Sa a Daniela de Sousa Reis
CHARACTERIZATION OF THE EFFECTS OF PROTON PUMP
INHIBITORS ON BONE METABOLISM: IN VITRO STUDY IN CO-
CULTURES OF OSTEOCLASTS AND BREAST CANCER CELLS
AND IN CULTURES OF OSTEOBLASTS
Disse ação de Candida u a ao g au de Mes e em Oncologia Molecula
subme ida ao Ins i u o de Ciências Biomédicas de Abel Salaza da Uni e sidade
do Po o, ealizada sob o ien ação do P o esso Dou o João Miguel Sil a e Cos a
Rod igues, p o esso auxilia con idado, e co-o ien ação da P o esso a Dou o a
Ma ia Helena Raposo Fe nandes, p o esso a ca ed á ica da Faculdade de
Medicina Den á ia da Uni e sidade do Po o
Po o, 2013
2
Ag adecimen os
Ag adeço aos meus o ien ado es, P o esso Dou o João Miguel da Cos a Rod igues e
P o esso a Dou o a Ma ia Helena Raposo Fe nandes po odos os ensinamen os, oda a
ajuda e disponibilidade pe manen e ao longo da elabo ação des e abalho.
Ao P o esso Dou o Manuel Teixei a (Ins i u o Po uguês de Oncologia), pela cedência
das linhas celula es de canc o da mama.
À Dou o a Paula Sampaio (Ins i u o de Biologia Molecula e Celula ), pela
disponibilidade na aquisição de imagens de mic oscopia con ocal.
À Técnica Mónica Ga cia (Faculdade de Medicina Den á ia da Uni e sidade do Po o),
pela disponibilidade e ajuda labo a o ial.
Aos meus colegas e amigos po odo o apoio e companhei ismo.
Aos meus amilia es e namo ado po odo o apoio e paciência
3
Resumo
Os inibido es de bombas de p o ões são uma classe de á macos amplamen e u ilizada
em á ias condições pa ológicas, como e luxo gas oeso ágico, dispepsia e úlce as
pép icas. Apesa de exis i em di e sas moléculas di e en es pe encen es a es a classe,
sabe-se que es as pa ilham á ias p op iedades uncionais e es u u ais, bem como
algumas ca ac e ís icas a macociné icas. O seu mecanismo de ação baseia-se na
inibição de H+/K+ ATPases, esponsá eis pela sec eção de ácido gás ico. Nes e
con ex o, uma ez que no p ocesso de eabso ção os os eoclas os solubilizam a ase
óssea mine al a a és da sec eção de ácido pa a a lacuna de eabso ção po bombas de
p o ões do ipo acuola , V-ATPases, os inibido es de bombas de p o ões su gem como
po enciais modelado es a macológicos da a i idade os eoclás ica, pa icula men e em
condições pa ológicas ca ac e izadas pela hipe ac i ação de os eoclas os, al como
acon ece nas me ás ases os eolí icas associadas ao canc o da mama.
Nes e abalho a alia am-se os e ei os celula es e molecula es de ês inibido es de
bombas de p o ões, nomeadamen e, omep azole, esomep azole e lansop azole, em
cul u as de células do canc o da mama, em co-cul u as de os eoclas os e células do
canc o da mama e, ainda, em cul u as de os eoblas os. As cul u as o am man idas na
p esença de di e en es concen ações de cada inibido du an e 21 dias. As células o am
ca ac e izadas aos dias 7, 14 e 21 a a és da p oli e ação/ iabilidade celula , con eúdo
p o eico, a i idade e colo ação his oquímica da os a ase alcalina e da os a ase ácida
esis en e ao a a a o, p esença de células mul inucleadas com anéis de ac ina e
exp essão de ece o es de i onec ina e calci onina, apop ose e capacidade de
eabso ção. Pos e io men e, as cul u as o am ambém a adas com inibido es das ias
MAPK/ERK, NFkB, PKC e JNK pa a se a aliado o en ol imen o des as ias de
sinalização in acelula es na espos a celula . Obse ou-se que os inibido es de bombas
de p o ões inibem a os eoclas ogénese em co-cul u as de os eoclas os e células do
canc o da mama, de o ma dependen e de dose e á maco es ado, en ol endo
al e ações signi ica i as nas di e sas ias de sinalização in acelula es es adas.
Ve i icou-se, ainda, que es es compos os êm, ambém, a capacidade de inibi o
c escimen o de cul u as de células do canc o da mama e a os eoblas ogénese. Os
inibido es de bombas de p o ões su gem, assim, como po enciais modelado es do
me abolismo ósseo no con ex o da me as ização óssea.
4
Abs ac
P o on pump inhibi o s a e a class o d ugs widely used in many pa hological
condi ions, such as gas oesophageal e lux, dyspepsia and pep ic ulce s. Al hough he e
a e se e al di e en molecules belonging o his class, i is well known ha hey sha e
many unc ional and s uc u al p ope ies as well as some pha macokine ic
cha ac e is ics. The mechanism o ac ion elies in he inhibi ion o H+/K+ ATPases,
esponsible o gas ic acid sec e ion. In his con ex , since in he eso p ion p ocess
os eoclas s solubilize he bone mine al phase h ough he sec e ion o acid o he
eso p ion lacuna by p o on pumps o he acuola ype, V-ATPases, p o on pump
inhibi o s appea as po en ially pha macological modula o s o os eoclas ic ac i i y,
pa icula ly in pa hological si ua ions cha ac e ized by hipe ac i a ion o os eoclas s, such
as os eoly ic me as ases associa ed wi h b eas cance .
In his s udy we e assessed he cellula and molecula e ec s o h ee p o on pump
inhibi o s, namely, omep azole, esomep azole and lansop azole, in cul u es o b eas
cance cells, in co-cul u es o os eoclas s and b eas cance cells and in os eoblas s
cul u es. The cul u es we e main ained in he p esence o di e en concen a ions o each
inhibi o o 21 days. Cells we e cha ac e ized a day 7, 14 and 21 o cellula
p oli e a ion/ iabili y, o al p o ein con en , alkaline phospha ase and a a a e esis an
acid phospha ase ac i i y and his ochemical s aining, p esence o mul inuclea ed cells
wi h ac in ings and exp ession o i onec in and calci onin ecep o s, apop osis and
eso bing abili y. La e , he cul u es we e ea ed wi h inhibi o s o he MAPK/ERK, NFkB,
PKC and JNK pa hways o assess he in ol emen o hese in acellula signaling
pa hways in he cellula esponse. Resul s e ealed ha p o on pump inhibi o s inhibi
os eoclas ogenesis in co-cul u es o os eoclas s and b eas cance cells, depending on
he iden i y and dose o he es ed molecule and in ol ing signi ican changes in he
se e al in acellula signaling pa hways es ed. Mo eo e , i was seen ha hese
compounds also ha e he abili y o inhibi cell g ow h in b eas cance cell cul u es and
os eoblas ogenesis. Thus, p o on pump inhibi o s appea as po en ial modula o s o bone
me abolism in he con ex o bone me as asis.
5
Index
Lis o abb e ia ions……………………………………………………………………………….7
1. In oduc ion………………………………………………………………………………………9
1.1 Bone issue…………………………………………………………………………………..9
1.1.1 S uc u al and unc ional cha ac e is ics…………………………………………...9
1.1.2 Bone cells……………………………………………………………………………10
1.1.3 Os eoclas ogenesis…………………………………………………………………12
1.1.4 Ho monal con ol……………………………………………………………………13
1.1.5 Signaling pa hways…………………………………………………………………14
1.2 B eas cance ………………………………………………………………………………17
1.2.1 Epidemiology and pa hology………………………………………………………17
1.2.2 Risk ac o s…………………………………………………………………………..18
1.2.3 Molecula ypes o b eas cance ………………………………………………….18
1.2.4 The apies…………………………………………………………………………….20
1.3 Bone me as asis in b eas cance ……………………………………………………….24
1.3.1 Bone me as asis…………………………………………………………………….24
1.3.2 Types o bone me as ases…………………………………………………………26
1.3.3 Molecula ypes o b eas cance and me as a ic beha io …………………….28
1.3.4 T ea men ……………………………………………………………………………29
1.4 P o on pump inhibi o s…………………………………………………………………….30
1.4.1 Mechanism o ac ion………………………………………………………………..30
1.4.2 Ad e se e ec s……………………………………………………………………...32
1.4.3 PPIs e sus H2RAs…………………………………………………………………33
1.4.4 Seconda y e ec s o PPI in bone me abolism…………………………………...34
2. Ma e ials and me hods………………………………………………………………………..36
2.1 Cell cul u es………………………………………………………………………………..36
2.1.1 Isola ion o pe iphe al blood mononuclea cells (PBMC)……………………….36
2.1.2 B eas cance cell lines cul u es (T47D and SK-BR-3)…………………………36
2.1.3 Co-cul u es o SK-BR-3 and T47D and PBMC…………………………………..36
2.1.4 Human e al os eoblas ic cell line (hFOB) cul u e……………………………….37
2.2 Cha ac e iza ion o cell cul u es………………………………………………………….38
2.2.1 To al p o ein quan i ica ion…………………………………………………………38
2.2.2 TRAP ac i i y quan i ica ion………………………………………………………..38
2.2.3 His ochemical s aining o TRAP…………………………………………………..38
2.2.4 ALP ac i i y quan i ica ion………………………………………………………….39
2.2.5 His ochemical s aining o ALP……………………………………………………..39

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2.2.6 Cellula p oli e a ion/ iabili y……………………………………………………….39
2.2.7 Immuno luo escence s aining and isualiza ion by CLSM……………………..39
2.2.8 Apop osis quan i ica ion……………………………………………………………40
2.2.9 Calcium phospha e eso bing abili y……………………………………………...40
2.2.10 S a is ical analysis…………………………………………………………………40
