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).
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