S udy o New The apeu ic S a egies o Comba B eas Cance Using D ug Combina ions Ana Co eia 1,2, Dany Sil a 1, Alexand a Co eia 3,4,5, Manuel Vilano a 3,4,5 , Fá ima Gä ne 2,3,6 and Nuno Vale 1,2,3,6,* 1Labo a o y o Pha macology, Depa men o D ug Sciences, Facul y o Pha macy, Uni e si y o Po o, Rua de Jo ge Vi e bo Fe ei a 228, 4050-313 Po o, Po ugal; [email p o ec ed] (A.C.); [email p o ec ed] (D.S.) 2 Depa men o Molecula Pa hology and Immunology, Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o, Rua de Jo ge Vi e bo Fe ei a 228, 4050-313 Po o, Po ugal; [email p o ec ed] 3i3S, Ins i u o de In es igação e Ino ação em Saúde, Uni e si y o Po o, Rua Al edo Allen 208, 4200-135 Po o, Po ugal; [email p o ec ed] (A.C.); [email p o ec ed] (M.V.) 4 Depa men o Imuno-Physiology and Pha macology, Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o, Rua de Jo ge Vi e bo Fe ei a 228, 4050-313 Po o, Po ugal 5Ins i u e o Molecula and Cell Biology (IBMC) o he Uni e si y o Po o, Rua Al edo Allen 208, 4200-135 Po o, Po ugal 6 Ins i u e o Molecula Pa hology and Immunology o he Uni e si y o Po o (IPATIMUP), Rua Júlio Ama al de Ca alho 45, 4200-135 Po o, Po ugal *Co espondence: [email p o ec ed]; Tel.: +351-220-428-606 Recei ed: 22 No embe 2018; Accep ed: 11 Decembe 2018; Published: 14 Decembe 2018   Abs ac : Cance is a disease ha a ec s and kills millions o people wo ldwide. B eas cance , especially, has a high incidence and mo ali y, and is challenging o ea . Due o i s high impac on he heal h sec o , oncological he apy is he subjec o an in ense and e y expensi e esea ch. To imp o e his he apy and educe i s cos s, s a egies such as d ug epu posing and d ug combina ions ha e been ex ensi ely s udied. D ug epu posing means gi ing new use ulness o d ugs which a e app o ed o he he apy o a ious diseases, bu , in his case, a e no app o ed o cance he apy. On he o he hand, he pu pose o combining d ugs is ha he esponse ha is ob ained is mo e ad an ageous han he esponse ob ained by he single d ugs. Using d ugs wi h po en ial o be epu posed, combined wi h 5- luo ou acil, he aim o his p ojec was o in es iga e whe he his combina ion led o he apeu ic bene i s, compa ing wi h he isola ed d ugs. We s a ed wi h a sc eening o he mos p omising d ugs, wi h e apamil and i aconazole being chosen. Se e al cellula iabili y s udies, cell dea h and p oli e a ion s udies, mainly in MCF-7 cells (Michigan Cance Founda ion-7, human b eas adenoca cinoma cells) we e pe o med. S udies we e also ca ied ou o unde s and he e ec o he d ugs a he le el o possible he apeu ic esis ance, e alua ing he epi helial-mesenchymal ansi ion. Combining all he esul s, he conclusion is ha he combina ion o e apamil and i aconazole wi h 5- luo ou acil had bene i s, mainly by dec easing cell iabili y and p oli e a ion. Fu he mo e, he combina ion o i aconazole and 5- luo ou acil seemed o be he mos e ec i e, being an in e es ing ocus in u u e s udies. Keywo ds: b eas cance ; d ug epu posing; d ug combina ions; 5- luo ou acil; cell iabili y; epi helial-mesenchymal ansi ion 1. In oduc ion Cance is he second-leading cause o dea h behind hea disease [ 1 ], accoun ing o an es ima ion o 9.6 million dea hs in 2018 [2]. Biomolecules 2018,8, 175; doi:10.3390/biom8040175 www.mdpi.com/jou nal/biomolecules Biomolecules 2018,8, 175 2 o 23 When dealing wi h his disease, he e a e h ee impo an p io i ies, which a e, in o de o impo ance: p e en ion, ea ly de ec ion and o al e adica ion. The ea men o cance , pa icula ly, is a e y complex issue, wi h h ee usual modes: su ge y, adio he apy and pha maco he apy [3]. Conce ning pha maco he apy, he ideal d ug is one ha selec i ely kills he neoplas ic cells, minimizing ad e se e ec s. Howe e , he di e ences be ween no mal and neoplas ic cells eside, usually, in a quan i a i e way, such as g ea e o lesse ac i a ion o signaling pa hways. The e o e, pha maco he apy o cance is di icul o achie e in a o ally success ul way [ 4 ]. Ne e heless, su i al o oncological pa ien s has imp o ed signi ican ly in he las yea s, mainly because o mul idisciplina y ca e, imp o ed chemo he apeu ic agen s, in oduc ion o a ge ed he apy, and he inco po a ion o pallia i e ca e se ices [ 5 ]. Howe e , despi e he ad ances men ioned abo e, many pa ien s s ill ail he apy, explained by he p esence o an in a umo al he e ogenei y and se e al d ug esis ance mechanisms, such as de egula ion o apop osis, ac i a ion o p osu i al signaling and epi helial-mesenchymal ansi ion (EMT) [ 6 ]. In ense pha macological esea ch is being made abou oncological he apy, o igina ing an explosion o cos s. Despi e his big in es men , he e is li le ou pu o a huge pha ma esea ch and de elopmen spending. Thus, he e is a equi emen o mo e e ec i e cance d ugs. To ace his p oblem, an in e es ing app oach named d ug epu posing is being inc easingly applied [ 7 ]. Highligh ing he impo ance o his app oach, he global ma ke o d ug epu posing eached € 20.7 billion in 2015 and is p ojec ed o each € 26.6 billion by 2020 [ 8 ]. Addi ionally, ano he impo an esponse o he p oblems in cance he apy encompasses he use o d ug combina ion he apies [9]. D ug epu posing is a me hodology o iden i y a new indica ion o al eady exis en d ugs. I allows lowe cos s and a sho e ime un il app o al o he d ug, han de eloping a d ug de no o, because in o ma ion ega ding side e ec s, pha macokine ics and in e ac ion wi h o he d ugs has been collec ed [ 10 ]. Cy os a ic and/o cy o oxic ac i i y wi hin a wide ange o d ug classes o he han cance has been demons a ed in se e al s udies. The e o e, he s udy o he a senal o d ugs app o ed o non-cance indica ions migh o e e ec i e ea men op ions o cance pa ien s. Commonly, ideal d ugs o epu posing sha e se e al cha ac e is ics: hey should be well-known d ugs, o en a ailable as gene ics, he oxicology p o ile o he d ug mus be good, he e should ha e a plausible mechanism o ac ion, ele an o he condi ion in ques ion, and e idence o e icacy a physiological dosing [ 9 ]. Many cu en ly used d ugs ha e, a leas , some ac ions ha may be use ul in cance ea men . Pa icula ly, a lo o o -pa en d ugs ha e shown some e idence o an icance e ec s, in which abou 50% a e suppo ed by ele an human da a and 16% a e suppo ed by da a om a leas one posi i e clinical ial [ 11 ]. B ie ly, o oncologic pu poses, d ugs like aspi in, i aconazole, e apamil, chlo oquine and all- ans e inoic acid ha e shown an icance ac i i y in, a leas , one andomized clinical ial [12,13]. Conce ning d ug combina ion app oach, i is known ha a disease is in e p e ed as a se o molecula pa hways ha in e connec , ha ing a bigge suscep ibili y o he simul aneous ac ion o se e al d ugs. This makes possible o s udy d ug combina ions in g ea e dep h [ 14 ]. Combining d ugs has se e al ad an ages: dec eased oxici y, be e e icacy, dec eased dosage a an equal o inc eased le el o e icacy, and coun e d ug esis ance [ 15 ]. Due o hese ad an ages, d ug combina ions ep esen an in e es ing and inc easingly used app oach ha has become a s anda d o he ea men o a wide ange o diseases, such as cance and in ec ious diseases [ 14 ]. Pa icula ly in cance , nume ous clinical ials es ing combina ions ha include chemo he apy d ugs, adia ion he apy, ho monal he apies, molecula ly a ge ed he apies, and immuno he apies a e being ca ied ou [ 16 ], wi h a c escen ocus on he combina ion o cy o oxic chemicals and bio he apies [17]. Combining epu posed pha maceu ical agen s wi h o he chemo he apeu ic agen s has also shown p omising esul s, use ul when adi ional an icance mono he apy has ailed o p o ide a sa e and ole able ea men o cance pa ien s [ 18 ]. An example o hese kinds o combina ions is ni oglyce in in combina ion wi h he chemo he apeu ics ino elbine and cispla in. In his case, Biomolecules 2018,8, 175 3 o 23 one andomized Phase II ial demons a ed imp o ed o e all su i al o pa ien s wi h non-squamous cell lung cance [18]. B eas cance is a collec ion o b eas diseases ha ha e dis inc his opa hologies, gene ic, genomic a ia ions, and clinical ou comes [ 19 ], being he second mos common cance wo ldwide, he mos equen cance in women and he i h cause o dea h om cance o e all. In he pas wo decades, he a es o b eas cance mo ali y ha e declined by app oxima ely 30%, wi h co esponding imp o emen s in i e-yea o e all su i al a es o 90%. Howe e , despi e hese ad ances, me as a ic b eas cance emains a challenge o ea , wi h an es ima ed i e-yea o e all su i al a e o only 23% [20]. The choices o ea men egimens o b eas cance is a e y complex issue, being di icul o ha e a uni e sally accep ed ea men , since each case is a case and he choice o he bes he apy should be ailo ed o each pe son. Thus, a lo o expe ise is necessa y o e alua e he bes he apy o each case [21]. Fo ea ly-s age b eas cance , su ge y is conside ed he gold-s anda d ea men [ 22 ]. On he o he side, he adju an ea men ’s main goal is o ea me as a ic disease. This ea men consis s o adia ion he apy and sys emic he apy (including a a ie y o chemo he apeu ic, ho monal and biologic agen s) [ 23 ]. The e a e a lo o app o ed d ugs o b eas cance and, in addi ion, he e a e a g ea a ie y o d ugs ha ha e he po en ial o be epu posed o i s ea men [ 24 ]. Howe e , o da e, no epu posed d ugs ha e been app o ed o he ea men o his kind o cance . An example o a po en ial epu posed d ug is he be a-blocke p op anolol. A s udy showed ha his be a blockade educed umo p oli e a ion by 66%, in ea ly s age b eas cance , by accessing Ki67. Chlo oquine is ano he example, wi h e y p omising esul s in b eas cell lines and Phase I ials, ei he alone o in combina ion wi h o he d ugs [25]. Despi e a lo o app o ed d ugs and po en ial d ugs o epu pose, b eas cance emains a majo heal hca e issue. Highly quali ied heal hca e p o ide s a e necessa y o p ope su gical ea men . Addi ionally, ad anced ea men app oaches ha in ol e adia ion a e di icul o achie e in de eloping coun ies, pa icula ly. Adequa e sys emic ea men s, managemen o po en ial se e e e ec s and new a ge ed he apies a e cos ly, and he mos sophis ica ed ea men s equi e ad anced and cos ly pa hology. In addi ion, he p oblem o d ug esis ance, ans e sal o mos o he cance ypes, emains an impo an issue o s udy and comba [26]. Thus, his p ojec aimed o in es iga e he e ec o d ugs wi h po en ial o be epu posed o b eas cance he apy, in combina ion wi h an al eady used d ug in his ype o he apy (5- luo ou acil), so ha he combina ion o hese d ugs p esen ed bene i s, when compa ed o he single d ugs o he combina ion. 5-Fluo ou acil (5-FU) was chosen as he e e ence d ug o his s udy, mainly because i is a po en ial d ug o be used in combina ion egimens in b eas cance he apeu ics, wi h he majo aim o imp o ing i s e icacy, as well as i s known oxicological p o ile. I is a he e ocyclic a oma ic o ganic compound wi h a s uc u e simila o ha o he py imidine molecules o DNA and RNA. This d ug and hei me aboli es a e esponsible o RNA and DNA damage, ac ing on S phase o cell cycle [27]. S udies add essing combina ions o d ugs using d ugs wi h non-cance indica ions ha e shown posi i e esul s in ela ion o cance he apy, no exclusi ely o b eas cance , bu also o o he kinds o cance [ 28 ]. By in eg a ing all he ob ained esul s, i was no o ious ha he chosen d ug combina ions showed a endency o be mo e e ec i e han he mos e ec i e d ug o each combina ion. Possibly, hese d ug combina ions may ac p ima ily in he a es o cell p oli e a ion. Also, i is impo an o highligh ha he collec i e o all esul s poin ed ou ha he combina ion o 5-FU and i aconazole was he mos p omising combina ion. Biomolecules 2018,8, 175 4 o 23 2. Ma e ials and Me hods 2.1. D ug Solu ions Fo he ea men o cells wi h he se e al d ugs unde s udy (5-FU, e apamil, i aconazole, isoniazid, ac ine, aspi in, cime idine, chlo oquine, losa an, all om Sigma-Ald ich (Sigma-Ald ich Quimica, S.L., Sin a, Po ugal) and p a as a in, om Cayman Chemical Company, Ann A bo , MI, USA), all he compounds we e dissol ed in au ocla ed wa e , excep i aconazole, ha was dissol ed in dime hyl sul oxide (DMSO), since i did no p esen solubili y in wa e . A s ock solu ion o each compound was p epa ed a a concen a ion o 10 mM and, excep o i aconazole, wo s ock solu ions wi h a concen a ion o 25 mM and 50 mM we e p epa ed, because his d ug was dissol ed in DMSO, which has signi ican oxici y o cells a e a pe cen age o 0.2%, a pe cen age ha was ne e exceeded in his wo k. All hese s ock solu ions we e conse ed on he eeze a − 26 ◦ C. Depending on he pu pose o he assay, es compounds we e used in concen a ions ha ange om 1 µ M o 100 µ M, dissol ed in cul u e medium igh be o e con ac wi h cells. The espec i e concen a ions used in each assay a e p esen ed in he Resul s sec ion. The es compounds applied o he cells a y wi h he pu pose o he expe imen , also speci ied in he abo e-men ioned sec ion. 2.2. Cell Cul u e The expe imen al wo k was ca ied ou mainly in he MCF-7 cell line (ATCC, Ame ican Type Cul u e Collec ion, Manassas, VA, USA). Addi ional expe imen s we e ca ied ou in MCF-10A (ATCC—Ame ican Type Cul u e Collec ion). MCF-7 and MCF-10A cells we e incuba ed a 37 ◦ C in a humidi ied a mosphe e wi h 95% o ai and 5% o CO2. MCF-7 cells we e cul i a ed in Dulbecco’s Modi ied Eagle Medium (DMEM), supplemen ed wi h 10% e al bo ine se um (FBS) and 1% o a mix u e o penicillin/s ep omycin (1000 U/mL; 10 mg/mL). MCF-10A we e cul i a ed in DMEM/F-12, supplemen ed wi h he same supplemen s desc ibed abo e plus 2 µ g/mL o human insulin (No oNo disk, Bags æ d, Denma k), 20 ng/mL o epide mal g ow h ac o (EGF) and 1 µ M o hyd oco isone. Fo main enance, he cells we e cul u ed in a monolaye , being subcul u ed 2–3 days pe week, and 1 day pe week in he case o MCF-10A, he las cell line g owing slowly. All he expe imen s we e ca ied ou wi h cells o 70–80% con luence. 2.3. MTT Reduc ion Assay MCF-7 and MCF-10A cells we e pla ed in 96-well pla es wi h a seeding densi y o 3.0 × 10 4 cells/mL, main ained a an incuba o a 37 ◦ C, o 24 h. A e his ime, di e en ea men s we e added o he cells a di e en concen a ions, o 48 h o 72 h, depending on he pu pose o he expe imen . The cells we e main ained a 37 ◦ C du ing he e e ed ime and, a e ha , he cell medium was emo ed and 100 µ L o (4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide (MTT) solu ion (0.5 mg/mL in PBS) was added o each well. Then, he cells we e incuba ed a 37 ◦ C o 3 h in a ligh -p o ec ed manne . A he end o his ime, MTT solu ion was emo ed and 100 µ L/well o DMSO was added, wi h he pu pose o solubiliza ion o o mazan c ys als. The las s ep consis ed o abso bance eadings a 570 nm in an au oma ed mic opla e eade (Sine gy HT, BioTek Ins umen s, Winooski, VT, USA). 2.4. Immunocy ochemis y MCF-7 cells we e seeded in T25 cm 2 lasks wi h a densi y o 4.7 × 10 4 cells/mL, con e ed om he densi y used in 96-well pla es (3.0 × 10 4 cells/mL). A e ha , cells we e incuba ed o 24 h a 37 ◦ C. Then, he di e en es compounds we e added and ac ed o 48 h. A e his ime, he cells we e included in cell blocks (one o each di e en ea men ) and di e en slides we e made om each block. Then, depa a iniza ion by subme ging he slides wice in xylene was pe o med, o 5 min each ime, ollowed by hyd a ion in alcohol a dec easing concen a ions (100%, 95% and 70% alcohol) un il insing in wa e . A e ha , an igen e ie al (unmasking) was pe o med by using a Re ie al Biomolecules 2018,8, 175 5 o 23 Solu ion (10% in wa e ), 20 min in a wa e -ba h a 100 ◦ C. The nex s ep consis ed in endogenous pe oxidase block, by incuba ion o he slides in a Pe oxidase Block Solu ion o 5 min. Then, he slides we e washed wice in TBS o 5 min. Incuba ion wi h p o ein block o 5 min ollowed, and he slides we e washed in TBS 2 × o 5 min. A e wa ds, he slides we e incuba ed wi h he mouse an ibodies an i-cy oke a in (pan) (1:1200 in BSA 5%), an i-E-cadhe in (1:50 in BSA 5%) and an i- imen in (1:500 in BSA 5%), o e nigh , a 4 ◦ C. The slides we e washed wice in TBS, o 5 min and he pos p ima y was added, ollowing an incuba ion ime o 30 min. Once again, he slides we e washed wice in TBS, o 5 min. A e ha , hey we e incuba ed o 30 min wi h he Polyme , washed wice in TBS o 5 min and, o each slide, 150 µ L o a solu ion o 50 µ L o DAB Ch omogen o 1 mL o DAB Subs a e Bu e was added o each slide. Finally, he slides we e insed in wa e , coun e s ained in hema oxylin du ing 1 min, washed again o 5 o 10 min, dehyd a ed (a inc easing concen a ions o alcohol, 70%, 95% and 2 × 100%), diaphanized (2 × in xylene), and he sec ions we e moun ed. The slides we e obse ed on a Nikon Eclipse E600 mic oscope, coupled o a digi al came a (Nikon Digi al Sigh DS-Fi2, Tokyo, Japan). The images we e ea ed wi h Imaging So wa e NIS-Elemen s AR Ve sion 4.30.0 (Nikon, Tokyo, Japan). 2.5. Annexin V-FITC and P opidium Iodide S aining MCF-7 cells we e seeded in 96-well pla es wi h a seeding densi y o 1.0 × 10 6 cells/mL, incuba ed a 37 ◦ C o 24 h. A e his ime, he di e en ea men s we e added o he cells ha we e incuba ed o 3 o 8 h. Then, cells we e ypsinized, washed wi h Hank’s balanced sal solu ion (HBSS, 2%FBS), cen i uged o 5 min, 400 × g, and s ained wi h he eBioscience Annexin V Apop osis De ec ion Ki Fluo escein Iso hiocyana e (FITC) (Wal ham, MA, USA), as ollows: Annexin V FITC (1 µ L pe well) in Binding Bu e (50 µ L pe well), 15 min a oom empe a u e, in he da k. Then, cells we e ans e ed o a es ube and 2 µ L o p opidium iodide (PI) we e added o each sample, 5 min be o e analysis in a low cy ome e (Beckman Coul e Epics XL and BD FACSCan o II; B ea, CA, USA and, San Jose, CA, USA, espec i ely). The acqui ed da a was analyzed using FlowJo (V10) analysis so wa e (Ashland, OR, USA). 