3. Resul s………………………………………………………………………………………….41
3.1 Assessmen o he e ec s o di e en PPI in cul u es o SK-BR-3 and T47D………41
3.1.1 Cha ac e iza ion o SK-BR-3 cul u es…………………………………………….41
3.1.2 Cha ac e iza ion o T47D cul u es ………………………………………………..42
3.2 Assessmen o he os eoclas ogenic e ec s o di e en PPI in co-cul u es o PBMC
and b eas cance cell lines……………………………………………………………………..44
3.2.1 Cha ac e iza ion o PBMC + SK-BR-3 co-cul u es………………………………44
3.2.2 Cha ac e iza ion o PBMC + T47D co-cul u es………………………………….48
3.3 Assessmen o he e ec s o di e en PPI in cul u es o hFOB………………………52
3.3.1 Cha ac e iza ion o hFOB cul u es (-DEX)……………………………………….52
3.3.2 Cha ac e iza ion o hFOB cul u es (+DEX)………………………………………55
3.3.3 Cha ac e iza ion o he in acellula e ec s in ol ed in cellula esponse…....57
4. Discussion……………………………………………………………………………………...62
5. Conclusion……………………………………………………………………………………..66
Re e ences………………………………………………………………………………………..67
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Lis o abb e ia ions
α-MEM - α-minimal essen ial medium
ALP – alkaline phospha ase
BMP – bone mo phogene ic p o ein
BP - bisphosphona e
CA2 – ca bonic anhyd ase II
cAMP – cyclic adenosine monophospha e
CaSR – calcium-sensing ecep o
CATK – ca hepsin K
CLSM – con ocal lase scanning mic oscopy
CSF – colony-s imula ing ac o
CTR – calci onin ecep o
DMEM/F12 - Dulbecco’s Modi ied Eagle’s Medium/HAM’s Nu ien Mix u e F12
ECM – ex acellula ma ix
EGF – epide mal g ow h ac o
ER – es ogen ecep o
ERK - ex acellula signal egula ed kinase
FGF - ib oblas s g ow h ac o
GMCSF - g anulocy e mac ophage colony-s imula ing ac o
HER2/e B2 - human epide mal g ow h ac o ecep o 2
hFOB – human e al os eoblas ic cell line
HR - ho monal ecep o
H2RA - his amine 2 ecep o an agonis
IGF - insulin-like g ow h ac o
IL - in e leukin
JNK - C-jun N- e minal kinase
MAPK - mi ogen ac i a ed p o ein kinases
M-CSF – mac ophage colony s imula ing ac o
MITF - mic oph halmia associa ed ansc ip ion ac o
MSC - mesenchymal s em cells
NF-kB - Nuclea ac o kappa B
OPG – os eop o ege in
PBMC – phe iphe al blood mononuclea cells
PDGF - pla ele -de i ed g ow h ac o
PGE2 – p os aglandin E2
PKC – p o ein kinase C
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pNPP - pa a-ni ophenilphospha e
PPI – p o on pump inhibi o
PR – p oges e one ecep o
PTH - pa a hy oid ho mone
PTH P - pa a hy oid ho mone ela ed p o ein
RANK – ecep o ac i a o o nuclea ac o kappa B
RANKL - ecep o ac i a o o nuclea ac o kB ligand
TGF-β - ans o ming g ow h ac o -be as
TNF – umo nec osis ac o
TRAF – umo nec osis ac o ecep o associa ed ac o
TRAP - a a a e esis an acid phospha ase
VEGF - ascula endo helial g ow h ac o
VNR – i onec in ecep o
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1. In oduc ion
1.1 Bone issue
1.1.1 S uc u al and unc ional cha ac e is ics
Bone is a specialized connec i e issue ha is cons an ly emodeling and epai ing
i sel . Wi h a mic os uc u e de eloped o enable and suppo a maximum s eng h
associa ed wi h a minimal mass, bone issue can be di ided in wo componen s: an
o ganic componen , composed essen ially by bone cells and an ex acellula ma ix
cons i u ed by ype I collagen (90% o o al p o ein), p o eoglycans and o he p o eins,
which con e s lexibili y; and an ino ganic, o mine alized, componen , composed mainly
by calcium and phospha e sal s in he o m o hyd oxyapa i e (Ca10(P04)6(OH)2),
ep esen ing abou 60% o bone weigh , and also small amoun s o silicon, sodium and
magnesium, which con e s igidi y [1, 3, 18].
In addi ion o p esen ing a se ies o mechanical unc ions such as, p o iding o m,
p o ec ion and suppo , he bone also ac s as a ese oi , s o ing and eleasing ions in
o de o main ain a cons an concen a ion in body luids, and abso bing and accumula ing
oxins and me als p e en ing i s ad e se e ec s in o he issues [2, 20].
I s mul icellula base comp ises essen ially h ee dis inc ypes o cells: os eoblas s
(cells esponsible o he o ma ion o new bone), os eoclas s (cells specialized in bone
eso p ion), and os eocy es, which a e os eoblas comple ely embedded in bone ma ix
ha a e hough o play a key ole as mechanosenso s (Fig. 1) [2, 20].
Figu e 1. Bone emodeling p ocess. Os eoclas s a e ac i a ed and he eso p ion p ocess
begins. Reso p ion phase akes app oxima ely 10 days. Then, os eoblas s p ecu so s a e ec ui ed,
p oli e a e and di e en ia e. Os eoblas s p oduce bone mine alized ma ix in a much longe
p ocess ha can ake se e al mon hs.
16
and ac in emodeling. S imula ion o he s ess-ac i a ed p o ein kinase, p38, esul s in he
downs eam ac i a ion o he mic oph halmia associa ed ansc ip ion ac o (MITF), which
con ols he exp ession o he genes encoding TRAP and CATK, equi ed o os eoclas ic
unc ion [4, 7].
Figu e 6. Signaling pa hways in ol ed on os eoclas ogenic di e en ia ion. Binding o RANKL
o i s ecep o RANK induces a ious in acellula signaling cascades, such as MAPK (JNK, ERK,
MAPK 14) and NF-κB. Se e al ansc ip ion ac o s ha e been ound c ucial o os eoclas
di e en ia ion downs eam o RANKL/RANK signaling. (Adap ed om Edwa ds, J. R. and Mundy,
G. R.)

17
1.2 B eas cance
1.2.1 Epidemiology and pa hology
B eas cance is by a he mos equen cance among women (small occu ence in
men) wi h an es ima ed 1.38 million new cance cases diagnosed in 2008 (23% o all
cance s), and anks second o e all (10.9% o all cance s). I is now he mos common
cance bo h in de eloped and de eloping egions and i is second only o lung cance as a
cause o cance mo ali y.
I is es ima ed ha in he yea o 2008, he incidence a e o b eas cance in Po ugal
was 60 pe 100,000 and he mo ali y a e o 13.5 pe 100,000 [GLOBOCAN 2008 IARC].
Nine y- i e pe cen o b eas cance s a e ca cinomas. Ca cinomas can be di ided in o:
in si u ca cinoma and in asi e o in il a ing ca cinoma, and hese may a ise ei he in
duc s o lobules. Mos ca cinomas show duc al di e en ia ion (90%). O he malignan
lesions a e associa ed wi h mesenchymal neoplasms, such as cys osa coma phyllodes,
angiosa coma, among o he s [38, 39].
Figu e 7. The emale b eas . The ana omic s uc u es and lesions o he emale b eas .
(h p://medicinembbs.blogspo .p /2010/12/b eas -ana omy.h ml)
Benign lesions include hype plasia, cys ic changes, adenomas, papillomas and
ib oadenomas, he mos common benign umo o he b eas . Pa hologies o he adipose
issue can also occu , called auma ic a nec osis (Fig. 7) [38, 39].
18
1.2.2 Risk ac o s
The e a e se e al isk ac o s conside ed o he de elopmen o b eas cance .
Ob iously, he age and he occu ence o a p io b eas cance o benign lesion a e wo o
hem, bu he e is also a numbe o li es yle, en i onmen al and ho monal ac o s ha a e
p obably in ol ed (unbalanced die , lack o exe cise, ea ly mena che, ho mone
eplacemen he apy, nullipa i y, …) [38, 39,40].
Women wi h a amily his o y o b eas cance should ob ain as much in o ma ion as
possible abou hose ela i es, including age a onse and ype o cance . The isk o
b eas cance de elopmen ela ed o amily his o y inc eases wi h he numbe o a ec ed
ela i es, speci ic lineage and age a diagnosis [38, 39, 40].
Abou 5-10% o b eas cance is hough o be linked o changes in ce ain genes. The
mos common a e hose o he BRCA1 and BRCA2 genes. Women wi h mu a ions in
hese genes ha e a highe isk o de eloping b eas and o a ian cance du ing hei li es
[38, 39, 40].
Despi e ad ances, 20-30% o pa ien s wi h ea ly b eas cance s expe ience elapse
wi h dis an me as a ic disease. Risk o ecu ence is in luenced by s age a ini ial
p esen a ion and he unde lying biology o he umo . Tumo size, nodal in ol emen ,
g ade, lympho ascula in asion, and es ogen ecep o (ER) and human epide mal g ow h
ac o ecep o 2 (HER2/e B2) s a us a e all independen isk ac o s o elapse [38, 39,
59].
1.2.3 Molecula ypes o b eas cance
The he e ogenei y o b eas cance s makes hem bo h a ascina ing and challenging
solid umo o diagnose and ea .
Un il e y ecen ly, pe sonalized cance medicine in b eas cance elied on only wo
p edic i e ma ke s, ER and HER2/e B2. B eas cance is now ecognized no as a single
disease wi h a iable mo phology, bu as a leas ou molecula ly dis inc neoplas ic
diso de s: luminal-A b eas cance (low p oli e a i e ER posi i e), luminal-B b eas cance
(high p oli e a i e ER posi i e), HER2-posi i e b eas cance (ampli ica ion and high
exp ession o he ERBB2 gene and se e al o he genes o he ERBB2 amplicon) and
iple-nega i e o basal-like b eas cance (ER/p oges e one ecep o nega i e and HER-
2-nega i e) (Fig. 8) [41, 42].
