Cance s 2014, 6, 67-78; doi:10.3390/cance s6010067
cance s
ISSN 2072-6694
www.mdpi.com/jou nal/cance s
A icle
26,26,26,27,27,27-Hexadeu e a ed-1,25-Dihyd oxy i amin D3
(1,25D-d6) As Adju an o Chemo he apy in B eas Cance
Cell Lines
Samuel Seoane 1,†, Ma ia A. Be mudez 1,†, Juan Sendon-Lago 1, Anxo Ma inez-O doñez 1,
So aya Abdul-Hadi 2, Miguel Maes o 3, An onio Mou iño 3 and Roman Pe ez-Fe nandez 1,*
1 Depa men o Physiology-CIMUS, Endoc ine Oncology Labo a o ies (P1L3), A da. Ba celona s/n,
Campus Vida-Uni e si y o San iago de Compos ela, San iago de Compos ela 15782, Spain;
E-Mails: [email p o ec ed] (S.S.); [email p o ec ed] (M.A.B.);
[email p o ec ed] (J.S.-L.); an[email p o ec ed] (A.M.-O.)
2 Uni e si y o Pue o Rico, Recin o de Rio Pied as, A da. Ba bosa-Ponce de Leon, San Juan 23301,
Pue o Rico; E-Mail: [email p o ec ed]
3 Depa men o O ganic Chemis y, School o Chemis y, Resea ch Labo a o y Ignacio Ribas, A da.
das Ciencias s/n, Uni e si y o San iago de Compos ela, San iago de Compos ela 15782, Spain;
E-Mails: [email p o ec ed] (M.M.); [email p o ec ed] (A.M.)
† These au ho s con ibu ed equally o his wo k.
* Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed];
Tel.: +34-8818-15421; Fax: +34-8818-15403.
Recei ed: 28 Oc obe 2013; in e ised o m: 2 Decembe 2013 / Accep ed: 16 Decembe 2013 /
Published: 27 Decembe 2013
Abs ac : I has been demons a ed ha 1,25-dihyd oxy i amin D3 (1,25D) and
some o i s analogues ha e an i umo ac i i y. 1,25D labeled wi h deu e ium
(26,26,26,27,27,27-hexadeu e a ed 1,25-dihyd oxy i amin D3, o 1,25D-d6) is commonly
used as in e nal s anda d o 1,25D liquid ch oma og aphy-mass spec ome y (LC-MS)
quan i ica ion. In he p esen s udy using human b eas cance cell lines, he biological
ac i i y o 1,25D-d6 adminis e ed alone and in combina ion wi h wo commonly used
an ineoplas ic agen s, 5- luo ou acil and e oposide, was e alua ed. Using an MTT assay,
low cy ome y, and wes e n blo s, ou da a demons a ed ha 1,25D-d6 has e ec s simila
o he na u al ho mone on cell p oli e a ion, cell cycle, and apop osis. Fu he mo e, he
combina ion o 1,25D-d6 and e oposide enhances he an i umo al e ec s o bo h
compounds. In e es ingly, he an i umo al e ec is highe in he mo e agg essi e
OPEN ACCESS
Cance s 2014, 6
68
MDA-MB-231 b eas cance cell line. Ou da a indica e ha 1,25D-d6 adminis e ed alone
o in combina ion wi h chemo he apy could be a good expe imen al me hod o accu a ely
quan i ying ac i e 1,25D le els in cul u es o in biological luids, on bo h in i o b eas
cance cell lines and in i o animal expe imen al models.
Keywo ds: 1,25-dihyd oxy i amin D3; 26,26,26,27,27,27-hexadeu e a ed 1,25-
dihyd oxy i amin D3; i amin D; deu e a ed i amin D; chemo he aphy; b eas cance
1. In oduc ion
In addi ion o i s well known ole in calcium homeos asis, nume ous s udies ha e demons a ed ha
1,25-dihyd oxy i amin D3 (1,25D) and i s analogues a ec s o he physiological o pa hological
p ocesses, such as egula ion o he p oli e a ion and di e en ia ion o a ious cell ypes. This has led
o he s udy o i s p ope ies in se e al p ocesses such as cance [1–4]. In b eas cance 1,25D and
some o i s analogues inhibi cycle p og ession in MCF-7 human b eas adenoca cinoma cells by
a es ing hem in he G0/G1 phase [5,6]. Fu he mo e, his ho mone also induces apop osis, leading o
mo phological and biochemical e ec s such as cell sh inkage, ch oma in condensa ion and DNA
agmen a ion. In u n, his inhibi s umo cell g ow h and con ibu es o umo supp ession [1,7,8].