2.6. CFSE Labeling o Cells MCF-7 cells we e esuspended in PBS wi h 0.1% BSA a a densi y o 2.0 × 10 6 cells/mL. The CellT aceTM ca boxy luo escein succinimydil es e (CFSE) Cell P oli e a ion Ki (Molecula P obes, In i ogen, Ca lsbad, CA, USA) was used o cell labelling. A CFSE s ock solu ion (5 mM in DMSO) s o ed a − 20 ◦ C was hawed and dilu ed in PBS wi h 0.1% BSA o a inal concen a ion o 10 µ M. Nex , 2.0 × 10 6 cells/mL we e labelled by he addi ion o he same olume o he p epa ed CFSE s aining solu ion and incuba ed a 37 ◦ C o 10 min. The s aining solu ion was neu alized wi h h ee olumes o cold comple e medium (DMEM, 10% FBS) and incuba ed o 5 min on ice. Then, cells we e cen i uged a 400 × g o 5 min, he supe na an was emo ed, and he cells we e washed one mo e ime wi h comple e RPMI medium. The cell pelle was esuspended in comple e medium a a densi y o 1.0 × 10 6 cells/mL and cells we e seeded in 96-well pla es o 3 h. A e ha , he medium was aspi ed and es compounds, dissol ed in he cul u e medium, we e added o cells, ha we e incuba ed a 37 ◦ C o app oxima ely 72 h. The inal s ep consis ed o washing and esuspension o cul u ed cells in HBSS (2% FBS). Fi e min be o e eading, 2 µ L o PI we e added o each cy ome e ube ( ha ep esen s each condi ion) o dead cell exclusion. Finally, cell p oli e a ion was de e mined by low cy ome y (Beckman Coul e Epics XL, B ea, CA, USA) and he da a was analyzed using FlowJo (V10) analysis so wa e. 2.7. S a is ical Analysis S a is ical analysis was pe o med in all expe imen s, only in he case o a numbe o independen expe imen s equal o bigge han 3 (n ≥ 3). The esul s a e exp essed as a i hme ic mean ± s anda d e o o he mean (SEM), excep in one case, whe e esul s a e exp essed as a i hme ic mean ± s anda d Biomolecules 2018,8, 175 6 o 23 de ia ion (SD), explici in he sub i les o he g aphs. Di e ences be ween ea ed cells and co esponding un ea ed con ol we e es ed using one-way ANOVA ollowed by Dunne ’s es . Di e ences be ween he d ug combina ion and he espec i e indi idual d ug o ha combina ion ha p oduces mo e ad an ageous e ec s in e ms o cell iabili y educ ion we e es ed by S uden ’s - es . Di e ences we e conside ed o be signi ican when p alue < 0.05. One-way ANOVA ollowed by Dunne ’s es and S uden ’s - es we e pe o med by using SigmaPlo 12.0 (San Jose, CA, USA) and G aphPad P ism 7 (San Diego, CA, USA), espec i ely. I is impo an o no e ha , o all expe imen s, no di e ences we e obse ed be ween con ol wi h/wi hou DMSO. 3. Resul s 3.1. D ug Sc eening in MCF-7 Cells Based on se e al s udies abou po en ial d ugs o epu pose [ 10 , 12 , 29 , 30 ] and in e es s o he in es iga ional g oup, nine d ugs, each in combina ion wi h he e e ence d ug (5-FU), we e es ed in MCF-7 cell line, in o de o make an ini ial sc eening o po en ial d ugs o be used in combina ion wi h 5-FU. Each d ug was used in a concen a ion o 50 µ M, hus, in a a io o 1:1 when combined wi h 5-FU, being in con ac wi h cells o 72 h. The esul s we e ob ained by MTT me hodology (Figu e 1). Biomolecules 2018, 8, x FOR PEER REVIEW 6 o 23 (San Jose, CA, USA) and G aphPad P ism 7 (San Diego, CA, USA), espec i ely. I is impo an o no e ha , o all expe imen s, no di e ences we e obse ed be ween con ol wi h/wi hou DMSO. 3. Resul s 3.1. D ug Sc eening in MCF-7 Cells Based on se e al s udies abou po en ial d ugs o epu pose [10,12,29,30] and in e es s o he in es iga ional g oup, nine d ugs, each in combina ion wi h he e e ence d ug (5-FU), we e es ed in MCF-7 cell line, in o de o make an ini ial sc eening o po en ial d ugs o be used in combina ion wi h 5-FU. Each d ug was used in a concen a ion o 50 μM, hus, in a a io o 1:1 when combined wi h 5-FU, being in con ac wi h cells o 72 h. The esul s we e ob ained by MTT me hodology (Figu e 1). *** ** ** Figu e 1. E ec o d ugs and espec i e d ug combina ions on he iabili y o MCF-7 cells. Each d ug and each combina ion we e added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± s anda d e o o he mean (SEM) and ep esen he iabili y o cells (% o con ol) o 3–4 independen expe imen s (n = 3, 4). ### p < 0.001 s. con ol; ** p < 0.01 and *** p < 0.001 s. single d ug o he combina ion wi h mo e e ec on cell iabili y educ ion. 5-FU: 5- luo ou acil. In his pa icula sc eening assay, he c i e ion o he choice o d ug combina ions o he con inui y o he p ojec was ha he combina ion o d ugs was mo e ad an ageous in e ms o educ ion o cell iabili y han he wo d ugs in he combina ion, whe e he po en ially epu posed d ug was mo e e icien han 5-FU. The combina ion was mo e e e i e han d ugs sepa a ed. Analyzing he ob ained esul s, i was possible o obse e ha chlo oquine was mo e e ec i e in e ms o cell iabili y educ ion han all he o he d ugs and d ug combina ions (6.5 ± 0.4% o cellula iabili y). Thus, as he aim o his wo k was o s udy a bene icial d ug combina ion in compa ison wi h indi idual d ugs o he combina ion, chlo oquine was excluded om he nex s eps. Impo an ly, he combina ions o 5-FU wi h aspi in, losa an, cime idine, p a as a in, isoniazid and ac ine did no show an ad an age in e ms o educ ion o cell iabili y, ela i e o bo h single d ugs o he combina ion, being also excluded om his s udy. Howe e , wo d ug combina ions we e ad an ageous: 5-FU combined wi h e apamil and i aconazole, chosen o he con inui y o his p ojec . The exposu e o MCF-7 cells o 5-FU combined wi h e apamil and i aconazole, o 72 h o con ac wi h cells, esul ed in a cell iabili y educ ion (in compa ison wi h he d ug wi h mo e e ec on iabili y educ ion o ha combina ion, he po en ial epu posed d ug) o 23% and 17%, Figu e 1. E ec o d ugs and espec i e d ug combina ions on he iabili y o MCF-7 cells. Each d ug and each combina ion we e added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± s anda d e o o he mean (SEM) and ep esen he iabili y o cells (% o con ol) o 3–4 independen expe imen s (n= 3, 4). ### p< 0.001 s. con ol; ** p< 0.01 and *** p< 0.001 s. single d ug o he combina ion wi h mo e e ec on cell iabili y educ ion. 5-FU: 5- luo ou acil. In his pa icula sc eening assay, he c i e ion o he choice o d ug combina ions o he con inui y o he p ojec was ha he combina ion o d ugs was mo e ad an ageous in e ms o educ ion o cell iabili y han he wo d ugs in he combina ion, whe e he po en ially epu posed d ug was mo e e icien han 5-FU. The combina ion was mo e e e i e han d ugs sepa a ed. Analyzing he ob ained esul s, i was possible o obse e ha chlo oquine was mo e e ec i e in e ms o cell iabili y educ ion han all he o he d ugs and d ug combina ions (6.5 ± 0.4% o cellula iabili y). Thus, as he aim o his wo k was o s udy a bene icial d ug combina ion in compa ison wi h indi idual d ugs o he combina ion, chlo oquine was excluded om he nex s eps. Impo an ly, he combina ions o 5-FU wi h aspi in, losa an, cime idine, p a as a in, isoniazid and ac ine did no show an ad an age in e ms o educ ion o cell iabili y, ela i e o bo h single d ugs o he combina ion, being also excluded om his s udy. Howe e , wo d ug combina ions we e ad an ageous: 5-FU combined wi h e apamil and i aconazole, chosen o he con inui y o his Biomolecules 2018,8, 175 7 o 23 p ojec . The exposu e o MCF-7 cells o 5-FU combined wi h e apamil and i aconazole, o 72 h o con ac wi h cells, esul ed in a cell iabili y educ ion (in compa ison wi h he d ug wi h mo e e ec on iabili y educ ion o ha combina ion, he po en ial epu posed d ug) o 23% and 17%, espec i ely. Wi h 5-FU + e apamil, cell iabili y was 12.1 ± 4.4%, whe eas wi h 5-FU + i aconazole was 24.5 ±5.2%. In bo h cases, he di e ences we e conside ed s a is ically signi ican . 3.2. Compa ison o Cellula Viabili y be ween MCF-7 and MCF-10A Cell Lines To compa e he e ec s o he chosen d ug combina ions in a umo al cell line (MCF-7) and a non- umo al cell line (MCF-10A), bo h cell lines we e exposed o 50 µ M o each d ug, o 72 h. The esul s, o each cell line, we e ob ained by MTT me hodology (Figu e 2). Biomolecules 2018, 8, x FOR PEER REVIEW 7 o 23 espec i ely. Wi h 5-FU + e apamil, cell iabili y was 12.1 ± 4.4%, whe eas wi h 5-FU + i aconazole was 24.5 ± 5.2%. In bo h cases, he di e ences we e conside ed s a is ically signi ican . 