19
Al hough he immedia e addi ional clinical alue o his molecula classi ica ion is limi ed
by i s close co ela ion o adi ional me hods o es ing o ER and HER2, he iden i ica ion
o gene ic abe a ions ha unde lie molecula ly dis inc sub ypes o b eas cance has
e ealed new he apeu ic a ge s and has eshaped b eas cance clinical ial design [41,
42].
Figu e 8. Molecula ypes o b eas cance . To dis inguish he molecula ype o cance , i s
HER2 memb ane ecep o mus be assessed and only a e es o nuclea ho mone ecep o . Ki
67 deno es nuclea an igen ki-67, a ma ke o cell p oli e a ion. (Adap ed om So i iou, C. and
Pusz ai, L.)
Es ogen ecep o /p oges e one ecep o posi i e (ER/PR+) umo s ha e a be e
p ognosis and a be e esponse o ea men han ecep o -nega i e umo s. Two hi ds o
women wi h diagnosed b eas cance ha e ER/PR+ umo s. These umo s a e highly
esponsi e o an i-es ogen he apeu ic s a egies. Howe e , despi e he widesp ead use
o ho monal adju an he apy, a qua e o women wi h ER+ disease will elapse. Since
he ea lies s udies o he in insic molecula sub ypes in b eas cance , he de ining
ea u e o luminal- B b eas cance has been i s poo ou come compa ed wi h he luminal-
A sub ype. Se e al s udies ha e sugges ed ha luminal-B b eas cance is ela i ely
insensi i e o endoc ine he apy compa ed wi h luminal-A [43, 49].
HER2 is o e exp essed in 15-20% o b eas cance , and i is associa ed wi h a highly
agg essi e beha io . HER amily is cons i u ed by ou membe s, HER1, HER2, HER3
and HER4. An in acellula y osine kinase domain exis s o HER1, HER2 and HER4 and
phospho yla ion o hese domains by homodime iza ion o he e odime iza ion induces
bo h cell p oli e a ion and su i al signaling [61]. HER2 is he p e e ed dime iza ion
B eas Cance
HER2 Nega i e
ER/PR Nega i e
Basal like
ER/PR posi i e
Good di e en ia ion
low Ki 67
Luminal A
Poo di e en ia ion
high Ki 67
Luminal B
HER2 Posi i e
ER posi i e o
ER nega i e
HER2 like
20
pa ne o he o he HER amily membe s. One o he o he downs eam e ec s o hese
eac ions is he p oduc ion o ascula endo helial g ow h ac o (VEGF) suppo ing
angiogenesis [58].
The a ailabili y o he an i-HER2 monoclonal an ibody he apy has signi ican ly imp o ed
he p ognosis o pa ien s wi h HER2 posi i e b eas cance bo h in ea ly and ad anced
disease s ages.
T iple-nega i e b eas cance s in pa icula a e di icul o de ine. This umo subg oup
does no espond o ho monal he apies o He 2- a ge ed he apies due o he lack o
exp ession o hese a ge s. These umo s a e associa ed wi h a poo p ognosis.
The sub ypes mos in need o he apeu ic ad ances a e iple nega i e b eas cance
and luminal-B b eas cance , whe e he apeu ic esis ance is common and whe e
ad ances in molecula p o iling ha e iden i ied p omising new he apeu ic a ge s [43, 49].
1.2.4 The apies
B eas cance su ge y has changed d ama ically o e he pas 20 yea s. Wi h he
eme gence o b eas conse ing he apy, many women now ha e he op ion o p ese ing
a cosme ically accep able b eas wi hou sac i icing su i al. Conse ing he apy e e s o
su gical emo al o he umo wi hou emo ing excessi e amoun s o no mal b eas
issue. The aims o his he apy a e o p o ide a cance ope a ion equi alen o
mas ec omy and a cosme ically accep able b eas , wi h a low a e o ecu ence in he
ea ed b eas . The main obs acle o widesp ead accep ance and u iliza ion o conse ing
he apy is he isk o in-b eas ecu ence. Depending o se e al ac o s, such as umo
size, mul i ocali y and possibili y o adio he apy used, mas ec omy may be he me hod o
choice. Rega dless o he me hod used, axilla y lymph node dissec ion is always
manda o y o e alua ion [39, 40].
Radia ion adju an he apy is known o subs an ially educe he isk o ecu ence and
dec ease b eas cance mo ali y, bo h when gi en a e mas ec omy and a e b eas -
conse ing su ge y. Radia ion he apy is gene ally gi en o e a 5- o 6-week ime span,
wi h ca e aken o y o a oid damage o he hea o lungs. The only usual changes wi h
b eas adia ion a e skin e y hema and possibly some ansien lymphedema [39].
Adju an sys emic he apy e e s o he adminis a ion o chemo he apy, ho mone
he apy and/o monoclonal an ibodies used ollowing p ima y su ge y o b eas cance .
The pu pose is o elimina e o delay he subsequen appea ance o clinically occul
21
mic ome as ases. The o iginal egimen o chemo he apy used was cyclophosphamide,
me ho exa e, 5- luo ou acil (CMF). The ea e , many o he egimes ha e been used. A
majo de e minan o he choice o adju an he apy is whe he an indi idual b eas cance
exp esses ho monal ecep o s (HR). Ho mone he apy bene i s pa ien s wi h ho mone
ecep o -posi i e b eas cance , bu no hose wi h ho mone ecep o nega i e disease
[39].
Ta ge ing he ER is he oldes molecula a ge ed he apy app oach, and he
widesp ead use o he selec i e ER modula o amoxi en in b eas cance is esponsible
o majo imp o emen s in cu e a es, quali y o li e and disease p e en ion o he pas 25
yea s [39, 40].
Tamoxi en, a selec i e es ogen ecep o modula o (SERM), inhibi s he g ow h o
b eas cance cells by compe i i e an agonism o es ogen a he ER, inhibi ing bo h
ansloca ion and nuclea binding o he ER. Tamoxi en and i s me aboli es bind o he ER,
and subsequen ly occu s ansloca ion o his complex o he nucleus and binding o he
es ogen- esponse elemen . This binding p e en s ansc ip ional ac i a ion o es ogen-
esponsi e genes (Fig. 8). Howe e , i s ac ions a e complex and i has also a pa ial
es ogen agonis ac i i y. Addi ionally, he e a e ma ked di e ences be ween he an i
p oli e a i e p ope ies o amoxi en and i s me aboli es [40, 57].
Thei long- e m e icacy, howe e , is limi ed by elapse o disease and de elopmen o
esis ance. Labo a o y and clinical da a ha e demons a ed ha ER-posi i e b eas
cance s ha o e exp ess HER2 a e associa ed wi h esis ance o amoxi en and o
ho monal he apy in gene al. Despi e con inuous exp ession o ER a elapse in ei he
locally ecu en o seconda y me as a ic umo s, up o hal o pa ien s wi h HR-posi i e
p ima y b eas cance who de elop me as a ic disease do no espond o i s -line
endoc ine ea men (de no o esis ance), and he emainde will e en ually elapse
despi e an ini ial esponse (acqui ed esis ance) [40].
Se e al ac o s may con ibu e o amoxi en esis ance in b eas cance , including
a iable exp ession o es ogen ecep o alpha and be a iso o ms, in e e ence wi h
binding o co-ac i a o s and co- ep esso s, al e na i ely spliced ER mRNA a ian s and
ER modula o s exp ession, such as epide mal g ow h ac o (EGF) and i s ecep o
(EGFR1, also called HER1), as well as he ype 2 EGFR, also called HER2. Eme ging
s udies also sugges ha ela i e esis ance o amoxi en may be ela ed o inhe i ance o

22
ce ain d ug me abolizing CYP2D6 geno ypes ha a e associa ed wi h a educed
ac i a ion o amoxi en o i s ac i e me aboli e endoxi en [40, 44, 60].
O he ho monal he apeu ic agen s include a oma ase inhibi o s ha in e e e wi h he
enzyme a oma ase, which plays a c i ical ole in he p oduc ion o es ogen in
pos menopausal women. A oma ase is an enzyme ha na u ally con e s es ogen om
and ogen. In p emenopausal women, mos o he es ogen is p oduced in he o a ies, bu
in pos menopausal women, mos es ogen is syn hesized in pe iphe al issue om
con e sion o and ogens. In con as o amoxi en, hese compounds lack pa ial agonis
ac i i y (Fig. 9). Examples o his class include anas ozole, le ozole and exemes ane [40,
45].
Figu e 9. Compa ison o he mechanisms o ac ion o es adiol, amoxi en and a oma ase inhibi o s.
Es adiol binds o ER, leading o dime iza ion, con o ma ional change and binding o es ogen esponse
elemen s (ERE) ups eam o es ogen esponsi e genes including hose esponsible o p oli e a ion.
Tamoxi en compe es wi h es adiol o ER binding whe eas A oma ase inhibi o s educe he syn hesis o
es ogen om and ogenic p ecu so s. (Adap ed om Johns on, S. R. D. and Dowse , M.)
App oxima ely 25-30% o b eas cance umou s display ampli ica ion o he HER2
gene o o e exp ession o i s p o ein p oduc , and his is associa ed wi h an ad e se
p ognosis. T as uzumab is a humanized monoclonal an ibody di ec ed agains he
ex e nal domain o he ecep o wi h clinical ac i i y as a single agen in pa ien s whose
cance s o e exp ess HER2 (Fig. 10). O e exp ession o he ecep o is associa ed wi h
inc eased disease ecu ence and wo se p ognosis. Ta ge ing bo h HER2 wi h
23
as uzumab and VEGF wi h be acizumab in combina ion wi h chemo he apy has become
a u he miles one o molecula a ge ed he apy. Howe e , in insic and acqui ed
esis ance o endoc ine and/o cy os a ic ea men s is s ill a common ea u e ha limi s
he bene i s o hese no el he apeu ic s a egies. In umo s ha a e HER2 and ER
posi i e, HER2 signaling is dominan . This esis ance can be pa ially o e come by
combining an i-es ogen and an i-HER2 he apies [40, 57, 61].