Mos o he abo e men ioned 1,25D-induced changes in gene exp ession and o he pa ame e s ha e
been cha ac e ized by Muñoz e al. in se e al mamma y umo cell lines [9]. They showed ha in
human b eas cance cells wi h simila le els o i amin D ecep o (VDR) exp ession 1,25D ea men
induces p o ound changes in pheno ype (i.e., mo phology, cy oa chi ec u e, size), p oli e a ion,
sensi i i y o apop o ic s imuli, adhesi eness, mig a ion, in asion, and he exp ession o ma ke genes
associa ed wi h he inhibi ion o myoepi helial cha ac e is ics and wi h dec eased malignancy. All o
his may con ibu e o he i amin’s p o ec i e ac ion agains neoplasia.
1,25D and i s analogues can also enhance, ei he syne gis ically o addi i ely, he an i umo
p ope ies o se e al an ineoplas ic agen s [10]. These p ope ies ha e been demons a ed speci ically
in b eas cance cells by combining 1,25D wi h DNA-damaging agen s (i.e., cispla in and
doxo ubicin) [11,12], mic o ubule-dis up ing agen s (i.e., pacli axel) [13], opoisome ase inhibi o s
(i.e., e oposide) [14] and wi h an ime aboli es (i.e., 5- luo ou acil) [14].
Howe e , he dosage o 1,25D and analogues is a c i ical ac o . Conce n o e possible side-e ec s
such as hype calcemia [10,15,16] may ha e led ce ain clinical s udies o use low dosages ha
p oduced disappoin ing esul s [17–22]. The e o e, he abili y o de e mina e i s le els in pa ien s may
play an impo an ole in ea men ou come. Deu e a ed o ms o i amin D a e commonly
used as in e nal s anda ds o plasma quan i ica ions by liquid ch oma og aphy-mass spec ome y
(LC-MS) [23,24]. Thus, adminis a ion o 1,25D-d6 o a mix u e o 1,25D-d6 and non-labeled 1,25D
could allow accu a ely quan i ying i s concen a ion and bio-a ailabili y in an expe imen al
model. One o hese deu e a ed analogs o i amin D is he 26,26,26,27,27,27-hexadeu e a ed
1,25-dihyd oxy i amin D3 (1,25D-d6) (Figu e 1A,B).
To compa e he e ec s o 1,25D and i s deu e a ed o m (1,25D-d6) on human b eas cance cells,
hese compounds we e adminis e ed alone and in combina ion wi h wo an ineoplas ic agen s,
Cance s 2014, 6
69
5- luo ou acil and e oposide, and hen ci o oxici y, cell cycle, apop osis, and h ee-dimensional cell
g ow h was e alua ed.
Figu e 1. S uc u es o (A) 1,25-dihyd oxy i amin D3 (1,25D) and (B) i s
26,26,26,27,27,27-hexadeu e a ed analogue (1,25-dihyd oxy i amin D3-d6; 1,25D-d6).
2. Resul s and Discussion
To e alua e cell iabili y a e 1,25D, 1,25D-d6, 5- luo ou acil, and e oposide adminis a ion, he
human b eas cance lines we e ea ed as desc ibed in he Expe imen al sec ion, ollowed by an MTT
assay a 48 h. We ound ha 1,25D-d6 p oduced e ec s simila o he na u al ho mone (1,25D),
inducing signi ican ly dec eased cell iabili y in all cell lines, in ela ion o con ol cells (p = 0.001 in
MCF-7 cells, p = 0.0007 in SKBR-3 cells, and p = 0.002 in MDA-MB-231 cells; Figu e 2A–C).
Combina ion o 1,25D-d6 wi h e oposide enhanced he e ec on cell iabili y o each compound in all
cell lines (1,25D-d6 + e oposide s. 1,25D-d6 o e oposide, MCF-7 cells: p = 0.0002 and p = 0.002,
espec i ely; SKBR-3 cells: p = 0.003 and p = 0.02, espec i ely; and MDA-MB-231 cells: p = 0.000006
and p = 0.003, espec i ely).