3.2. Compa ison o Cellula Viabili y be ween MCF-7 and MCF-10A Cell Lines To compa e he e ec s o he chosen d ug combina ions in a umo al cell line (MCF-7) and a non- umo al cell line (MCF-10A), bo h cell lines we e exposed o 50 μM o each d ug, o 72 h. The esul s, o each cell line, we e ob ained by MTT me hodology (Figu e 2). Figu e 2. E ec o 5-FU, e apamil (A), i aconazole (B) and espec i e combina ions wi h 5-FU on he iabili y o MCF-7 cells (le , black ba s) and MCF-10A cells ( igh , whi e ba s). Each d ug and combina ion we e added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM and ep esen he iabili y o cells (% o con ol) o 3–4 independen expe imen s (n = 3, 4). ### p < 0.001 and # p < 0.05 s. espec i e con ols. I was obse ed ha , in gene al, he d ugs had no o e y li le e ec s on he iabili y o MCF- 10A cells, con as ing wi h he e ec s on iabili y o MCF-7 cells. Taking in o accoun he wo combina ions, 5-FU combined wi h i aconazole led o he lowes alues o cell iabili y in MCF-10A, 63.6 ± 8.5%, whe eas 5-FU combined wi h e apamil led o simila alues o cellula iabili y: 86.3 ± 3.2%. All he single d ugs and combina ions led o e ec s on cell iabili y educ ion o MCF-7 cells much mo e p onounced, compa ed wi h MCF-10A cell line. 3.3. Concen a ion-E ec Cu es and IC50 De e mina ion o he D ugs in MCF-7 Cells In o de o de e mine he IC50 (hal maximal inhibi o y concen a ion) alues o 5-FU, e apamil and i aconazole on he iabili y o MCF-7 cells, cells we e exposed o inc easing concen a ions o hese d ugs (1, 3, 5, 10, 25, 50 and 100 μM), con e ed in loga i hm o concen a ions. The d ugs we e in con ac wi h cells o a ime o 48 h, and cellula iabili y was de e mined by MTT educ ion assay. The ob ained alues allowed he c ea ion o concen a ion-e ec cu es (Figu es 3–5) o calcula e IC50 alues (Table 1). The IC50 o he d ugs was calcula ed by using he no malized da a (be ween 0% and 100%). Thus, i was de ined as he concen a ion o he d ug ha inhibi s 50% o cellula iabili y. Howe e , his me hod o calcula ion o IC50 has limi a ions, as epo ed in he Discussion Sec ion. This alue was calcula ed using G aphPad P ism 7 so wa e and he op ion “Analyze Da a, nonlinea eg ession (Cu e i )”. A B Figu e 2. E ec o 5-FU, e apamil ( A ), i aconazole ( B ) and espec i e combina ions wi h 5-FU on he iabili y o MCF-7 cells (le , black ba s) and MCF-10A cells ( igh , whi e ba s). Each d ug and combina ion we e added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM and ep esen he iabili y o cells (% o con ol) o 3–4 independen expe imen s (n= 3, 4). ### p< 0.001 and # p< 0.05 s. espec i e con ols. I was obse ed ha , in gene al, he d ugs had no o e y li le e ec s on he iabili y o MCF-10A cells, con as ing wi h he e ec s on iabili y o MCF-7 cells. Taking in o accoun he wo combina ions, 5-FU combined wi h i aconazole led o he lowes alues o cell iabili y in MCF-10A, 63.6 ± 8.5%, whe eas 5-FU combined wi h e apamil led o simila alues o cellula iabili y: 86.3 ± 3.2%. All he single d ugs and combina ions led o e ec s on cell iabili y educ ion o MCF-7 cells much mo e p onounced, compa ed wi h MCF-10A cell line. 3.3. Concen a ion-E ec Cu es and IC50 De e mina ion o he D ugs in MCF-7 Cells In o de o de e mine he IC 50 (hal maximal inhibi o y concen a ion) alues o 5-FU, e apamil and i aconazole on he iabili y o MCF-7 cells, cells we e exposed o inc easing concen a ions o hese d ugs (1, 3, 5, 10, 25, 50 and 100 µ M), con e ed in loga i hm o concen a ions. The d ugs we e in con ac wi h cells o a ime o 48 h, and cellula iabili y was de e mined by MTT educ ion assay. The ob ained alues allowed he c ea ion o concen a ion-e ec cu es (Figu es 3–5) o calcula e IC 50 alues (Table 1). The IC 50 o he d ugs was calcula ed by using he no malized da a (be ween 0% and 100%). Thus, i was de ined as he concen a ion o he d ug ha inhibi s 50% o cellula iabili y. Howe e , his me hod o calcula ion o IC 50 has limi a ions, as epo ed in he Discussion Sec ion. This alue was calcula ed using G aphPad P ism 7 so wa e and he op ion “Analyze Da a, nonlinea eg ession (Cu e i )”. Biomolecules 2018,8, 175 8 o 23 Biomolecules 2018, 8, x FOR PEER REVIEW 8 o 23 Figu e 3. Concen a ion-e ec cu es ob ained o 5-FU. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o h ee independen expe imen s. The le cu es ep esen he iabili y o cells (% o con ol), whe eas he igh cu es ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less a ec ed he cell iabili y, and all o he da a poin s we e no malized o his alue, being 0% de ined as he concen a ion ha a ec ed mo e he cell iabili y. ### p < 0.001 and # p < 0.05 s. con ol. Figu e 4. Concen a ion-e ec cu es ob ained o e apamil. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o h ee independen expe imen s. The le cu e ep esen s he iabili y o cells (% o con ol), whe eas he igh cu e ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less a ec ed he cell iabili y, and all o he da a poin s we e no malized o his alue, being 0% de ined as he concen a ion ha a ec ed mo e he cell iabili y. ### p < 0.001 and # p < 0.05 s. con ol. Figu e 5. Concen a ion-e ec cu es ob ained o i aconazole. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o i e independen expe imen s. The le cu e ep esen s he iabili y o cells (% o con ol), whe eas he igh cu e ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less Cell Viabili y (% o con ol) Cell Viabili y (% o maximal esponse) Figu e 3. Concen a ion-e ec cu es ob ained o 5-FU. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o h ee independen expe imen s. The le cu es ep esen he iabili y o cells (% o con ol), whe eas he igh cu es ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less a ec ed he cell iabili y, and all o he da a poin s we e no malized o his alue, being 0% de ined as he concen a ion ha a ec ed mo e he cell iabili y. ### p< 0.001 and # p< 0.05 s. con ol. Biomolecules 2018, 8, x FOR PEER REVIEW 8 o 23 Figu e 3. Concen a ion-e ec cu es ob ained o 5-FU. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o h ee independen expe imen s. The le cu es ep esen he iabili y o cells (% o con ol), whe eas he igh cu es ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less a ec ed he cell iabili y, and all o he da a poin s we e no malized o his alue, being 0% de ined as he concen a ion ha a ec ed mo e he cell iabili y. ### p < 0.001 and # p < 0.05 s. con ol. Figu e 4. Concen a ion-e ec cu es ob ained o e apamil. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o h ee independen expe imen s. The le cu e ep esen s he iabili y o cells (% o con ol), whe eas he igh cu e ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less a ec ed he cell iabili y, and all o he da a poin s we e no malized o his alue, being 0% de ined as he concen a ion ha a ec ed mo e he cell iabili y. ### p < 0.001 and # p < 0.05 s. con ol. Figu e 5. Concen a ion-e ec cu es ob ained o i aconazole. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o i e independen expe imen s. The le cu e ep esen s he iabili y o cells (% o con ol), whe eas he igh cu e ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less Cell Viabili y (% o con ol) Cell Viabili y (% o maximal esponse) Figu e 4. Concen a ion-e ec cu es ob ained o e apamil. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o h ee independen expe imen s. The le cu e ep esen s he iabili y o cells (% o con ol), whe eas he igh cu e ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less a ec ed he cell iabili y, and all o he da a poin s we e no malized o his alue, being 0% de ined as he concen a ion ha a ec ed mo e he cell iabili y. ### p< 0.001 and # p< 0.05 s. con ol. Biomolecules 2018, 8, x FOR PEER REVIEW 8 o 23 Figu e 3. Concen a ion-e ec cu es ob ained o 5-FU. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o h ee independen expe imen s. The le cu es ep esen he iabili y o cells (% o con ol), whe eas he igh cu es ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less a ec ed he cell iabili y, and all o he da a poin s we e no malized o his alue, being 0% de ined as he concen a ion ha a ec ed mo e he cell iabili y. ### p < 0.001 and # p < 0.05 s. con ol. Figu e 4. Concen a ion-e ec cu es ob ained o e apamil. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o h ee independen expe imen s. The le cu e ep esen s he iabili y o cells (% o con ol), whe eas he igh cu e ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less a ec ed he cell iabili y, and all o he da a poin s we e no malized o his alue, being 0% de ined as he concen a ion ha a ec ed mo e he cell iabili y. ### p < 0.001 and # p < 0.05 s. con ol. Figu e 5. Concen a ion-e ec cu es ob ained o i aconazole. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o i e independen expe imen s. The le cu e ep esen s he iabili y o cells (% o con ol), whe eas he igh cu e ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less Cell Viabili y (% o con ol) Cell Viabili y (% o maximal esponse) Figu e 5. Concen a ion-e ec cu es ob ained o i aconazole. The d ug was added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM o i e independen expe imen s. The le cu e ep esen s he iabili y o cells (% o con ol), whe eas he igh cu e ep esen s a no maliza ion be ween 0 and 1 (0% and 100%), whe e 100% was de ined as he concen a ion o he d ug ha less a ec ed he cell iabili y, and all o he da a poin s we e no malized o his alue, being 0% de ined as he concen a ion ha a ec ed he cell iabili y mo e. ## p< 0.01 and # p< 0.05 s. con ol. Biomolecules 2018,8, 175 9 o 23 Table 1. Ob ained IC 50 alues o 5-FU, e apamil and i aconazole on he iabili y o MCF-7 cells, wi h he co esponding 95% con idence in e als. D ug IC50 (µM) 5-FU 11.79 Ve apamil 29.49 I aconazole 2.08 3.4. E ec o 5-FU Fixed in I s IC50 Value and Va ia ion o he Repu posed D ugs Concen a ion in Values a ound Thei IC50 Values, on Viabili y o MCF-7 Cells The IC 50 alue ob ained o 5-FU (app oxima ely 11.8 µ M) was ixed, and he concen a ion o he o he wo d ugs a ied by alues a ound he ob ained IC 50 alue o each d ug (app oxima ely 29 and 6 µ M o e apamil and i aconazole, espec i ely). I is impo an o no e ha he IC 50 alue ob ained o i aconazole was ini ially 6 µ M and his was he alue o IC 50 conside ed o his d ug. Howe e , in a inal phase o he wo k, he expe imen o gene a ing a concen a ion-e ec cu e o i aconazole was