Figu e 10. Po encial mechanisms o ac ion o ans uzumab. Clea age o he ex acellula
domain o HER2 lea es a memb ane-bound phospho yla ed p95, which can ac i a e signal-
ansduc ion pa hways. Binding o as uzumab o a jux amemb ane domain o HER2 educes he
elease o he ex acellula domain, he eby educing p95. T as uzumab may educe HER2
signaling by physically inhibi homodime iza ion o he e odime iza ion. T as uzumab may also
ec ui immune e ec o cells and o he componen s o an ibody-dependen cell-media ed
cy o oxici y, leading o umo -cell dea h. O he mechanisms such as ecep o down- egula ion
h ough endocy osis ha e been sugges ed. (Adap ed om Hudis, C. A.)
24
1.3 Bone me as asis in b eas cance
1.3.1 Bone me as asis
Mos pa ien s wi h cance die no because o he p ima y umou , bu a he because i
has sp ead o o he si es. I is di icul o de e mine, p ecisely, how equen ly di e en
umou s me as asize o bone. Pa ien s wi h ad anced b eas and p os a e cance s almos
always de elop bone me as ases. Mo eo e , he chances a e high ha , in pa ien s who
a e o iginally diagnosed wi h b eas o p os a e cance s, he bulk o he umou bu den a
he ime o dea h will be in bone [46]. Me as asis o bone occu s in he la e s ages o
umo p og ession, in a mul is ep p ocess [50, 51].
Because umo me as asis is a complex p ocess in ol ing indi idual disc e e s eps,
in e up ion o one o mo e o hese s eps can inhibi he me as a ic p ocess. Each o
hese s eps ep esen s cellula in e ac ions caused by speci ic de e minan s o bo h he
umo and he issue. The s eps in ol ed in he shedding o he umo cells om he
p ima y si e in ol e de achmen o umo cells om adjacen cells, ollowed by in asion o
adjacen issue in he p ima y o gans. The cells hen en e he umo capilla ies
(s imula ed by speci ic angiogenesis ac o s p oduced by he umo ) and ia hese
capilla ies each he gene al ci cula ion. The s eps in ol ed in en e ing he umo blood
essels a he p ima y si e a e simila o hose in ol ed in exi ing he ascula u e in he
bone ma ow ca i y. These s eps include he a achmen o he umo cells o basemen
memb ane, he sec e ion o p o eoly ic enzymes ha enable umo cells o dis up ha
memb ane, and he mig a ion o he umo cells h ough i . Tumo cells ha me as asize o
he skele on adhe e o he endos eal su ace and colonize bone (Fig. 11) [50, 51].
Figu e 11. The s eps in ol ed in umou -cell me as asis om a p ima y si e o he skele on.
The p ima y malignan neoplasm p omo es new blood- essel o ma ion, and hese blood essels
25
ca y he cance cells o capilla y beds in bone. Agg ega es o umou cells and o he blood cells
e en ually o m embolisms ha a es in dis an capilla ies in bone. These cance cells can hen
adhe e o he ascula endo helial cells o escape he blood essels. As hey en e he bone, hey
a e exposed o ac o s o he mic oen i onmen ha suppo g ow h o me as ases. (Adap ed om
Mundy, G. R.)
Al hough he dis ibu ion o me as ases in dis an o gans can be p edic ed by he
ana omic dis ibu ion o blood low om he p ima y si e in 30% o cases, speci ic
p ope ies o he umo cell and ea u es a he me as a ic si e de e mine whe e he
me as asis occu s in he majo i y o cases [51].
The me as asis o umo cells o speci ic si es in he skele on is no a simple and
andom e en de e mined solely by blood low. Ra he , i is a complex p ocess ha is
dependen on speci ic p ope ies o he umo cells and on ac o s in he bone
mic oen i onmen ha a o me as asis. Tumo cells mos equen ly a ec he hea ily
ascula ized a eas o he skele on, pa icula ly he ed bone ma ow o he axial skele on
and he p oximal ends o he long bones, he ibs, and he e eb al column [51].
Physical ac o s wi hin he bone mic oen i onmen , including low oxygen le els, acidic
pH, and high ex acellula calcium concen a ions, may also enhance umo g ow h.
Hypoxia is a majo con ibu o o umo me as asis, egula ing sec e ed p oduc s ha
d i e umo -cell p oli e a ion and sp ead. Hypoxia also con ibu es o esis ance o
adia ion and chemo he apy in p ima y umo s. Solid umo s a e pa icula ly p one o
hypoxia because hey p oli e a e apidly, ou g owing he mal o med umo ascula u e,
which is unable o mee he inc easing me abolic demands o he expanding umo .
Hypoxia egula es no mal ma ow hema opoiesis and chond ocy e di e en ia ion. Cance
cells capable o su i ing a low oxygen le els can h i e in he hypoxic bone
mic oen i onmen and pa icipa e in he icious cycle o bone me as asis [50].
Acidosis o he bone mic oen i onmen also po en ia es he icious cycle o bone
me as asis. Ex acellula pH is igh ly egula ed wi hin bone and has signi ican e ec s on
os eoblas and os eoclas unc ion wi h os eoclas s being maximally s imula ed a pH
le els o <6.9. Os eoblas mine aliza ion and bone o ma ion is signi ican ly a ec ed by
acid. Tumo me as asis leads o localized egions o acidosis wi hin he skele on.
Inc eased glycolysis and lac ic acid p oduc ion by p oli e a ing cance cells (due o he
hypoxic condi ions) and dec eased bu e ing capaci y o he in e s i ial luid con ibu e o
32
PPIs a e lipophilic and a e inac i e in he neu al en i onmen o he bloods eam. A e
abso p ion, PPIs c oss he plasma memb ane, en e and accumula e in he sec e o y
canaliculi o pa ie al cells, whe e hey a e p o ona ed by acid and hen con e ed in o hei
ac i e o m, sul enamide. The ac i a ed sul enamide eac s co alen ly wi h he cys eine
esidues on he ex acellula su ace o he p o on pump, inhibi ing gas ic acid sec e ion
(Fig. 15) [67].
Figu e 15. PPIs ac i a ion. PPIs such as omep azole concen a e in he acidic sec e o y
canaliculi o he pa ie al cell, whe e hey a e ac i a ed and gene a e a sulphenamide. The
sulphenamide in e ac s co alen ly wi h he sulphyd yl goups o cys eine esidues in he
ex acellula domain o he ATPase he eby inhibi ing i s ac i i y. (Adap ed om Olbe, L. e al)
PPIs a e e y speci ic o he inhibi ion o gas ic p o on pumps because hey a e
ac i a ed only in he acidic en i onmen o he s omach, whe eas hey a e no ac i a ed by
he simila enzyme ound in he colon and he kidney [67]. Di e ences in cys eine binding
p ope ies among he PPIs may, a leas pa ly, unde lie he di e ences among hem in
he du a ion o he inhibi ion o gas ic acid sec e ion. Cu en ly a ailable benzimidazole-
based PPIs ha e simila hal -li es o 1–2 h. As hese agen s a e usually p esc ibed once
daily, his means ha he e is e ec i ely no ci cula ing PPI p esen a he end o he
dosing in e al. A PPI wi h an ex ended hal -li e migh ha e a p olonged an i-sec e o y
e ec wi h consequen he apeu ic ad an ages [65].
1.4.2 Ad e se e ec s
As men ioned abo e, he e a e common ad e se e en s ha occu in 1-4% o pa ien s.
The mos common a e headache, abdominal pain, gas oin es inal al e a ions (nausea
and dia hea, o example), a igue, communi y-acqui ed pneumonia, acu e in e s i ial
neph i is and alle gy- ela ed p oblems. Howe e , he knowledge o he ad e se e en s o
newly ma ke ed d ugs is limi ed, and i is only a e widesp ead clinical use ha he side-
e ec p o ile o a d ug is pe o med mo e asse i ely. The p esence o side e ec s on

33
p o on pump inhibi o s is g ea es o omep azole, since his d ug has been on he ma ke
o a longe pe iod [68, 70, 73].
PPIs educe gas ic acidi y, which is necessa y o ac i a e pepsinogen in o pepsin,
which, by i s u n is impo an o he beginning o he diges ion o die a y p o eins and o
elease i amin B12 om ood. PPIs used o a sho - e m may minimally educe he
abso p ion o p o ein-bound i amin B12. Elde ly pa ien s who al eady ha e gas ic
a ophy may p esen lowe le els o i amin B12 in se um wi h long e m PPIs used.
Howe e , s udies ha e shown ha PPIs used o a long- e m in younge pa ien s do no
educe se um i amin B12 concen a ions. In pa ien s long- e m ea ed wi h high dose o
PPIs duodenal abso p ion o o ganic and non-o ganic i on may be educed. Ne e heless,
his e ec is small, and PPIs a e no associa ed wi h an inc eased isk o la en i on
de iciency o i on de iciency [68, 70, 73].
Fo he p o on pump inhibi o s omep azole, lansop azole, pan op azole and
esomep azole, ce ain hepa ic isoenzyme pa hways, no ably cy och ome P450
(CYP2C19), ha e an impo an ole in he d ug me abolism. In pa icula , among pa ien s
who a e geno ypically apid, o ex ensi e, me abolize s, p o on pump inhibi o s wi h
signi ican CYP2C19 me abolism end o yield lowe plasma le els and, hus, ha e low
e icacy. The con ibu ion o he CYP2C19 pa hway a ies in he me abolism o he
di e en p o on pump inhibi o s [69].
1.4.3 PPI’s e sus H2RAs
Despi e he e iciency o he ac ion o he H2RAs, he e we e pa ien s whose acid-
ela ed diso de s ailed o espond o o equi ed e y high doses o hese d ugs.
Fu he mo e, he phenomenon o H2RA ole ance was ecognized, wi h implica ions on
possible ailu es o ch onic/main enance H2RA he apy. PPIs we e subsequen ly
de eloped, and, in mos cases, ha e been ound o be supe io o H2RAs in hei acid
supp essing abili y. They also elie e oesophagi is symp oms and heal e osions mo e
e ec i ely han H2RAs. In con as o H2RAs o an icholine gic agen s, which only
pa ially inhibi his amine-, gas in- o ace ylcholine- s imula ed acid sec e ion, PPIs inhibi
acid sec e ion in esponse o all s imula o y agen s (Fig. 16). Consequen ly, PPIs a e
excep ionally e ec i e o he con ol o acid p oduc ion. Mo eo e , PPIs ele a e pH abo e
3-4 4 o longe ime pe iods han do H2RAs[69].