Figu e 2. Cell iabili y in (A) MCF-7 cells; (B) SKBR-3 cells, and (C) MDA-MB-231
cells 48 h a e adminis a ion o placebo (con ol), 100 nM 1,25D, 100 Nm 1,25D-d6, 500 nM
e oposide, 200 nM 5- luo ou acil, and he combina ion o hese subs ances a he same
doses. The alues ep esen means ± SD om h ee independen de e mina ions. a = p < 0.001
s. con ol cells; b = p < 0.01 s. con ol cells; c = p < 0.05 s. con ol cells; ns = no signi ican .
0
0.1
0.2
0.3
0.4
0.5
MTT Me aboliza ion
AMCF-7 cells
Con ol
(500 nM) E oposide
(200 nM) Fluo ou acil
(100 nM) 1,25D
(100 nM) 1,25D-d6
1,25D + E oposide
1,25-d6+ E oposide
1,25D + Fluo ou acil
1,25D-d6 + Fluo ou acil
b a b a ans b b
Cance s 2014, 6
70
Figu e 2. Con .
As shown in Figu e 2C, dec eased cell iabili y is g ea e in he mo e agg essi e b eas cance
MDA-MB-231 cell line han in MCF-7 o SKBR-3 cells. Adminis a ion o 5- luo ou acil plus ei he
1,25D o 1,25D-d6 does no signi ican ly educe cell iabili y as compa ed wi h adminis a ion o
each compound alone (Figu e 2A–C). Dec eased cell iabili y in he mo e agg essi e cell line
MDA-MB-231 a e ea men wi h e oposide plus 1,25D o 1,25D-d6 could no be explained by
i amin D ecep o (VDR) le els because hese cells had lowe VDR exp ession, compa ed wi h he
o he less agg essi e MCF-7 and SKBR-3cell lines [25]. Lowe VDR exp ession has also been
demons a ed in in asi e umo s as compa ed wi h no mal issue [26]. VDR le els a e e y impo an
o inducing 1,25D-dependen an i-p oli e a i e e ec s on b eas cance cells, bu o e exp ession o
VDR in MDA-MB-231 cells does no es o es he sensi i i y o he 1-hyd oxy i amin D5 analogue,
as occu s in cells wi h high le els o VDR, sugges ing ha o he ac o s a e in ol ed in
1,25D-media ed inhibi ion o cell p oli e a ion [27].
Gi en ha ou esul s show ha MDA-MB-231 cells a e he mos esponsi e o ea men wi h
1,25D-d6 plus e oposide, we ocused ou nex expe imen s on his cell line and his an ineoplas ic
agen . The lowe cell iabili y in MDA-MB-231 cells a e ea men wi h 1,25D and 1,25D-d6
plus e oposide could be explained by educed cell p oli e a ion o inc eased cell dea h. The e o e, we
nex in es iga ed he e ec o bo h ho mones and e oposide on cell cycle and apop osis. I is well
known ha e oposide is cell-cycle dependen and phase speci ic, inducing G2/M a es and ac i a ion
o p53 [28,29]. Ou esul s co obo a e his da a. Figu e 3A shows ha adminis a ion o e oposide
inc eased he G2/M phase in ela ion o con ol cells (p < 0.001). In addi ion, Wes e n blo indica ed
ha e oposide also inc eased cyclin A, cyclin B, and p53 exp ession (Figu e 3B), sugges ing ha
B
SKBR-3 cells
0
0.1
0.2
0.3
0.4
0.5
MTT Me aboliza ion
Con ol
(500 nM) E oposide
(200 nM) Fluo ou acil
(100 nM) 1,25D
(100 nM) 1,25D-d6
1,25D + E oposide
1,25D-d6+ E oposide
1,25D + Fluo ou acil
1,25D-d6+ Fluo ou acil
b b a a ans b b
C
0
0.1
0.2
0.3
0.4
0.5
0.6
MTT Me aboliza ion
MDAMB-231 cells
Con ol
(500 nM) E oposide
(200 nM) Fluo ou acil
(100 nM) 1,25D
(100 nM) 1,25D-d6
1,25D + E oposide
1,25D-d6+ E oposide
1,25D + Fluo ou acil
1,25D-d6+ Fluo ou acil
b b a a ab c b
Cance s 2014, 6
71
dec eased cell p oli e a ion is due, a leas in pa , o al e a ions in cell cycle. Adminis a ion o
e oposide plus 1,25D o 1,25D-d6 modi ies nei he G2/M phase o he cell cycle o p53, cyclin A, and
cyclin B exp ession as compa ed wi h he adminis a ion o e oposide alone (Figu e 3A,B).