epea ed and a new alue o IC50 (2.08 µM) was ob ained. The d ugs we e in con ac wi h cells o a ime o 48 h and cellula iabili y was de e mined by MTT educ ion assay (Figu e 6). Biomolecules 2018, 8, x FOR PEER REVIEW 9 o 23 a ec ed he cell iabili y, and all o he da a poin s we e no malized o his alue, being 0% de ined as he concen a ion ha a ec ed he cell iabili y mo e. ## p < 0.01 and # p < 0.05 s. con ol. Table 1. Ob ained IC50 alues o 5-FU, e apamil and i aconazole on he iabili y o MCF-7 cells, wi h he co esponding 95% con idence in e als. D ug IC50 (μM) 5-FU 11.79 Ve apamil 29.49 I aconazole 2.08 3.4. E ec o 5-FU Fixed in I s IC50 Value and Va ia ion o he Repu posed D ugs Concen a ion in Values a ound Thei IC50 Values, on Viabili y o MCF-7 Cells The IC50 alue ob ained o 5-FU (app oxima ely 11.8 μM) was ixed, and he concen a ion o he o he wo d ugs a ied by alues a ound he ob ained IC50 alue o each d ug (app oxima ely 29 and 6 μM o e apamil and i aconazole, espec i ely). I is impo an o no e ha he IC50 alue ob ained o i aconazole was ini ially 6 μM and his was he alue o IC50 conside ed o his d ug. Howe e , in a inal phase o he wo k, he expe imen o gene a ing a concen a ion-e ec cu e o i aconazole was epea ed and a new alue o IC50 (2.08 μM) was ob ained. The d ugs we e in con ac wi h cells o a ime o 48 h and cellula iabili y was de e mined by MTT educ ion assay (Figu e 6). Figu e 6. E ec o e apamil (A) and i aconazole (B) combined wi h 5-FU on he iabili y o MCF-7 cells. Each d ug and d ug combina ion we e added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM, and ep esen he iabili y o cells (% o con ol) o h ee independen expe imen s. ### p < 0.001, ## p < 0.01 and # p < 0.05 s. con ol. The lowes alues o cellula iabili y we e ob ained o concen a ions o 55 and 3 μM, o e apamil and i aconazole, (10.4 ± 0.6%; 34.9 ± 2.7%) espec i ely. A endency o lowe alues o cellula iabili y wi h an inc ease o e apamil concen a ion was obse ed, whe eas wi h i aconazole, i seemed ha abo e 3 μM, he concen a ion o he d ug had li le o no e ec on educ ion o he cellula iabili y. Clea ly, e apamil appea ed o be he d ug which was mo e a ec ed by di e ences in concen a ion. 3.5. E ec o Concen a ions o 11.8, 55 and 3 μM o 5-FU, Ve apamil and I aconazole, Respec i ely, on he Viabili y o MCF-7 Cells, o 48 and 72 h Fu he , we es ed he e ec o 5-FU, e apamil, i aconazole and he espec i e combina ions o he las wo d ugs wi h 5-FU on he iabili y o MCF-7 cells, using 5-FU in a concen a ion o 11.8 μM (IC50 alue), and e apamil and i aconazole in a concen a ion o 55 and 3 μM, espec i ely, ac ing on cells o a ime o 48 h o 72 h (Figu e 7). The concen a ions used o he epu posed d ugs we e A B Figu e 6. E ec o e apamil ( A ) and i aconazole ( B ) combined wi h 5-FU on he iabili y o MCF-7 cells. Each d ug and d ug combina ion we e added in esh medium, in sex aplica es. Resul s a e p esen ed as mean ± SEM, and ep esen he iabili y o cells (% o con ol) o h ee independen expe imen s. ### p< 0.001, ## p< 0.01 and # p< 0.05 s. con ol. The lowes alues o cellula iabili y we e ob ained o concen a ions o 55 and 3 µ M, o e apamil and i aconazole, (10.4 ± 0.6%; 34.9 ± 2.7%) espec i ely. A endency o lowe alues o cellula iabili y wi h an inc ease o e apamil concen a ion was obse ed, whe eas wi h i aconazole, i seemed ha abo e 3 µ M, he concen a ion o he d ug had li le o no e ec on educ ion o he cellula iabili y. Clea ly, e apamil appea ed o be he d ug which was mo e a ec ed by di e ences in concen a ion. 3.5. E ec o Concen a ions o 11.8, 55 and 3 µM o 5-FU, Ve apamil and I aconazole, Respec i ely, on he Viabili y o MCF-7 Cells, o 48 and 72 h Fu he , we es ed he e ec o 5-FU, e apamil, i aconazole and he espec i e combina ions o he las wo d ugs wi h 5-FU on he iabili y o MCF-7 cells, using 5-FU in a concen a ion o 11.8 µ M (IC 50 alue), and e apamil and i aconazole in a concen a ion o 55 and 3 µ M, espec i ely, ac ing on cells o a ime o 48 h o 72 h (Figu e 7). The concen a ions used o he epu posed d ugs we e selec ed aking in o accoun he expe imen desc ibed in Sec ion 3.4, being he concen a ions ha ga e lowe alues o cellula iabili y in combina ion wi h 5-FU ixed on i s IC50 alue. Biomolecules 2018,8, 175 16 o 23 Biomolecules 2018, 8, x FOR PEER REVIEW 15 o 23 esul s a e exp essed as mean luo escence in ensi y (MFI) due o CFSE s aining. Highe alues o luo escence in ensi y mean less cell p oli e a ion. Figu e 14. Flow cy ome y ga ing s a egy used in he ca boxy luo escein succinimydil es e (CFSE) p oli e a ion assay o MCF-7 cells. (A) The ga e sepa a ed MCF-7 cells om cellula deb is, whe eas in (B) single cells a e ga ed, hus excluding agg ega es. (C) Dead cells we e excluded by PI inco po a ion, and (D) ep esen s cell luo escence due o CFSE s aining in non- ea ed (con ol) o 5- FU + Ve apamil ( ea ed cells), as indica ed. (A) (B) Figu e 15. Cell luo escence due o CFSE s aining. In all g aphs, he black line indica es con ol (s ained), he g ey line indica es uns ained con ol and he blue line ep esen s 5-FU. (A) The ed line indica es he combina ion o 5-FU + e apamil and he o ange line indica es e apamil. (B) Red and o ange lines ep esen 5-FU + i aconazole and i aconazole, espec i ely. Analyzing he ob ained esul s, i was possible o conclude ha he wo d ug combina ions p omo ed a mo e p onounced a es on cell di ision, compa ed o he indi idual d ugs o each combina ion. Pa icula ly, wi h 5-FU + e apamil, MFI alues we e 162.3 ± 48.9% and wi h 5- FU+i aconazole hese alues we e 175.4 ± 50.1%, compa ed wi h alues o 154.9 ± 30.2% (5-FU) and Figu e 16. Flow cy ome y ga ing s a egy used in he ca boxy luo escein succinimydil es e (CFSE) p oli e a ion assay o MCF-7 cells. ( A ) The ga e sepa a ed MCF-7 cells om cellula deb is, whe eas in ( B ) single cells a e ga ed, hus excluding agg ega es. ( C ) Dead cells we e excluded by PI inco po a ion, and ( D ) ep esen s cell luo escence due o CFSE s aining in non- ea ed (con ol) o 5-FU + Ve apamil ( ea ed cells), as indica ed. Analyzing he ob ained esul s, i was possible o conclude ha he wo d ug combina ions p omo ed a mo e p onounced a es on cell di ision, compa ed o he indi idual d ugs o each combina ion. Pa icula ly, wi h 5-FU + e apamil, MFI alues we e 162.3 ± 48.9% and wi h 5-FU+i aconazole hese alues we e 175.4 ± 50.1%, compa ed wi h alues o 154.9 ± 30.2% (5-FU) and 169.16 ± 49.5% (i aconazole) ob ained wi h he indi idual d ugs wi h mo e e ec on cell p oli e a ion a es o each combina ion, espec i ely. 5-Fluo ou acil combined wi h i aconazole was he d ug combina ion ha led o he highes MFI alues. I is impo an o no e ha high SEM alues we e ob ained, since he wo expe imen s esul ed in e y di e en alues. Howe e , analyzing g aph C, i was possible o conclude ha bo h expe imen s showed he same endency o esul s, in which d ug combina ions a e ad an ageous ela i e o he isola ed d ugs. 4. Discussion In his s udy, he wo po en ial epu posed d ugs in he d ug sc eening assay we e e apamil and i aconazole, combined wi h 5-FU, since hey we e hose ha showed be e esponses (inc eased educ ion o cellula iabili y), when compa ed o bo h indi idual d ugs o each combina ion. In pa icula , e apamil is a calcium channel blocke , mainly a L- ype calcium channel blocke , inhibi ing he ansmemb ane lux o calcium ions, being u ilized clinically o ea ca diac a hy hmias, angina and ca diomyopa hies [ 31 , 32 ]. Besides he known ole o ion channels in specialized exci able cells, hese channels also play c i ical oles in cance pa hophysiology by se e al mechanisms, con olling cance cell p oli e a ion by egula ing se e al key su i al signaling pa hways and memb ane po en ial [ 33 ]. Ve apamil is also known o be a i s -gene a ion inhibi o o P-glycop o ein, an impo an p o ein p esen in he cell memb ane, ha pumps a wide a ie y o subs ances ou o cells [ 34 ]. Thus, when combined wi h chemo he apeu ic agen s, his d ug may help o p omo e in acellula d ug accumula ion. This has been demons a ed in non-small cell lung cance , colo ec al ca cinoma, leukemia and neu oblas oma cell lines [ 30 ]. Pa icula ly in b eas cance , se e al s udies epo ed bene icial e ec s o e apamil. Fo example, his d ug shown an i-p oli e a i e e ec in b eas cance in a mouse model [ 35 ]. Po en ia ion o amoxi en ac i i y by e apamil, in a human b eas Biomolecules 2018,8, 175 17 o 23 cance cell line (MCF-7), has also been epo ed [ 36 ]. Howe e , he e a e con adic o y epo s abou he an icance p ope ies o e apamil. Fo example, in human epide moid ca cinoma cells, e apamil did no inhibi he g ow h o cells [ 33 ]. Addi ionally, he cu en use o calcium-channel blocke s, such as e apamil, o 10 o mo e yea s was associa ed wi h highe isks o duc al and lobula b eas cance . Thus, mo e esea ch is needed o con i m hese indings and o e alua e po en ial unde lying biological mechanisms o e apamil in cance [37]. Ve apamil is an L- ype calcium channel blocke , bu i is also a blocke o o he calcium channels. Conce ning i aconazole, i is a b oad-spec um an i- ungal agen ha inhibi