34
Figu e 16. Gas ic acid sec e ion by pa ie al cell. Gas ic acid sec e ion is a p ocess egula ed
by h ee ypes o ecep o s on he pa ie al cell (his amine, gas in and ace ylcholine). Ac i a ion o
hese ecep o s leads o ac i a ion o he gas ic acid p o on pump, H+K+-ATPase, which egula es
acid anspo and is he inal common pa hway o acid sec e ion o hese h ee ecep o s. In
con as o H2RAs which only pa ially block hese ecep o s, PPIs di ec ly block he ac ion o
H+K+-ATPase, supp essing gas ic acid sec e ion, ega dless o he s imulus. (Adap ed om Olbe,
L. e al.)
1.4.4 Seconda y e ec s o PPI in bone me abolism
Long- e m PPI he apy and i s a endan po en acid supp essi e p ope ies ha e
gene a ed g ea conce n ega ding o he po en ial e ec s on calcium abso p ion and bone
me abolism. Se e al epidemiological s udies ha e sugges ed an associa ion be ween PPI
use and he isk o ac u es o he hip, o o he si es, including spine, w is s and o ea ms.
Some po en ial mechanisms by which PPI he apy may lead o ac u es ha e been
iden i ied. Fi s , he small in es ine’s abili y o abso b inges ed calcium depends on pH.
Wi hou an acidic en i onmen in he gas oin es inal ac , calcium may be e ained in he
ood ma ix p e en ing abso p ion. Second, impai ed calcium abso p ion may lead o
compensa o y seconda y hype pa a hy oidism. Seconda y hype pa a hy oidism e e s o
he inc ease in ci cula ing le els o pa a hy oid ho mone, which, in an a emp o ec i y he
de ici in calcium abso p ion, inc ease he a e o os eoclas ic ac i i y [66, 72]. Finally, PPI
may di ec ly in e e e wi h os eoclas s ac i i y [74]. S udies ha e shown ha omep azole
inhibi s bone eso p ion in i o and in i o in humans by inhibi ing os eoclas ic acuola
H+ ATPase ac i i y. A educed bone eso p ion should lead o inc eases in bone densi y,
howe e , bone eso p ion is necessa y o he no mal bone de elopmen , eplacing old
bone and epai ing mic o ac u es. Thus, a dec ease in os eoclas ac i i y can a ec bone
s uc u e and, hus, p edispose pa ien s o ac u es [66, 70, 72, 74].
35
Taken oge he , hese s udies a e limi ed by a low magni ude o associa ion and
inabili y o assess po en ial con ounding ac o s [79]. Despi e all hese ese a ions, i
seems likely ha PPI may a ec signi ican ly bone me abolism, pa icula ly in some
ulne able indi iduals, such as pos menopausal women, whe e PPI use con ibu es o an
inc eased isk o ac u es a a ious si es, in a manne ela ed o he dose and du a ion o
exposu e o he d ug [66, 70, 74, 80].
In his con ex , since in he eso p ion p ocess os eoclas s solubilize he bone mine al
phase h ough he sec e ion o acid o he eso p ion lacuna by p o on pumps o he
acuola ype, V-ATPases, PPIs appea as po en ially pha macological modula o s o
os eoclas ic ac i i y, pa icula ly in pa hological si ua ions cha ac e ized by hipe ac i a ion
o os eoclas s such as os eoly ic me as ases associa ed wi h b eas cance .
36
2. Ma e ials and me hods
2.1 Cell Cul u es
2.1.1 Isola ion o Pe iphe al blood mononuclea cells (PBMC)
The cells we e isola ed om blood o male heal hy dono s wi h ages be ween 25 and
35 yea s old. The blood was dilu ed wi h PBS (phospha ase bu e solu ion) supplemen ed
wi h 2 mM EDTA (1:2) and applied on op o 4ml o Ficoll-PaqueTM PREMIUM (GE
Heal hca e Bioscience). A e cen i uga ion a 400 g o 30 minu es a oom empe a u e,
PBMC we e collec ed and washed wice wi h PBS supplemen ed wi h 2 mM EDTA wi h
cen i uga ions a 300 g o 10 minu es a 4ºC in each. PBMC we e seeded a
1.5x106cells/cm2.
2.1.2 B eas cance cell lines cul u e (T47D and SK-BR-3)
Human b eas cance cell lines, es ogen- ecep o posi i e T47D and es ogen-nega i e
SK-BR-3, we e main ained in α-Minimal Essen ial Medium (α-MEM) supplemen ed wi h
10% ( / ) e al bo ine se um, 100 IU/mL penicillin, 2.5 µg/mL s ep omycin, 2.5 µg/mL
ampho e icin B and 50 µg/mL asco bic acid. Cul u es we e pe o med a 37ºC in a 5%
CO2 humidi ied a mosphe e. A abou 70-80% con luence, cells we e de ached wi h 0.05%
ypsin and 0.5 mM EDTA, and sepa a ely seeded a 104cells/cm2.
Cell cul u es we e main ained in α-MEM supplemen ed wi h 10% ( / ) e al bo ine
se um, 100 IU/mL penicillin, 2.5 µg/mL s ep omycin, 2.5 µg/mL ampho e icin B and 50
µg/mL asco bic acid. Cul u es we e ea ed wi h di e en PPI (omep azole, esomep azole
and lansop azole), a a concen a ion ange o 10-9-10-3 M. Cul u e medium was changed
once a week and PPI we e enewed a each medium change. Cell cul u es we e
main ained o 21 days in a 5% CO2 humidi ied a mosphe e a 37ºC.
Cul u es we e assessed o cellula iabili y/ p oli e a ion and apop osis.
2.1.3 Co-cul u e o SK-BR-3 and T47D and PBMC
SK-BR-3 and T47D cell lines we e sepa a ely seeded a 102 cells/cm2 and 104cells/cm2,
espec i ely. Cul u es we e incuba ed o 24h a 37ºC in α-MEM supplemen ed wi h 10%
( / ) e al bo ine se um, 100 IU/mL penicillin, 2.5 µg/mL s ep omycin, 2.5 µg/mL
37
ampho e icin B and 50 µg/mL asco bic acid. A e ha ime, PMBC we e added a
1.5x106cells/cm2.
Cell cul u es we e main ained in α-MEM supplemen ed wi h 30% ( / ) human se um
( om he same dono whe e PBMC we e ob ained), 100 IU/mL penicillin, 2.5 µg/mL
s ep omycin, 2.5 µg/mL ampho e icin B and 2 mM L-glu amine. Cul u es we e ea ed
wi h di e en PPI (omep azole, esomep azole and lansop azole), a a concen a ion ange
o 10-7-10-3 M. Cul u e medium was changed once a week and PPI we e enewed a each
medium change. Cell cul u es we e main ained o 21 days in a 5% CO2 humidi ied
a mosphe e a 37ºC.
Cul u es we e assessed o o al p o ein con en , TRAP ac i i y, numbe o TRAP
posi i e cells, p esence o mul inuclea ed cells wi h ac in ings and exp essing VNR and
CTR by con ocal lase scanning mic oscopy (CLSM) and calcium phospha e eso bing
abili y. In o de o assess he in acellula mechanisms in ol ed, when indica ed, he
medium was also supplemen ed wi h se e al signalling pa hway inhibi o s, namely, 1µM
U0126 (MAPK/ERK pa hway inhibi o ), 10µM PDTC (NF-kB pa hway inhibi o ), 5µM GO
6983 (PKC pa hway inhibi o ) and 10µM SP (JNK pa hway inhibi o ).
2.1.4 Human e al os eoblas ic cell line (hFOB) cul u e
The hFOB cells we e cul u ed in Dulbecco’s Modi ied Eagle’s Medium/HAM’s Nu ien
Mix u e F12 (DMEM/F12) supplemen ed wi h 10% ( / ) e al bo ine se um, 100 IU/mL
penicillin, 2.5 µg/mL s ep omycin, 2.5 µg/mL ampho e icin B, 50 µg/mL asco bic acid and
0,6% gen amicin. Cul u es we e pe o med a 37ºC in a 5% CO2 humidi ied a mosphe e.
A abou 70-80% con luence, cells we e de ached wi h 0.05% ypsin and 0.5 mM EDTA,
and seeded a 104cells/cm2.
Cell cul u es we e main ained in DMEM/F12 supplemen ed wi h 10% ( / ) e al bo ine
se um, 100 IU/mL penicillin, 2.5 µg/mL s ep omycin, 2.5 µg/mL ampho e icin B, 50 µg/mL
asco bic acid and 0,6% gen amicin, in absence and p esence o 1% dexame hasone.
Cul u es we e ea ed wi h di e en PPI a a concen a ion ange o 10-7-10-3 M. Cul u e
medium was changed once a week and PPI we e enewed a each medium change. Cell
cul u es we e main ained o 21 days in a 5% CO2 humidi ied a mosphe e a 37ºC.
Cul u es we e assessed o cellula iabili y/ p oli e a ion, o al p o ein con en , ALP
ac i i y and his ochemical s aining and isualized by CLSM. I was also ca ied ou a

38
cha ac e iza ion o he in acellula mechanisms in ol ed, o ha , cells we e seeded a
104cells/cm2 and main ained in DMEM/F12 as desc ibed abo e. When indica ed, he
medium was also supplemen ed wi h se e al signalling pa hway inhibi o s, namely, 1µM
U0126 (MAPK/ERK pa hway inhibi o ), 10µM PDTC (NF-kB pa hway inhibi o ), 5µM GO
6983 (PKC pa hway inhibi o ) and 10µM SP (JNK pa hway inhibi o ).