Figu e 3. (A) Flow cy ome y analysis o cell cycle in MDA-MB-231 cells a e 48 h o
ea men wi h placebo (con ol), 100 nM 1,25D, 100 nM 1,25D-d6, 500 nM e oposide, and
1,25D o 100 nM 1,25D-d6 (100 nM) + e oposide (E o, 500 nM). The alues ep esen
means ± SD om h ee independen de e mina ions.*** = p < 0.001; (B) Wes e n blo o
p53, cyclin A, cyclin B, cyclin D and GAPDH (used as loading con ol) om p o ein
ex ac s ob ained om MDA-MB-231 cells ea ed as in (A). Numbe s show he
quan i ica ion o p o ein exp ession a e no maliza ion o GAPDH. A ep esen a i e
expe imen is shown.
Ou nex s ep was o e alua e cell dea h by low cy ome y. As shown in Figu e 4A, adminis a ion
o e oposide alone o he MDA-MB-231 cells induced an inc eased ea ly (PI−/Ann+: 9.5 ± 3.5%) and
la e (PI+/Ann+: 25.8 ± 6.2%) apop osis wi h espec o con ol cells (3.5 ± 2.8%, no signi ican , and
14.0 ± 1.0%, p = 0.03, espec i ely). Adminis a ion o 1,25D plus e oposide (PI−/Ann+: 12.4 ± 2.5%,
PI+/Ann+: 22.1 ± 2.7%) signi ican ly enhances he e ec on apop osis wi h espec o adminis a ion
o 1,25D (PI−/Ann+: 4 ± 2.6%, p = 0.02, PI+/Ann+: 14.1 ± 1.2%, p = 0.009, espec i ely), bu no
wi h espec o adminis a ion o e oposide alone. Adminis a ion o 1,25D-d6 plus e oposide
enhances, bu no signi ican ly, ea ly apop osis wi h espec he adminis a ion o each compound
(1,25D-d6 + e oposide e sus 1,25D-d6 o e oposide, p = 0.081 and p = 0.3, espec i ely). We also used
Wes e n blo o analyse he exp ession le els o se e al p o eins in ol ed in he apop osis pa hway.
Ou da a indica es ha no signi ican changes we e ound in he an i-apop o ic Bcl-2 and he
p o-apop o ic Bid p o eins a e adminis a ion ei he alone o in combina ion o e oposide, 1,25D, and
1,25D-d6, in ela ion o con ol cells (Figu e 4B). Adminis a ion o e oposide alone o combined ei he
wi h 1,25D, o 1,25D-d6 inc eased he p o-apop o ic Bak p o ein. In addi ion, a isible inc ease in Bax,
clea ed PARP, and ac i e caspase-3 p o eins a e e oposide plus 1,25D, o 1,25D-d6 was obse ed, as
compa ed wi h con ol cells and cells ea ed wi h 1,25D, 1,25D-d6, o e oposide alone (Figu e 4B).
Thus, ou da a seems o indica e a inc eased cell dea h when he MDA-MB-231 cells a e ea ed wi h
B
Cyclin A
Cyclin B
Cyclin D
p53
GAPDH
19 20 21 26 29 29
29 32 35 47 53 55
14 13 13 36 33 37
48 45 41 30 30 32
A
0
20
40
60
80
100
120
Cell pe cen age
Go/G1
G2/M
S
*** *** ***

Cance s 2014, 6
72
e oposide plus 1,25D o 1,25D-d6. This cell dea h was clea ly ela ed o an inc eased ea ly apop osis,
as shown by PI−/annexin V+ cells, and induc ion o p o-apop o ic ma ke s, such as ac i e caspase-3,
clea ed PARP, Bax and Bak. Se e al epo s ha e desc ibed he an i- umo e ec s o 1,25D on cance
cells by egula ing key media o s o apop osis [10], sugges ing ha he mos p obable mechanisms
a e h ough he down egula ion o he an i-apop o ic p o ein Bcl-2 [30], and dis up ion o
mi ochond ial unc ion, which is associa ed wi h Bax ansloca ion o mi ochond ia, cy och ome c
elease, and p oduc ion o eac i e oxygen species [31]. In addi ion, 1,25D-induced apop osis ia
caspase-independen mechanisms has also been desc ibed [32]. Ou esul s suppo he abo e
men ioned caspase-dependen mechanism when ei he 1,25D o 1,25D-d6 a e combined wi h e oposide.