s lanos e ol 14- α -deme hylase, being used o ea ungal in ec ions and o p ophylaxis in immunosupp essi e diso de s [ 29 , 38 ]. S udies ha e shown ha his d ug possesses an ineoplas ic ac i i y and also has a syne gis ic ac ion when combined wi h chemo he apeu ic agen s. Al hough i showed p omising an icance ac i i y in se e al ypes o cance , i s p ecise an icance mechanism has emained elusi e [ 29 , 39 ]. Howe e , i is known ha i ac s ia se e al mechanisms o p e en umo g ow h, including inhibi ion o he Hedgehog pa hway, p e en ion o angiogenesis, dec eased endo helial cell p oli e a ion, and cell cycle a es . Also, in o a ian and b eas cance , in i o s udies con i m ha i aconazole inhibi s P-glycop o ein, hus e e sing esis ance con e ed by his e lux pump [ 29 ]. In b eas cance , a pilo ial e alua ed he pha macokine ics o his d ug when adminis e ed o 13 pa ien s wi h me as a ic b eas cance . The conclusions we e ha as he plasma le els o i aconazole inc eased, highe le els o h ombospondin-1, which inhibi s angiogenesis, we e de ec ed [ 9 ]. In ano he s udy, i aconazole inhibi ed MCF-7 and SKBR-3 cells p oli e a ion ia induc ion o cell dea h and G0/G1 cell cycle a es [40]. Despi e hese d ugs showing a leas minimal e idence o some cance ac i i y, hei mechanisms o ac ion o his condi ion a e no ye ully de ined. I is also impo an o no e ha in his esea ch, because d ugs ha e been s udied in he con ex o combina ion wi h 5-FU, he mechanism by which hey ac in he cell may be di e en om ha which would be obse ed wi h he indi idual d ugs, since he combina ion may ac oge he in a di e en way in each o i s indi idual componen s. Thus, a deep s udy o hese mechanisms may ep esen an impo an s ep in b eas cance he apy, as well as in o he ypes o cance . The da a ob ained in his wo k allowed o conclude ha he wo selec ed d ug combina ions ha e po en ial bene icial e ec s on b eas cance he apy. Thus, analyzing he ob ained esul s along his esea ch, i was clea ha cellula iabili y was educed in compa ison wi h indi idual d ugs o he combina ion, in he g ea majo i y o cases. This educed cellula iabili y obse ed by MTT assay, as well as he cellula damage obse ed in cells ea ed wi h d ug combina ions, namely p esence o damage in cell memb anes and cellula acuoles in he cy oplasm (Figu e 11) may e lec e en s such as cell dea h o cell p oli e a ion a es . The p elimina y esul s ob ained by CFSE s aining o cells indica ed ha all d ug combina ions showed a endency o s op cell p oli e a ion. Thus, i can be hypo hesized ha he d ug combina ions may ac p ima ily in he a es o cell p oli e a ion. The combina ion 5-FU + i aconazole being he combina ion o d ugs ha mos a ec s his pa ame e . Besides he da a ob ained by CFSE s aining o cells, he da a ob ained by he compa ison o MCF-7 and MCF-10A cell lines also suppo ed his hypo hesis; once he d ugs had a much mo e p onounced e ec on MCF-7 han MCF-10A cells, he las , he p oli e a ion dec ease and, hus, di ided less. Addi ionally, when compa ing he esul s ob ained o 48 and 72 h- ea men s, he cell iabili y was mo e educed a 72 h, sus aining again he hypo hesis ha he d ug combina ions ac on cell p oli e a ion; in 72 h, hey ha e mo e ime o p oli e a e (and, maybe, inco po a e he d ug), a he han in 48 h. 5-Fluo ou acil ac s mainly on he S phase o cell cycle, hus, an explana ion abou his endency o d ug combina ions o s op p oli e a ion may be, o example, a po en ia ion o he 5-FU ac ion by he epu posed d ugs o a p omo ion o he accumula ion o 5-FU inside he cells. Fu he s udies o es his hypo hesis will be ele an . E en hough no e y conclusi e (possible mainly because o he small numbe o expe imen s), annexin V/PI s aining o cells showed ha apop osis was no obse ed a 3 h o ea men , excep Biomolecules 2018,8, 175 18 o 23 in he case o 5-FU + i aconazole (126.8 ± 23.9%), bu was obse ed in he majo i y o cases (single d ugs and combina ions) a 8 h, sugges ing ha wi h la e imes, he obse a ion o apop o ic e en s is clea e . The same happened conce ning la e apop osis/nec osis, whe e an inc ease in hese alues was obse ed a 8 h o ea men , compa ing o 3 h. Also, i was expec ed ha in he condi ions whe e apop osis was e i ied a 3 h (only e i ied wi h 5-FU + i aconazole), he 8 h end would be an inc ease in la e apop osis, which was no e i ied, p obably because his p ocess ook e en longe han 8 h o occu . The imeline o apop o ic- ela ed biochemical e en s depends on a huge numbe o ac o s, such as cell cul u e condi ions, he cell line used, d ug concen a ion/s imulus in ensi y and exposu e ime [ 41 ]. In cell cul u e, apop o ic signs may be accomplished in less han wo hou s [ 42 ]. Howe e , he e a e also s udies ha epo ha , in a gene al way, cul u ed cells induced o unde go apop osis exhibi signs o apop o ic e en s wi hin 5 o 10 h [ 41 ]. Combining all esul s ob ained by his echnique, i was no o ious ha he e was a g ea e endency o cell dea h a 8 h han a 3 h, ei he by apop osis o by nec osis. Howe e , in his momen , i is no possible o conclude ha a ce ain combina ion ac s ia apop osis and/o nec osis, being c ucial o inc ease he numbe o independen expe imen s o ob ain mo e sus ainable esul s ha can allow be e conclusions. Also, conce ning his assay, i would be use ul o es a di e en ime poin s, namely la e imes. On c ossing da a om p oli e a ion and cell dea h assays, he seemingly mo e plausible conclusion is ha d ug combina ions ac essen ially on pa hways ela ed o cell p oli e a ion a es . A e his a es , he cells end o accumula e damage (Figu e 11) and, consequen ly die. Hence, i is plausible ha he highes cell dea h alues we e ob ained o 8 h. This inc ease is likely o con inue wi h inc easing d ug exposu e ime. Ano he s udy ca ied ou in he scope o his wo k was he e alua ion o he EMT o possible d ug esis an cells. Epi helial-mesenchymal ansi ion is an impo an p ocess in no mal de elopmen bu also plays a c i ical ole du ing umo p og ession and malignan ans o ma ion, leading o he acquisi ion o in asi e and me as a ic p ope ies in cance cells [43]. S udies ha e epo ed ha he acquisi ion o 5-FU esis ance induces epi helial-mesenchymal ansi ions h ough he Hedgehog signaling pa hway in HCT-8 colon cance cells [ 44 ]. Also, EMT was associa ed wi h acqui ed esis ance o 5-FU in HT-29 colon cance cells [ 45 ]. Addi ional s udies also p o ed ha chemo esis ance o 5-FU induces epi helial-mesenchymal ansi ion ia up egula ion o Snail in MCF-7 human b eas cance cells [ 46 ]. Thus, o s udy his ansi ion o s a e, he p esence o E-cadhe in, cy oke a ins AE1/AE3 and imen in was s udied. The down egula ion o E-cadhe in is a molecula signa u e o EMT. This molecule is an adhesion molecule ha is p esen in he plasma memb ane o mos epi helial cells [ 47 ]. Cy oke a ins a e also ma ke s o an epi helial pheno ype. This is a p o ein wi h an in e media e ilamen made o ke a in, ha ing impo an oles in cell di e en ia ion [ 48 , 49 ]. Pa icula ly, cy oke a in AE1/AE3 is a mix u e o wo di e en clones o an i-cy oke a in monoclonal an ibodies, AE1 and AE3, ha de ec high and low molecula weigh ke a ins. By combining hese wo eagen s, a single eagen wi h a b oad spec um o eac i i y agains a huge di e si y o cy oke a ins is ob ained [ 49 ]. Rela i e o imen in, i is ubiqui ously exp essed in no mal mesenchymal cells, being known o main ain cellula in eg i y and p o iding esis ance agains s ess. O e exp ession o imen in in cance co ela es well wi h accele a ed umo g ow h, in asion, and poo p ognosis [50]. E-cadhe in’s down egula ion is an impo an leading e en o EMT and is conside ed a hallma k o his ansi ion. I s down egula ion can lead o dec eased exp ession and/o o ganiza ion o addi ional epi helial ma ke s. Concomi an ly, inc eased exp ession o mesenchymal p o eins (such as imen in), as well as o ex acellula ma ix emodeling enzymes occu s oge he wi h ac in cy oskele on eo ganiza ion [ 51 ]. Thus, his seems o be a p ocess in which se e al e en s occu concomi an ly. Analyzing he images ob ained by immunocy ochemis y, i was clea ha no EMT was obse ed, in any o he cases. Howe e , in he case o 5-FU combined wi h e apamil, he e seemed o be a e y weak s aining o E-cadhe in. The eason o his inding is unknown, since he e a e no s udies Biomolecules 2018,8, 175 19 o 23 wi h his d ug combina ion ega ding EMT in cance cells, pa icula ly in b eas cance cells. Thus, his combina ion may ha e some ac ion on E-cadhe in o e en p oduce a ansi ion om epi helial o mesenchymal s a e, i s obse ed by a loss o E-cadhe in; his may no be ully obse ed due o a sho ime o d ug ac ion. Thus, i could be s a ed ha 48 h was no enough ime o he ansi ion o he mesenchymal s a e o occu . Howe e , s udies show ha his ansi ion is obse ed ea lie . Fo example, in a s udy using he MCF-7 cell line o s udy whe he EGF induced EMT, i was ound ha he loss o E-cadhe in, as well as inc eased exp ession o imen in, began o be obse ed abou 4–8 h a e he s imulus [ 52 ]. In ano he s udy, EMT occu ed on day se en a e he s imulus, in his case wi h g anulocy e mac ophage colony-s