26,26,26,27,27,27-Hexadeuterated-1,25-Dihydroxyvitamin D3 (1,25D-d6) As Adjuvant of Chemotherapy in Breast Cancer Cell Lines

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.