2.2 Cha ac e iza ion o cell cul u es
2.2.1 To al p o ein quan i ica ion
Cellula p o ein con en was de e mined by B ad o d’s me hod. Cell cul u es we e
washed wice wi h PBS and solubilized wi h 0.1 M NaOH. Samples we e ea ed wi h
Coomassie® P o ein Assay Reagen (Fluka) and incuba ed o 2 minu es a oom
empe a u e. The abso bance was quan i ied a 595 nm in an ELISA pla e eade (Sine gy
HT, Bio eck). Resul s we e exp essed as mg/mL.
2.2.2 TRAP ac i i y quan i ica ion
TRAP ac i i y was assessed by pa a-ni ophenilphospha e (pNPP) hyd olysis me hod.
Cell cul u es we e washed wice wi h PBS and solubilized wi h 0.1% (V/V) T i on X-100.
Samples we e incuba ed o 1 hou a 37ºC wi h 10 mM pNPP p epa ed in 0.04 M a a ic
acid and 0.09 M ci a e a pH 4.8. A e incuba ion, he eac ion was s opped wi h 5 M
NaOH and he abso bance was measu ed a 400 nm in an ELISA pla e eade (Syne gy
HT, Bio eck). Resul s we e no malized wi h o al p o ein con en and exp essed as
nmol/min/µgp o ein.
2.2.3 His ochemical s aining o TRAP
A days 14 and 21, cell cul u es we e washed wice wi h PBS and ixed wi h 3.7% ( / )
o maldehyde o 15 minu es a oom empe a u e. A e new wash wi h PBS, cells we e
s ained o TRAP wi h Acid Phospha ase Leukocy e (TRAP) ki (SIGMA), acco ding o
manu ac u e ’s ins uc ions. Sho ly, cells we e incuba ed wi h naph ol AS-BI 0.12 mg/mL,
in he p esence o 6.76 nM a a e and 0.14 mg/mL Fas Ga ne GBC, o 1 hou a 37ºC
in he da k. A e incuba ion, cell laye s we e washed, s ained wi h hema oxilin and
isualized by ligh mic oscopy. TRAP posi i e mul inuclea ed cells (pu ple/da k ed) we e
coun ed.
39
2.2.4 ALP ac i i y quan i ica ion
ALP ac i i y was also de e mined by he pNPP hyd olysis me hod used o TRAP ac i i y
quan i ica ion. He e, a e solubiliza ion wi h 0.1% (V/V) T i on X-100, samples we e
incuba ed wi h 10 mM pNPP in a 0.15 M bica bona e bu e in 2.5 mM MgCl2 a pH 10.
The p ocedu e was he same as desc ibed o TRAP ac i i y.
2.2.5 His ochemical s aining o ALP
Cell cul u es we e washed wice wi h PBS and ixed wi h 1.5% glu a aldehyde in
cacodyla e bu e , o 10 minu es a oom empe a u e. A e ixa ion, cells we e incuba ed
wi h 30 mg/mL phospha ase naph hyl in 0.1 M T is bu e a pH 10, in he p esence o 30
mg/mL Fas Blue RR Sal , o 1 hou a 37ºC, in he da k. A e incuba ion, cell laye s we e
washed and isualized by ligh mic oscopy.
2.2.6 Cellula p oli e a ion/ iabili y
The celula p oli e a ion/ iabili y assessmen was pe o med by MTT (3-(4, 5-
dime hyl hiazol-2-yl)-2, 5-diphenyl e azolium b omide) assay. Cell cul u es we e
incuba ed wi h 5 mg/mL MTT, o 4 hou s a 37ºC. A e incuba ion, he medium was
emo ed and 100 µL dime hyl sul oxide (DMSO) was added. The abso bance was
quan i ied a 550nm in an ELISA pla e eade (Syne gy HT, Bio eck).
2.2.7 Immuno luo escence s aining and isualiza ion by CLSM
Cell cul u es we e washed wice wi h PBS and ixed wi h 3.7% ( / ) pa a o maldehyde,
o 15 minu es a oom empe a u e. A e ixa ion, cells we e washed again wi h PBS and
pe meabilized wi h 0.1% (V/V) T i on X-100 o 5 minu es. Cul u es we e hen s ained o
F-ac in wi h 5 U/mL Alexa Fluo ® 647-Phalloidin (In i ogen), o nucleus wi h 500 nM
p opidium iodide, and o i onec in ecep o s (VNR) and calci onin ecep o s (CTR) wi h
50 µg/mL mouse IgGs an i-VNR and IgGs an i-CTR (R&D Sys ems), espec i ely. An i-
VNR and an i-CTR de ec ion was pe o med wi h 2 µg/mL Alexa Fluo ® 488 an i-mouse
IgGs (In i ogen). Cul u es we e isualized by CLSM.
40
2.2.8 Apop osis quan i ica ion
Apop osis was analyzed h ough caspase 3 ac i i y quan i ica ion. Fo ha , b eas
cance cell cul u es we e washed wice wi h PBS and assessed o caspase 3 ac i i y wi h
EnzCheck® Caspase 3 Assay Ki # 2 (Molecula P obes), acco ding o manu ac u e ’s
ins uc ions. The luo escence was measu ed a 486/520 nm (exci a ion/emission) in an
ELISA pla e eade (Syne gy HT, Bio eck).
2.2.9 Calcium phospha e eso bing abili y
Co-cul u es o SK-BR-3 and T47D and PBMC we e pe o med on calcium phospha e
coa ed cul u e pla es (BD BioCoa ™ Os eologic™ Bone Cell Cul u e Pla es, BD
Biosciences), o 21 days. A e ha pe iod, cells we e bleached wi h 6% NaOCl and 5.2%
NaCl, and he emaining calcium phospha e laye s we e isualized by phase con as ligh
mic oscopy (Nikon TMS phase con as mic oscope). Reso p ion lacunae we e iden i ied
and o al eso bed a ea was quan i ied wi h ImageJ 1.41 so wa e. Resul s we e
p esen ed as a % o eso bed a ea.
2.2.10 S a is ical analysis
Da a p esen ed in his wo k a e he means o sepa a e expe imen s. Th ee eplicas o
each condi ion we e made o each expe imen and da a a e exp essed as he mean ±
s anda d de ia ion. Da a we e e alua ed using a wo-way analysis o a iance (ANOVA)
and no signi ican di e ences in he pa e n o he cell beha io we e obse ed. S a is ical
di e ences ound be ween con ol and expe imen al condi ions we e de e mined by
Bon e oni’s me hod. Fo alues o p ≤ 0.05, di e ences we e conside ed s a is ical
signi ican .
41
3. Resul s
3.1 Assessmen o he e ec s o di e en PPI in cul u es o SK-BR-3 and T47D
In he i s pa o his wo k, i was p e ended o e alua e he e ec s o PPI in cul u es o
SK-BR-3 and T47D. Each cul u e was ea ed wi h di e en concen a ions o omep azole,
esomep azole and lansop azole, and main ained o 21 days. Samples we e assessed a
days 7, 14 and 21.
3.1.1 Cha ac e iza ion o SK-BR-3 cul u es
3.1.1.1 Cellula p oli e a ion/ iabili y
Figu e 17. Cellula p oli e a ion/ iabili y o SK-BR-3 cul u es ea ed wi h di e en concen a ions o
omep azole, esomep azole and lansop azole. * Signi ican ly di e en om he con ol.
In gene al, SK-BR-3 cell cul u es main ained a cell p oli e a ion/ iabili y iden ical o
nega i e con ol o he lowes concen a ions o each PPI. Howe e , wi h he inc ease o
PPI concen a ions, cell p oli e a ion/ iabili y s a ed o dec ease. This dose-dependen
dec ease was seen o he h ee PPI s a ing om he concen a ion 10-5 M, wi h a
dec ease o abou 18.79%, 21.13% and 3.64% o omep azole, esomep azole and
lansop azole, espec i ely.
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
7
14
21
Abso bance (550nm)
Days
Omep azole
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
7
14
21
Days
Esomep azole
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
7
14
21
Days
Lansop azole
Nega i e Con ol
10-9M
10-8M
10-7M
10-6M
10-5M
10-4M
10-3M
*
*
*
*
*
*
*
*
*
48
The esul s o his ochemical s aining o TRAP we e consis en wi h hose ob ained o
TRAP ac i i y, showing mo e e iden ly ha GO6983 was he inhibi o ha less a ec ed
he cul u es, excep o he cul u es ea ed wi h lansop azole (da a no shown).
3.2.2 Cha ac e iza ion o PBMC + T47D co-cul u es
3.2.2.1 TRAP ac i i y
Figu e 26. TRAP ac i i y o PBMC + T47D co-cul u es ea ed wi h di e en concen a ions o
omep azole, esomep azole and lansop azole. * Signi ican ly di e en om he con ol.
PBMC + T47D co-cul u es p esen ed le els o TRAP ac i i y highe han hose ob ained
in PBMC + SK-BR-3 co-cul u es. TRAP ac i i y dec eased wi h he inc ease o each PPI
concen a ion. This dose-dependen inhibi ion was highe in he p esence o omep azole
and lansop azole. Compa ed o he con ol, he e ec s became s a is ically signi ican o
he concen a ion o 10-7 M omep azole (30.93%) 10-5 M o esomep azole (16.87%) and
10-7 M o lansop azole (34%). Again, co-cul u es ea ed wi h he highes concen a ions o
omep azole and lansop azole displayed null le els o TRAP ac i i y.
0
10
20
30
40
7 14 21
nmol/min.µg-1
Days
Omep azole
0
10
20
30
40
7 14 21
Days
Esomep azole
0
10
20
30
40
7 14 21
Days
Lansop azole
Nega i e con ol
10-7M
10-6M
10-5M
10-4M
10-3M
*
*
*
*
*
*
*
*
*

49
3.2.2.2 His ochemical s aining o TRAP
Figu e 27. P esence o TRAP+ mul inuclea ed cells on PBMC + T47D co-cul u es ea ed wi h
di e en concen a ions o omep azole, esomep azole and lansop azole. *Signi ican ly di e en
om he con ol.