imula ing ac o o colon cance [ 53 ]. Thus, i is no ed ha he obse a ion o EMT depends on he s imulus, as well as on he cell lines in which he s udy is aking place. An in e es ing u u e app oach could be he s udy o his ansi ion a di e en imes poin s. Be o e concluding, i is also impo an o highligh ha one o he ac o s ha should be conside ed in his esea ch wo k is he concen a ion o he d ugs used in mos o he expe imen s, which was based on he concen a ion-e ec cu es and, mo e speci ically, on he IC 50 alues ob ained. The IC 50 concep is no clea . Fo example, i would be ambiguous i he alues ha de ine 100% and 0% a e no clea ly de ined [ 54 , 55 ]. To sol e his p oblem, no malizing he da a in o de ha esponses a y be ween 0 and 100, o cing he bo om and op pla eaus o equal 0 and 100, espec i ely, may be a solu ion. Howe e , a no malized model should be applied only when he alues ha de ine 0 and 100 a e co ec ly de e mined [ 54 , 55 ]. In his p ojec , IC 50 alues o he h ee epu posed d ugs was calcula ed conside ing his ype o no maliza ion, since no pe ec ly de ined dose- esponse cu es wi h de ined pla eaus and whe e bo h maximal and minimal ac i i y we e e y explici . Ne e heless, he ob ained alue o he IC 50 o he e e ence d ug, 5-FU was consis en wi h ha ob ained in s udies o his d ug in MCF-7 using he same me hodology (MTT) [ 56 ]. Thus, conce ning ob ained IC 50 alues, i aconazole appea ed o be he mos po en d ug, since i p esen ed he lowes IC50 alue. Ano he impo an aspec is he ac ha only one a io o concen a ions (1:1) be ween he combina ions was es ed in he sc eening assay (Figu e 1), because i was impo an o ensu e ha he mechanisms o ac ion o bo h d ugs did no o e lap. In line wi h his, o example, in i o expe imen s iden i ied a s ong an agonism be ween i ino ecan and cispla in when hey we e adminis e ed a a 1:1 a io, bu in a a io o 4:1, a syne gis ic e ec was obse ed. Also, he combina ion o i ino ecan and loxu idine was syne gis ic a an equimola a io bu was s ongly an agonis ic a a 10:1 a io [ 57 ]. Thus, i is a ha d p ocess o op imize a ios o concen a ion in d ug combina ions. Finally, by in eg a ing all he ob ained esul s, i was no ed ha all d ug combina ions showed a endency o be mo e e ec i e han he mos e ec i e d ug o each combina ion. The bes esul s we e ob ained o 72-h ea men and, in mos cases, he e we e no accen ed di e ences be ween he di e en concen a ions es ed. Al hough he combina ion o 5-FU and e apamil was he one ha led o he lowes cell iabili y alues (app oxima ely 12%, a 72 h in concen a ions o 50 µ M o each d ug in he combina ion), he collec i e o all esul s poin ed ou ha he combina ion o 5-FU and i aconazole was he mos p omising combina ion. The esul s ob ained by MTT educ ion assay suppo his e idence (bo h a 48 h and a 72 h, ega dless o he concen a ion used, excep a 72 h, a concen a ions o 50 µ M o each d ug in he combina ion, in which case 5-FU + e apamil was highligh ed). Fu he mo e, analyzing he IC 50 alues ob ained, i aconazole was he d ug ha p esen ed he lowes alue, being appa en ly he mos po en . Addi ionally, CFSE s aining o cells showed ha 5-FU combined wi h i aconazole was he d ug combina ion ha p omo ed mo e ele a ed alues o MFI, indica ing a mo e p onounced p oli e a ion a es . S udies in MCF-10A cells also suppo ed his hypo hesis, since i aconazole was he only d ug which when combined wi h 5-FU and used indi idually showed signi ican ly di e en e ec s ela i e o he con ol, which may indica e inc eased oxici y. In addi ion o all his, i is impo an o men ion ha conce ning he wo po en ial epu posed d ugs used in his esea ch, i aconazole is he one ha is mos s udied in he ield o cance , showing p omising esul s. Addi ionally, his d ug was used in a lowe concen a ion and, as known, a success ul he apy is a he apy in which e icacy is Biomolecules 2018,8, 175 20 o 23 achie ed wi h he lowes possible d ug concen a ion. Thus, he wo d ug combina ions we e shown o be p omising in b eas cance he apy, e en hough e idence poin ed o i aconazole, and pa icula ly i s combina ion wi h 5-FU, a e y ele an objec o s udy o he he apy o b eas cance and, possibly, o he ypes o cance . 5. Concluding Rema ks Cance is a complex g oup o diseases ha in ol es se e al pa hways and di e en molecules, being e y challenging o ea . Oncological he apy, which includes b eas cance he apy, is inc easingly being s udied wi h he apeu ic s a egies such as d ug epu posing and d ug combina ion being la gely adop ed. In his esea ch, he combina ion o d ugs wi h po en ial o be epu posed ( e apamil, i aconazole) wi h a e e ence chemo he apeu ic d ug (5-FU) showed ad an ages o e indi idual d ugs, mainly by a es ing cell p oli e a ion and dec easing cellula iabili y. Thus, he d ug combina ions showed e idence o be p omising o he ea men o b eas cance . A u u e s udy on mo e esis an b eas cance cell lines (such as he iple nega i e line MDA-MB-231) and also on di e en cance cell lines co esponding o o he ypes o cance would be in e es ing. In i o s udies o applica ion in human he apy would also be ex emely impo an in a mo e ad anced line o wo k. Based on ecen li e a u e [ 58 – 61 ], we belie e ha op imizing d ug a ios could esul in be e an i umo e icacy han he single-d ug sys em and each d ug signi ican ly changes syne gy be ween he d ugs. Howe e , o all o his o be achie ed, a be e knowledge abou he mechanisms o ac ion unde lying hese d ug combina ions is equi ed, wi h a special ocus on 5-FU combined wi h i aconazole, since i was, globally, he mos p omising d ug combina ion. Au ho Con ibu ions: All o au ho s we e esponsible o he a icle and app o ed he o iginal d a p epa a ion. Funding: This wo k was inanced by FEDER—Fundo Eu opeu de Desen ol imen o Regional unds h ough he COMPETE 2020—Ope a ional P og amme o Compe i i eness and In e na ionalisa ion (POCI), Po ugal 2020, and Po uguese unds h ough FCT - Fundação pa a a Ciência e a Tecnologia, in he amewo k o he p ojec s “Ins i u e o Resea ch and Inno a ion in Heal h Sciences” (POCI-01-0145-FEDER-007274). NV also acknowledges suppo om FCT and FEDER (Eu opean Union), awa d numbe IF/00092/2014/CP1255/CT0004. Alexand a Co eia was suppo ed by FCT g an SFRH/BPD/91623/2012. Acknowledgmen s: Nuno Vale hanks FCT o he IF posi ion and Fundação Manuel An ónio da Mo a (FMAM, Po o, Po ugal) and P ize (Po ugal) o suppo o he Nuno Vale Resea ch G oup. The con en s o his a icle a e solely he esponsibili y o he au ho s and do no necessa ily ep esen he o icial iews o FCT, FMAM and P ize . Con lic s o In e es : The au ho s decla e no con lic o in e es . Re e ences 1. Malani, P.N. Ha ison’s P inciples o In e nal Medicine. JAMA 2012,308, 1813–1814. [C ossRe ] 2. Cance Resea ch UK. Wo ldwide Cance S a is ics. A ailable online: h ps://www.cance esea chuk.o g/h eal h-p o essional/cance -s a is ics/wo ldwide-cance #heading-One (accessed on 8 Augus 2018). 3. Cance The apy Ad iso . B eas Cance (In asi e) T ea men Regimens. A ailable online: h ps://www.canc e he apyad iso .com/b eas -cance /b eas -cance -in asi e- ea men - egimens/a icle/218154/ (accessed on 21 Ma ch 2018). 4. G een, R.J.; Ha is, N.D. Pa hology and The apeu ics o Pha macis s, 3 d ed.; Pha maceu ical P ess: London, UK, 2008. 5. Dawood, S.; Aus in, L.; C is o anilli, M. Cance s em cells: Implica ions o cance he apy. Oncology (Willis on Pa k) 2014,28, 1101–1107, 1110. [PubMed] 6. K eso, A.; Dick, J.E. E olu ion o he cance s em cell model. Cell S em Cell 2014 ,14, 275–291. [C ossRe ] [PubMed] 7. And esen, V.; Gje sen, B.T. D ug Repu posing o he T ea men o Acu e Myeloid Leukemia. F on . Med. 2017,4, 211. [C ossRe ] [PubMed] Biomolecules 2018,8, 175 21 o 23 8. He nandez, J.J.; Michael, P.; Lindsay, S.; Conno , Y.; Naheed, K.; Vijay, M.S.; S e en, V.M. Gi ing D ugs a Second Chance: O e coming Regula o y and Financial Hu dles in Repu posing App o ed D ugs as Cance The apeu ics. F on . Oncol. 2017,7, 273. [C ossRe ] [PubMed] 9. Pan zia ka, P.; Gau hie , B.; Lydie, M.; Vidula, S.; Vikas, P.S. The epu posing d ugs in oncology (ReDO) p ojec . Ecance medicalscience 2014,8, 442. [C ossRe ] 10. Ishida, J.; Konishi, M.; Ebne , N.; Sp inge , J. Repu posing o app o ed ca dio ascula d ugs. J. T ansl. Med. 2016,14, 269. [C ossRe ] 11. Ve baande d, C.; Meheus, L.; Huys, I.; Pan zia ka, P. Repu posing d ugs in oncology: Nex s eps. T ends Cance 2017,3, 543–546. [C ossRe ] 12. Be olini, F.; Sukha me, V.P.; Bouche, G. D ug epu posing in oncology—Pa ien and heal h sys ems oppo uni ies. Na . Re . Clin. Oncol. 2015,12, 732. [C ossRe ] 13. ClinicalT ials.go . A Phase III Randomized T ial o Me o min s. Placebo in Ea ly S age B eas Cance . A ailable online: h ps://clinical ials.go /c 2/show/NCT01101438 (accessed on 25 May 2018). 14. Foucquie , J.; Guedj, M. Analysis o d ug combina ions: Cu en me hodological landscape. Pha macol. Res. Pe spec . 2015,3, e00149. [C ossRe ] 15. Han, K.; Jeng, E.E.; Hess, G.T.; Mo gens, D.W.; Li, A.; Bassik, M.C. Syne gis ic d ug combina ions o cance iden i ied in a CRISPR sc een o pai wise gene ic in e ac ions. Na . Bio echnol. 