Figu e 4. (A) Flow cy ome y analysis using annexin-FICT and P opidium Iodide (PI)
indica ed inc eased apop osis (PI−/Ann+ and PI+/Ann+) o MDA-MB-231 cells 48 h a e
adminis a ion o e oposide, and e oposide plus 1,25D, o 1,25D-d6 as compa ed o con ol
cells. The alues ep esen means ± SD om h ee independen de e mina ions; (B)
Wes e n blo analyses o p o- (Bid, Bax, Bak, clea ed PARP, and ac i e caspase-3) and
an i- (Bcl-2) apop o ic p o eins in MDA-MB-231 cells ea ed as in Figu e 3A. Numbe s
show he quan i ica ion o p o ein exp ession a e no maliza ion o GAPDH. A
ep esen a i e expe imen is shown.
G oups
PI−/Ann−
PI−/Ann+
PI+/Ann+
PI+/Ann−
Con ol
79 ± 1.9%
3.5 ± 2.8%
14.0 ± 1.0%
2.2 ± 0.6%
1,25D
78.4 + 1.9%
4.0 ± 2.6%
14.1 ± 1.2%
1.7 ± 0.4%
1,25D-d6
75.2 ± 2.4%
4.0 ± 2.7%
16.4 ± 1.2%
1.9 ± 0.1%
E oposide
56.8 ± 2.8%
9.5 ± 3.5%
25.8 ± 6.2%
5.6 ± 0.1%
1,25D + E o
56.0 ± 7.6%
12.4 ± 2.5%
22.1 ± 2.7%
6.3 ± 3.1%
1,25D-d6 + E o
56.0 ± 14.1%
16.1 ± 8.6%
21.0 ± 7.5%
4.2 ± 0.3%
B
Bcl-2
Bid
Caspase 3
GAPDH
Bax
Bak
Pa p
68 61 65 66 69 68
46 46 49 44 46 54
23 25 23 25 37 37
47 49 43 59 67 63
6 5 6 7 19 20
36 39 49 58 71 69
A
Cance s 2014, 6
73
We also explo ed h ee-dimensional g ow h o MDA-MB-231 cells. Figu e 5 shows ha ea men
wi h e oposide p oduced a signi ican dec ease (p = 0.0007) in sphe e diame e compa ed wi h con ol
cells, and his e ec inc eased when e oposide was combined wi h 1,25D-d6 (p = 0.002).
Figu e 5. Th ee-dimensional g ow h o MDA-MB-231 cells 48 h a e ea men wi h
placebo (con ol), 100 nM 1,25D-d6, 500 nM e oposide, and 100 nM 1,25D-d6 plus 500 nM
e oposide. The alues ep esen means ± SD om h ee independen de e mina ions.
a = p< 0.05 s. con ol; b = p < 0.001 s. con ol; c = p < 0.01 s. e oposide.
P e ious s udies ha e epo ed ha some deu e a ed i amin D analogs ha e simila o lowe e ec s
han hei non-deu e a ed o ms on clonal g ow h and cell di e en ia ion in se e al leukemia cell lines,
in addi ion o s imula ing in es inal calcium abso p ion and bone calcium mobiliza ion [33]. Howe e ,
o he s udies ha e demons a ed deu e a ed 1,25D o be mo e po en a lowe concen a ions han
1,25D on g ow h pla e ca ilage [34]. A ecen s udy demons a ed ha , when deu e a ed, he gemini
i amin D analogues (de i a i es o 1,25D wi h wo chains emana ing a C20) showed highe
bioac i i y, p obably by s abilizing he ligand binding domain (LBD) o he VDR and by enhanced
coac i a o in e ac ions [35]. Ou da a do no e eal signi ican di e ences in bioac i i y be ween
1,25D and 1,25D-d6 when combined wi h e oposide. I has been demons a ed ha e oposide inhibi s
he abili y o 1,25D o cause accumula ion o 25-hyd oxy i amin D3 24-hyd oxylase mRNA(CYP24),
an enzyme ha ca abolizes his ho mone, hus inc easing he bioa ailabili y o 1,25D [36]. Al hough
we did no e alua e CYP24 mRNA le els, we ound ha e oposide in combina ion wi h 1,25D-d6 had
simila e ec s o 1,25D plus e oposide on cell iabili y, cell p oli e a ion, and apop osis. Thus, i
seems easonable o assume ha simila mechanisms a e in ol ed wi h 1,25D-d6, and o specula e ha
e oposide could also inc ease i s bioa ailabili y and p o iding i wi h simila biological ac i i y, a
leas in he mamma y umo cell lines used in ou expe imen al app oach.