PBMC + T47D co-cul u es showed a highe p esence o TRAP mul inuclea ed cells
han PBMC + SK-BR-3 co-cul u es. As obse ed o TRAP ac i i y, he numbe o TRAP+
mul inuclea ed cells dec eased wi h he inc ease o each PPI concen a ion, pa icula ly in
he p esence o omep azole and lansop azole.
3.2.2.3 Calcium phospha ase eso bing abili y
0
20
40
60
80
100
120
140
14
21
TRAP+
mul inuclea ed cells
Days
Omep azole
0
20
40
60
80
100
120
140
14
21
Days
Esomep azole
0
20
40
60
80
100
120
140
14
21
Days
Lansop azole
Nega i e con ol
10-7M
10-6M
10-5M
10-4M
10-3M
Nega i e con ol Omep azole (10-7 M)
Esomep azole (10-5 M) Lansop azole (10-7 M)
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
50
Figu e 28. Visualiza ion o calcium phospha ase eso bed a ea o PBMC + T47D co-cul u es
ea ed wi h 10-7 M omep azole, 10-5 M esomep azole and 10-7M lansop azole, by ligh mic oscopy.
Whi e ba s ep esen 600µm.
Figu e 29. Reso bed a ea o PBMC + T47D co-cul u es ea ed wi h 10-7 M omep azole, 10-5 M
esomep azole and 10-7 M lansop azole. *Signi ican ly di e en om he con ol.
Visualiza ion o calcium phospha ase eso bed a ea by ligh mic oscopy showed a
dec ease in calcium phospha ase eso bing abili y in he p esence o he h ee PPI. The
co-cul u e ha p esen ed he lowes a ea o eso p ion was he one ha was ea ed wi h
10-7 M omep azole, wi h a maximal inhibi ion o 44% compa ed o he con ol. In he
p esence o esomep azole and lansop azole he inhibi ion was abou 23% and 35%,
espec i ely. Globally, PBMC + T47D co-cul u es p esen ed a highe eso bing abili y
han PBMC + SK-BR-3 co-cul u es (53% e sus 30%).
3.2.2.4 Visualiza ion o cells wi h ac in ings and exp ession o VNR and CTR
0,00
10,00
20,00
30,00
40,00
50,00
60,00
70,00
% Reso p ion A ea
T47D + PBMC
Con ol
Omep azole
Esomep azole
Lansop azole
Nega i e con ol Omep azole (10-6 M)
Nega i e con ol Omep azole (10-6 M)
V
N
R
C
T
R
*
*
*
51
Figu e 30. Visualiza ion o cells wi h ac in ings and exp ession o VNR and CTR o PBMC + T47D
co-cul u es in absence and p esence o 10-6 M omep azole, by CLSM. Cells we e s ained blue o
ac in and g een o VNR and CTR. Whi e ba s ep esen 150µm.
Visualiza ion o PBMC + T47D co-cul u es by CSLM e ealed he p esence o
os eoclas ic cells in all analysed condi ions. The esul s we e somehow in line wi h he
da a ob ained om he p e ious analysis. Tha means, co-cul u es ea ed wi h 10-6 M
omep azole showed a dec ease in os eoclas ic cells compa ed o he nega i e con ol.
3.2.2.5 Cha ac e iza ion o he in acellula mechanisms in ol ed in he cellula
esponse
Cha ac e iza ion o he in acellula mechanisms in ol ed in os eoclas ogenesis was
also pe o med in PBMC + T47D co-cul u es. Fo ha , co-cul u es we e main ained o 21
days in he absence and p esence o 10-7 M omep azole, 10-5 M esomep azole and 10-7 M
lansop azole ( he lowes concen a ion o each PPI ha elici ed a signi ican an i-
os eoclas ogenic e ec ) and ea ed wi h in acellula signalling pa hway inhibi o s: 1µM
U0126 (MAPK/ERK inhibi o ); 10µM PDTC (NF-kB inhibi o ); 5µM GO 6983 (PKC
inhibi o ) and 10µM SP 600125 (JNK inhibi o ). Samples we e assessed a days 7, 14 and
21.
3.2.2.5.1 TRAP ac i i y
0
5
10
15
20
25
30
35
40
7
14
21
nmol/min.µg-1
Days
Con ol
0
5
10
15
20
25
30
35
40
7
14
21
Days
Omep azole
No inhibi o
U0126
PDTC
GO6983
SP 600125
0
5
10
15
20
25
30
35
40
7
14
21
nmol/min.µg-1
Days
Esomep azole
0
5
10
15
20
25
30
35
40
7
14
21
Days
Lansop azole
No inhibi o
U0126
PDTC
GO6983
SP 600125
*
*
*
*
*
*
*
*
*
*
52
Figu e 31. TRAP ac i i y on PBMC + T47D co-cul u es ea ed wi h 10-7 M omep azole, 10-5 M
esomep azole and 10-7 M lansop azole and supplemen ed wi h di e en in acellula signalling
pa hway inhibi o s. * Signi ican ly di e en om he con ol.
TRAP ac i i y o un ea ed and ea ed PBMC + T47D co-cul u es dec eased in he
p esence o all he es ed inhibi o s. In co-cul u es ea ed wi h omep azole and
esomep azole, wi h excep ion o U0126 ha equally a ec ed TRAP ac i i y, he p esence
o all he inhibi o s induced a less signi ican inhibi ion han in un ea ed cul u es. In he
p esence o lansop azole, TRAP ac i i y dec eases we e simila o hose seen on
un ea ed cul u es o hose supplemen ed wi h U0126, PDTC and GO6983, whe eas
SP600125 supplemen ed cul u es we e less a ec ed.
The esul s o his ochemical s aining o TRAP we e consis en wi h hose ob ained o
TRAP ac i i y. (da a no shown)
3.3 Assessmen o he e ec s o di e en PPI in cul u es o hFOB
Finally, in he las pa o his wo k, he aim was o e alua e he e ec s o PPI in
cul u es o hFOB. Cul u es we e pe o med in he absence and p esence o he
os eogenic induce dexame hasone (DEX) and supplemen ed wi h di e en
concen a ions o omep azole, esomep azole and lansop azole. Cell cul u es we e
main ained o 21 days and samples we e assessed a days 7, 14 and 21.
3.3.1 Cha ac e iza ion o hFOB cul u es (-DEX)
3.3.1.1 Cellula p oli e a ion/ iabili y
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
7
14
21
Abso bance (550)
Days
Omep azole
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
7
14
21
Days
Esomep azole
*
*
*
*
*
*
*
*
*
*
53
Figu e 32. Cellula p oli e a ion/ iabili y o hFOB cul u es ea ed wi h di e en concen a ions o
omep azole, esomep azole and lansop azole, in absence o DEX. * Signi ican ly di e en om he
con ol.
Cellula p oli e a ion/ iabili y o hFOB cul u es, main ained in he absence o DEX,
dec eased wi h he inc ease o each PPI concen a ion. This dose-dependen inhibi ion
was highe in he p esence o omep azole and lansop azole, wi h null le els o cellula
p oli e a ion/ iabili y o he highes concen a ion o lansop azole (10-3 M). The inhibi ion
became s a is ically signi ican in he p esence o 10-6 M omep azole and lansop azole
and 10-5 M esomep azole, wi h a co esponding inhibi ion o abou 17.78%, 31.30% and
16.69%.
3.3.1.2 ALP ac i i y
Figu e 33. ALP ac i i y o hFOB cul u es ea ed wi h di e en concen a ions o omep azole,
esomep azole and lansop azole, in absence o DEX. * Signi ican ly di e en om he con ol.
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
7
14
21
Days
Lansop azole
Nega i e con ol
10-7M
10-6M
10-5M
10-4M
10-3M
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
14
21
nmol/min.µg-1
Days
Omep azole
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
14
21
Days
Esomep azole
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
14
21
Days
Lansop azole
Nega i e con ol
10-7M
10-6M
10-5M
10-4M
10-3M
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*

54
As obse ed o cellula p oli e a ion/ iabili y, ALP ac i i y o hFOB cul u es, main ained
in he absence o DEX, dec eased wi h he inc ease o PPI concen a ion, pa icula ly in
he p esence o omep azole and lansop azole, wi h null le els o ac i i y o he highes
concen a ion (10-3 M). The inhibi ion became s a is ically signi ican in he p esence o 10-
6 M omep azole (39.46%), 10-6 M esomep azole (8.16%) and 10-6 M lansop azole
(25.85%).
3.3.1.3 His ochemical s aining o ALP
Figu e 34. His ochemical s aining o ALP in hFOB cul u es ea ed wi h 10-6 M omep azole,
esomep azole and lansop azole, in absence o DEX. Whi e ba s ep esen 200µm.
Visualiza ion o his ochemical s aining o ALP e ealed a cellula beha io consis en
wi h he da a ob ained om ALP ac i i y analysis. Cul u es ea ed wi h 10-6 M omep azole
and lansop azole displayed a signi ican dec ease in ALP exp ession, while he same
concen a ion o esomep azole did no ha e a so sha p e ec in he cellula esponse.
Nega i e Con ol Omep azole (10-6 M)
Esomep azole (10-6 M) Lansop azole (10-6 M)
55
3.3.2 Cha ac e iza ion o hFOB cul u es (+DEX)
3.3.2.1 Cellula p oli e a ion/ iabili y
Figu e 35. Cellula p oli e a ion/ iabili y o hFOB cul u es ea ed wi h di e en concen a ions o
omep azole, esomep azole and lansop azole, in he p esence o DEX. * Signi ican ly di e en om
he con ol.
Cellula p oli e a ion/ iabili y o hFOB cul u es, in p esence o DEX, dec eased wi h he
inc ease o each PPI concen a ion. This dose-dependen inhibi ion was highe in he
p esence o omep azole and lansop azole. The inhibi ion became s a is ically signi ican in
he p esence o 10-7 M omep azole, 10-5 M esomep azole and 10-4 M lansop azole, wi h a
co esponding inhibi ion o abou 13.92%, 12.66% and 31.65%.