2017 ,35, 463–474. [C ossRe ] [PubMed] 16. Na ional Cance Ins i u e. Combining The apies o Imp o e Ou comes. A ailable online: h ps://www.canc e .go /abou -nci/budge /plan/ ea ing/combining- he apies (accessed on 4 Augus 2018). 17. Lu, D.-Y. (Ed.) D ug combina ions. In Pe sonalized Cance Chemo he apy; Woodhead Publishing: Ox o d, UK, 2015; pp. 37–41. 18. Mokh a i, R.B.; Homayouni, T.S.; Baluch, N. Combina ion he apy in comba ing cance . Onco a ge 2017 , 8, 38022–38043. [C ossRe ] [PubMed] 19. Va go-Gogola, T.; Rosen, J.M. Modelling b eas cance : One size does no i all. Na . Re . Cance 2007 , 7, 659–672. [C ossRe ] [PubMed] 20. Tinoco, G.; Wa sch, S.; Gluck, S.; A ancha, K.; Mon e o, A.J. T ea ing b eas cance in he 21s cen u y: Eme ging biological he apies. J. Cance 2013,4, 117–132. [C ossRe ] [PubMed] 21. Ame ican Cance Socie y. T ea men o B eas Cance by S age. A ailable online: h ps://www.cance .o g/c ance /b eas -cance / ea men / ea men -o -b eas -cance -by-s age.h ml (accessed on 20 Augus 2018). 22. Mille , E.; Lee, H.J.; Lulla, A.; He nandez, L.; Goka e, P.; Lim, B. Cu en ea men o ea ly b eas cance : Adju an and neoadju an he apy. F1000Resea ch 2014,3, 198. [C ossRe ] [PubMed] 23. Medscape. B eas Cance T ea men & Managemen . 2018. A ailable online: h ps://emedicine.medscape.c om/a icle/1947145- ea men #showall (accessed on 6 Augus 2018). 24. An icance Fund. Repu posing Non-Cance D ugs o B eas Cance ? An Eme ging Oppo uni y. A ailable online: h ps://www.an icance und.o g/en/ epu posing-non-cance -d ugs-b eas -cance -eme ging-op po uni y (accessed on 7 Augus 2018). 25. Pan zia ka, P.; B yan, A.B.; C ispino, S.; Dicke son, E.B. P op anolol and b eas cance —A wo k in p og ess. Ecance medicalscience 2018,12, ed82. [C ossRe ] [PubMed] 26. Becke , S. A his o ic and scien i ic e iew o b eas cance : The nex global heal hca e challenge. In . J. Gynecol. Obs e . 2015,131, S36–S39. [C ossRe ] 27. Longley, D.B.; Ha kin, D.P.; Johns on, P.G. 5- luo ou acil: Mechanisms o ac ion and clinical s a egies. Na . Re . Cance 2003,3, 330–338. [C ossRe ] 28. Hu, Q.; Hou, Y.C.; Huang, J.; Fang, J.Y.; Xiong, H. I aconazole induces apop osis and cell cycle a es ia inhibi ing Hedgehog signaling in gas ic cance cells. J. Exp. Clin. Cance Res. 2017,36, 50. [C ossRe ] 29. Pounds, R.; Leona d, S.; Dawson, C.; Kehoe, S. Repu posing i aconazole o he ea men o cance . Oncol. Le . 2017,14, 2587–2597. [C ossRe ] 30. Zhao, L.U.; Zhao, Y.; Schwa z, B. Ve apamil inhibi s umo p og ession o chemo he apy- esis an panc ea ic cance side popula ion cells. In . J. Oncol. 2016,49, 99–110. [C ossRe ] [PubMed] 31. Simpson, W.G. The calcium channel blocke e apamil and cance chemo he apy. Cell Calcium 1985 , 6, 449–467. [C ossRe ] 32. McTa ish, D.; So kin, E.M. Ve apamil: An upda ed e iew o i s pha macodynamic and pha macokine ic p ope ies, and he apeu ic use in hype ension. D ugs 1989,38, 19–76. [C ossRe ] [PubMed] Biomolecules 2018,8, 175 22 o 23 33. Kale, V.P.; Amin, S.G.; Pandey, M.K. Ta ge ing ion channels o cance he apy by epu posing he app o ed d ugs. Biochim. E Biophys. Ac a (BBA) Biomemb . 2015,1848 P B, 2747–2755. [C ossRe ] [PubMed] 34. Lin, J.H.; Yamazaki, M. Role o P-glycop o ein in pha macokine ics: Clinical implica ions. Clin. Pha macokine . 2003,42, 59–98. [C ossRe ] [PubMed] 35. Taylo , J.M.; Simpson, R.U. Inhibi ion o cance cell g ow h by calcium channel an agonis s in he a hymic mouse. Cance Res. 1992,52, 2413–2418. [PubMed] 36. Gup a, V.; Kama h, N.; Tkalce ic, G.T.; Singh, S.V. Po en ia ion o amoxi en ac i i y by e apamil in a human b eas cance cell line. Biochem. Pha macol. 1994,47, 1701–1704. [C ossRe ] 37. Li, C.I.; Daling, J.R.; Tang, M.T.; Haugen, K.L.; Po e , P.L.; Malone, K.E. Use o an ihype ensi e medica ions and b eas cance isk among women aged 55 o 74 yea s. JAMA In e n. Med. 2013 ,173, 1629–1637. [C ossRe ] 38. UniP o . UniP o KB-Q16850 (CP51A_HUMAN). A ailable online: h ps://www.unip o .o g/unip o /Q168 50 (accessed on 22 Augus 2018). 39. Shim, J.S.; Liu, J.O. Recen Ad ances in D ug Reposi ioning o he Disco e y o New An icance D ugs. In . J. Boil. Sci. 2014,10, 654–663. [C ossRe ] 40. Wang, X.; Wei, S.; Zhao, Y.; Shi, C.; Liu, P.; Zhang, C.; Lei, Y.; Zhang, B.; Bai, B.; Huang, Y.; e al. An i-p oli e a ion o b eas cance cells wi h i aconazole: Hedgehog pa hway inhibi ion induces apop osis and au ophagic cell dea h. Cance Le . 2017,385, 128–136. [C ossRe ] 41. Sundquis , T.; Mo a ec, R.; Niles, A.B.; O’B ien, M.; Riss, T. Timing you apop osis assays. Cell No es 2006 , 16, 18–21. 42. Sa as e, A. Mo phologic c i e ia and de ec ion o apop osis. He z 1999,24, 189–195. [C ossRe ] [PubMed] 43. La ue, L.; Bellacosa, A. Epi helial–mesenchymal ansi ion in de elopmen and cance : Role o phospha idylinosi ol 30kinase/AKT pa hways. Oncogene 2005,24, 7443–7454. [C ossRe ] [PubMed] 44. Liu, Y.; Du, F.; Zhao, Q.; Jin, J.; Ma, X.; Li, H. Acquisi ion o 5- luo ou acil esis ance induces epi helial-mesenchymal ansi ions h ough he Hedgehog signaling pa hway in HCT-8 colon cance cells. Oncol. Le . 2015,9, 2675–2679. [C ossRe ] [PubMed] 45. Kim, A.Y.; Kwak, J.H.; Je, N.K.; Jung, Y.S. Epi helial-mesenchymal T ansi ion is Associa ed wi h Acqui ed Resis ance o 5-Fluo ocu acil in HT-29 Colon Cance Cells. Toxicol. Res. 2015 ,31, 151–156. [C ossRe ] [PubMed] 46. Zhang, W.; Feng, M.; Zheng, G.; Chen, Y.; Wang, X.; Pen, B.; Yin, J.; Yu, Y.; He, Z. Chemo esis ance o 5- luo ou acil induces epi helial-mesenchymal ansi ion ia up- egula ion o Snail in MCF7 human b eas cance cells. Biochem. Biophys. Res. Commun. 2012,417, 679–685. [C ossRe ] [PubMed] 47. Van Roy, F.; Be x, G. The cell-cell adhesion molecule E-cadhe in. Cell. Mol. Li e Sci. 2008 ,65, 3756–3788. [C ossRe ] 48. E win; E iwa i; Gunan i; Handha yani, E.; No iana, D. Changes in his opa hology and cy oke a in AE1/AE3 exp ession in skin g a wi h di e en ime on Indonesian local ca s. Ve . Wo ld 2017 ,10, 662–666. [C ossRe ] 49. P oPa h. Cy oke a in AE1/AE3. A ailable online: h p://p opa h.o g/companies/p ess-clippings/26-new sle e s/271-cy oke a in-ae1-ae3-no embe -2003 (accessed on 26 Augus 2018). 50. Sa elli, A.; Li, S. Vimen in in cance and i s po en ial as a molecula a ge o cance he apy. Cell. Mol. Li e Sci. 2011,68, 3033–3046. [C ossRe ] 51. Mo eno-Bueno, G.; Peinado, H.; Molina, P.; Olmeda, D.; Cubillo, E.; San os, V.; Palacios, J.; Po illo, F.; Cano, A. The mo phological and molecula ea u es o he epi helial- o-mesenchymal ansi ion. Na . P o oc. 2009,4, 1591–1613. [C ossRe ] 52. Kim, J.; Kong, J.; Chang, H.; Kim, H.; Kim, A. EGF induces epi helial-mesenchymal ansi ion h ough phospho-Smad2/3-Snail signaling pa hway in b eas cance cells. Onco a ge 2016 ,7, 85021–85032. [C ossRe ] [PubMed] 53. Chen, Y.; Zhao, Z.; Chen, Y.; Chen, G. An epi helial- o-mesenchymal ansi ion-inducing po en ial o g anulocy e mac ophage colony-s imula ing ac o in colon cance . Sci. Rep. 2017 ,7, 8265. [C ossRe ] [PubMed] 54. G aphPad So wa e. 50% o Wha ? How Exac ly a e IC50 and EC50 De ined? A ailable online: h ps: //www.g aphpad.com/suppo / aq/50-o -wha -how-exac ly-a e-ic50-and-ec50-de ined/ (accessed on 23 Augus 2018). Biomolecules 2018,8, 175 23 o 23 55. G aphPad So wa e. P os and Cons o No malizing he Da a. A ailable online: h ps://www.g aphp ad.com/guides/p ism/7/cu e- i ing/index.h m? eg_p os_and_cons_o _no malizing.h m (accessed on 23 Augus 2018). 56. He nandez-Va gas, H.; Balles a , E.; Ca mona-Saez, P.; on Kobbe, C.; Bañón-Rod íguez, I.; Es elle , M.; Mo eno-Bueno, G.; Palacios, J. T ansc ip ional p o iling o MCF7 b eas cance cells in esponse o 5-Fluo ou acil: Rela ionship wi h cell cycle changes and apop osis, and iden i ica ion o no el a ge s o p53. In . J. Cance 2006,119, 1164–1175. [C ossRe ] [PubMed] 57. Tolche , A.W.; Maye , L.D. Imp o ing combina ion cance he apy: The CombiPlex de elopmen pla o m. Fu u e Oncol. 2018,14, 1317–1332. [C ossRe ] [PubMed] 58. Vogus, D.R.; Pusulu i, A.; Chen, R.; Mi ago i, S. Schedule dependen syne gy o gemci abine and doxo ubicin: Imp o emen o in i o e icacy and lack o in i o - in i o co ela ion. Bioeng. T ansl. Med. 2018,3, 49–57. [C ossRe ] [PubMed] 59. Vogus, D.R.; E anus, M.A.; Pusulu i, A.; Ba ajas, V.; Zhang, M.; K ishnan, V.; Nowak, M.; Menega i, S.; Helgeson, M.E.; Squi es, T.M.; e al. A hyalu onic acid conjuga e enginee ed o syne gis ically and sequen ially deli e gemci abine and doxo ubicin o ea iple nega i e b eas cance . J. Con ol. Release 2017,267, 191–202. [C ossRe ] [PubMed] 60. Zhao, Z.; Lou, S.; Hu, Y.; Zhu, J.; Zhang, C. A nano-in-nano polyme -dend ime nanopa icle-based nanosys em o con olled mul id ug deli e y. Mol. Pha m. 2017,14, 2697–2710. [C ossRe ] [PubMed] 61. Lou, S.; Zhao, Z.; Dezo , M.; Lohnies, T.; Zhang, C. Mul i unc ional nanosys em o a ge ed and con olled deli e y o mul iple chemo he apeu ic agen s o he ea men o d ug- esis an b eas cance . ACS Omega 2018,3, 9210–9219. [C ossRe ] © 2018 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h p://c ea i ecommons.o g/licenses/by/4.0/).