In summa y, ou esul s seems o indica e ha adminis a ion o e oposide (and pe haps o he
an ineoplas ic agen s) plus 1,25D-d6, ins ead o he non-deu e a ed o m, could be a be e me hod o
accu a ely quan i ying i s concen a ion in b eas cance cell lines o in animal expe imen al models
in i o.
Con ol 1,25D-d6
1,25D-d6+E o
E o
100 mm0
2
4
6
8
10
12
14
16 Con ol
1,25D-d6
1,25D-d6 + E o
E oposide
3D cul u e ( ela i e uni s)
a
b
c
Cance s 2014, 6
74
3. Expe imen al Sec ion
3.1. Cell Cul u e and D ugs
The human b eas ca cinoma cell lines MCF-7, SK-BR-3, and MDA-MB-231, ob ained om he
Eu opean Collec ion o Cell Cul u es (Salisbu y, Wil s, UK), we e g own in 90 mm pe i dishes in
DMEM supplemen ed wi h 10% e al bo ine se um (FBS), 100 U/mL penicillin, 100 mg/mL
s ep omycin, and 2 mM L-glu amine in an ai -CO2 (95:5) a mosphe e a 37 °C. Con luen cells we e
washed wice wi h PBS and ha es ed by a b ie incuba ion wi h ypsin-EDTA solu ion (Sigma-Ald ich,
Mad id, Spain) in PBS. 1,25D and 1,25 D-d6 we e p o ided by P o s. An onio Mou iño and Miguel
Maes o (Uni e si y o San iago de Compos ela, San iago de Compos ela, Spain). 5-Fluo ou acil and
e oposide we e ob ained om Fe e Fa ma (Ba celona, Spain) and Te a Gené icos Española, S.L.
(Mad id, Spain). 5-Fluo ou acil and e oposide we e dissol ed in PBS and used a 200 nM and
500 nM, espec i ely.
3.2. T ea men s
Cell lines we e ea ed ei he wi h 1,25D o 1,25D-d6 a 100 nM. The wo an ineoplas ic agen s,
e oposide and 5- luo ou acil we e used a 500 nM and 200 nM, espec i ely. The same doses we e
used when combined wi h 1,25D o 1,25D-d6.
3.3. MTT Me aboliza ion
Cell iabili y expe imen s we e ca ied ou using MTT assay. Cells (2.5 × 104 cells/mL) we e
seeded in a olume o 0.5 mL in o 24-well issue cul u e pla es. MCF-7, SKBR-3, and MDA-MB231
cell lines we e ea ed a e 24 h as desc ibed abo e. The abso bance o he samples was eco ded 48 h
a e ea men a 570 nm in a mul iwell pla e eade (LB 940 Mi h as, Be hold Technologies, Bad
Wildbad, Ge many). Resul s we e plo ed o each cell line as he mean ± SD alues o quad uplica es
om a leas wo independen expe imen s.
3.4. Cell Cycle and Apop osis Assays
Cell cycle and apop osis assays we e ca ied ou by using a Gua a low cy ome e (Millipo e
Co po a ion, Bille ica, MA, USA). B ie ly, 2 × 105 cells/well we e cul u ed in DMEN. Fo y-eigh
hou s la e , cell we e ha es ed, ixed wi h 70% cold e hanol o 30 min, washed wi h PBS, and
incuba ed wi h ibonuclease (100 µg/mL), and p opidium iodide (PI, 50 µg/mL) o 60 min in
da kness, o cell cycle e alua ion. Apop osis analyses we e pe o med using Annexin V (Ann)-FITC.