3.3.2.2 ALP ac i i y
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
7
14
21
Abso ância (550)
Days
Omep azole
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
7
14
21
Days
Esomep azole
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
7
14
21
Days
Lansop azole
Nega i e con ol
10-7M
10-6M
10-5M
10-4M
10-3M
0
0,5
1
1,5
2
2,5
3
3,5
14
21
nmol/min.µg-1
Days
Omep azole
0
0,5
1
1,5
2
2,5
3
3,5
14
21
Days
Esomep azole
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
56
Figu e 36. ALP ac i i y o hFOB cul u es ea ed wi h di e en concen a ions o omep azole,
esomep azole and lansop azole, in he p esence o DEX. * Signi ican ly di e en om he con ol.
ALP ac i i y o hFOB cul u es, in he p esence o DEX, dec eased wi h he inc ease o
each PPI concen a ion, pa icula ly in he p esence o omep azole and lansop azole. The
inhibi ion became s a is ically signi ican s a ing om he concen a ion 10-6 M, wi h a
dec ease o abou 21.07%, 10.98% and 13.95% o omep azole, esomep azole and
lansop azole, espec i ely. Once again, co-cul u es ea ed wi h he highes
concen a ions o omep azole and lansop azole displayed null le els o TRAP ac i i y.
3.3.2.3 His ochemical s aining o ALP
Figu e 37. His ochemical s aining o ALP in hFOB cul u es ea ed wi h 10-6 M omep azole,
esomep azole and lansop azole, in he p esence o DEX. Whi e ba s ep esen 200µm.
The esul s o his ochemical s aining o ALP we e consis en wi h hose ob ained in ALP
ac i i y analysis, showing ha cul u es ea ed wi h omep azole and lansop azole had less
exp ession o ALP.
0
0,5
1
1,5
2
2,5
3
3,5
14
21
Days
Lansop azole
Nega i e con ol
10-7M
10-6M
10-5M
10-4M
10-3M
Nega i e Con ol Omep azole (10-6 M)
Esomep azole (10-6 M) Lansop azole (10-6 M)
*
*
*
57
3.3.2.4 Visualiza ion o hFOB cul u es by CLSM
Figu e 38. Visualiza ion o hFOB cul u es ea ed wi h 10-6 M omep azole, esomep azole and
lansop azole, in he p esence o DEX, by CLSM. Cells we e s ained g een o ac in and ed o
nuclei. Whi e ba s ep esen 60µm.
Visualiza ion o hFOB cul u es by CLSM e ealed he p esence o os eoblas ic cells in
all es ed condi ions. Resul s we e consis en wi h he da a ob ained om he p e ious
analysis, ha is, cul u es ea ed wi h omep azole and lansop azole showed a mo e
signi ican dec ease in he amoun o os eoblas ic cells compa ed o he nega i e con ol.
3.3.3 Cha ac e iza ion o he in acellula e ec s in ol ed in he obse ed cellula
esponse
Following he assessmen o he e ec s o PPI in cul u es o hFOB, i was also ca ied
ou a cha ac e iza ion o he in acellula mechanisms in ol ed in he obse ed cellula
beha io . Fo ha , cul u es we e main ained o 21 days in he absence and p esence o
10-6 M o each PPI and ea ed wi h in acellula signalling pa hway inhibi o s: 1µM U0126
(MAPK/ERK inhibi o ); 10µM PDTC (NF-kB inhibi o ); 5µM GO 6983 (PKC inhibi o ) and
10µM SP 600125 (JNK inhibi o ). Samples we e assessed a days 7, 14 and 21.
Nega i e con ol Omep azole (10-6 M)
Esomep azole (10-6 M) Lansop azole (10-6 M)
64
Following he assessmen o he os eoclas ogenic e ec s o he di e en PPI in co-
cul u es o PBMC and b eas cance cell lines, i was also ca ied ou a cha ac e iza ion o
he in acellula mechanisms in ol ed in his p ocess. Fo ha , i was used he lowes
common concen a ion o each PPI ha elici ed a signi ican an i-os eoclas ogenic e ec in
each co-cul u e. In SK-BR-3 co-cul u es i was used 10-6 M o he h ee PPI whe eas in
T47D co-cul u es we e used he ollowing concen a ions: 10-7 M omep azole, 10-5 M
esomep azole and 10-7 M lansop azole.
In con ol co-cul u es o PBMC and SK-BR-3, he os eoclas ogenic p ocess was highly
dependen on NFkB pa hway and signi ican ly dependen on MEK and JNK pa hways. I
was seen ha he p esence o omep azole seemed no o a ec he signaling pa hways o
co-cul u es, compa ed o con ol. In he p esence o esomep azole, os eoclas ogenesis
appea ed o be signi ican ly dependen on NFkB pa hway, while in he p esence o
lansop azole his p ocess appea ed o be signi ican ly dependen on PKC pa hway.
In con ol co-cul u es o PBMC and T47D, os eoclas di e en ia ion was highly
dependen on NFkB pa hway and signi ican ly dependen on PKC and JNK pa hway. I
was obse ed ha in he p esence o omep azole and esomep azole all he es ed
pa hways seemed o become less impo an in os eoclas ogenesis, compa ed o con ol.
On he o he hand, he p esence o lansop azole only a ec ed he JNK pa hway, making
his pa hway less impo an o he obse ed os eoclas ic esponse.
I was also obse ed ha he PPI es ed a e capable o di ec ly ac on os eoblas ic
hFOB cells. The p esence o dexame hasone, an os eogenic enhance , g ea ly induced
ALP ac i i y, as epo ed p e iously [89, 90, 91]. Cellula p oli e a ion/ iabili y o hFOB
cul u es main ained in he absence and p esence o DEX dec eased in he p esence o
he h ee PPI s a ing om he concen a ion 10-6 M. This inhibi ion was also seen o ALP
ac i i y, howe e , he pe cen age o inhibi ion was highe o ALP ac i i y compa ed o ha
ela ed o cell g ow h. This sugges s ha PPI e ec s a e mo e likely o be due o speci ic
changes on he cellula di e en ia ion a he han o dec eases on cellula iabili y due o
oxic mechanisms. The inhibi o y e ec s we e mo e p onounced in he p esence o
omep azole and lansop azole. As obse ed in he p e ious cul u e sys ems, he highes
es ed concen a ions o PPI appea ed o be oxic o he cells.
In compa ison wi h os eoclas s, signi ican ly less a en ion has been paid o ATPases in
os eoblas s. Os eoblas ic cells ha e been shown o exp ess some ypes o ATPases,
namely, Na+/K+-ATPase and Ca+-ATPase, howe e , he e is only indi ec e idence o he

65
p esence o acuola H+-ATPase in os eoblas s [86]. In i o s udies demons a ed ha
ba ilomycin A1 and concanamycin A, which a e po en and speci ic inhibi o s o H+-
ATPases, dose-dependen ly inhibi ed he g ow h and changed he mo phology o a
os eoblas cell lines [85, 86]. The p esen esul s a e in ag eemen wi h his la e s udy,
al hough he molecules do no ha e he same mechanism o ac ion.
I was also ca ied ou a cha ac e iza ion o he in acellula mechanisms in ol ed in he
os eoblas ic esponse.
In con ol hFOB cul u es main ained in he absence o DEX, cellula g ow h was highly
dependen on NFkB pa hway. The PKC and JNK pa hways seemed o be mo e impo an
o cellula g ow h in he p esence o omep azole and esomep azole, whe eas in he
p esence o lansop azole, cellula p oli e a ion/ iabili y appea ed o be signi ican ly
dependen on NFkB pa hway.
In con ol cul u es, ALP ac i i y was highly dependen on NFkB and signi ican ly
dependen on MEK and PKC pa hways. In he p esence o omep azole he enzyme
ac i i y appea ed o be signi ican ly dependen on JNK pa hway. In he p esence o
esomep azole PKC and JNK pa hways a e he mos impo an , while he MEK pa hway
seemed o s and ou in he p esence o lansop azole.
In con ol hFOB cul u es main ained in p esence o DEX, p oli e a ion/ iabili y was
highly dependen on NFkB pa hway. The pa hways ha appea ed o be mo e impo an
o cellula p oli e a ion/ iabili y in he p esence o omep azole we e he PKC and JNK. In
he p esence o esomep azole he MEK pa hway was mo e impo an , while in he
p esence o lansop azole cellula g ow h seemed signi ican ly dependen on PKC
pa hway.
In con ol cul u es, ALP ac i i y was highly dependen on MEK and PKC pa hways and
signi ican ly dependen on NFkB and JNK pa hways. As obse ed in he cul u es
main ained in he absence o DEX, he ALP ac i i y appea ed o be signi ican ly
dependen on JNK pa hway in he p esence o omep azole and esomep azole. The NFkB
pa hway was also impo an in he p esence o esomep azole. The enzyme ac i i y was
signi ican ly dependen on NFkB and PKC pa hways.
66
5. Conclusion
The PPI, omep azole, esomep azole and lansop azole, ha e he abili y o nega i ely
a ec he os eoclas ogenic p ocess on co-cul u es o os eoclas s and b eas cance cells,
a ec ing no only os eoclas ic cells di e en ia ion bu also hei eso bing abili y. This
inhibi ion was depending on he iden i y and concen a ion o he es ed molecule, and
in ol ed signi ican changes in di e en in acellula signaling pa hways. PPI also inhibi s
cellula p oli e a ion and iabili y o b eas cance cell cul u es. This inhibi ion appea ed o
be, a leas pa ially, due o an inc ease on apop osis. Simila ly, hese compounds induced
a dec ease on os eoblas ogenesis, an e ec ha migh con ibu e o e en ual dis up ion o
no mal bone cellula me abolic ac i i ies and consequen ly, lead o dele e ious e ec s o
hese d ugs in he bone issue. Globally, hese e ec s we e obse ed a concen a ions
iden ical o hose ound in human se um du ing he apeu ic use o hese molecules.
This s udy con ibu ed o a be e unde s anding o he ole o PPI in bone me abolism
in he con ex o b eas cance bone me as ases. In conclusion, PPI appea ed o ha e he
po en ial o con ol he des uc i e e ec s o os eoly ic me as ases, which migh open
doo s o new he apeu ic app oaches ega ding his pa hological condi ion.
67
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