Cells (2 × 105) we e ha es ed, washed wice wi h PBS, and esuspended in 1 × binding bu e (10 mM
Hepes (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2). 5 µL o FITC-Annexin V was added and
incuba ed o 15 min a oom empe a u e in da kness. Finally, 400 µL o 1 × binding bu e was added
o each ube, and analyzed.
Cance s 2014, 6
75
3.5. Wes e n Blo Assays
Wes e n blo ing o MDA-MB231 cells was pe o med as p e iously desc ibed [37]. B ie ly, 50 µg
o o al p o ein was subjec ed o SDS–PAGE elec opho esis. P o eins we e ans e ed o a
ni ocellulose memb ane, blocked, and immunolabeled o e nigh a 4 °C wi h a p ima y an ibody, and
hen incuba ed wi h he app op ia e seconda y an ibody. The signal was de ec ed wi h he Pie ce ECL
Wes e n blo ing subs a e (The mo Scien i ic, Rock o d, IL, USA) and isualized by pu ing he blo
in con ac wi h s anda d X- ay ilm ollowing he manu ac u e ’s ins uc ions. The ollowing
an ibodies we e used: an i-clea ed PARP, an i-Bcl-2, an i-Cyclin D, an i-p-53, and an i-GAPDH
(San a C uz Bio echnology, CA, USA), an i-ac i e Caspase-3, an i- Cyclin A, and an i-Cyclin B (BD
Biosciences, San Diego, CA, USA), and an i-Bid, an i-Bax and an i-Bak (Cell Signaling Technology,
Dan e s, MA, USA).
3.6. Th ee-Dimensional Cell Cul u e
Fo 3D cell cul u e, 12 mm co e slips we e coa ed wi h 60 µL o ice-cold Ma igel (BD
Biosciences) and incuba ed a 37 °C o 20 min o allow he Ma igel o solidi y. Cells we e ea ed o
5 min wi h 0.25% ypsin-EDTA solu ion (2.5 g/L o ypsin, 0.38 g/L o EDTA; Sigma, Mad id,
Spain). A single cell suspension con aining 5 × 103 cells pe 100 µL o cul u e medium, supplemen ed
wi h 2% ( ol/ ol) o Ma igel, was ca e ully placed on he co e slips on op o he solidi ied Ma igel
and incuba ed a 37 °C o 30 min. Co e slips we e hen placed in six-well pla es wi h 500 µL o
cul u e medium pe well. A e 10 days, cells we e ea ed wi h 1,25D-d6 and e oposide alone o in
combina ion o one week. Pho og aphs o he 3D cul u es we e aken wi h a Nikon Eclipse Ti-S
in e ed mic oscope (Izasa, Ba celona, Spain) equipped wi h a P ogRes C3 came a and he P ogRess
Cap u e P o 2.7 so wa e [38]. Quan i ica ion o he sphe e diame e s was made manually by acing a
s aigh line ac oss he diame e o he sphe e and sco ing i s alue as a bi a y leng h uni s. 50 sphe es
we e sco ed o each condi ion o calcula e he mean.
3.7. S a is ical Analysis
Each expe imen was pe o med a leas h ee imes. Values a e exp essed as mean ± s anda d
de ia ion. Means we e compa ed using one-way ANOVA, wi h he Tukey’s es o pos hoc
compa isons. p alues o less han 0.05 we e conside ed s a is ically signi ican . The MATLAB
R2011a Ve sion7.1 so wa e [39] was used o all calcula ions.
4. Conclusions
Ou da a indica es ha 1,25D-d6 had simila bioac i i y o he na u al ho mone 1,25D when
combined wi h he an ineoplas ic d ug, e oposide. Combined wi h 1,25D-d6, e oposide enhanced he
an i umo al ac i i y o each d ug on cell iabili y in he b eas cance cell lines MCF-7, SKBR-3 and
MDA-MB-231. In e es ingly, he an i umo e ec was highe in he mo e agg essi e cell line
MDA-MB-231. Ou esul s suppo ha 1,25D-d6 adminis e ed alone o in combina ion wi h
chemo he apy could be a good expe imen al me hod o accu a ely quan i ying i s concen a ion in
cul u e medium o biological luids.