Characterization of the Effects of Proton Pump inhibitors on bone Metabolism: in vitro study in cocultures of osteoclasts and Breast Cancer Cells and in Cultures of Osteoblasts

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 6 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 7 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 8 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 9 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 Re e ences 1. Boyle, W.J., Simone , W.S., and Lacey, D.L. (2003). Os eoclas di e en ia ion and ac i a ion. Na u e. 423: 337-342; 2. Da a, H.K.,Ng, W.F., Walke , J.A., Tuck, S.P., and Va anasi, S.S. (2008). The cell biology o bone me abolism. J. Clin. Pa hol. 61: 577-587; 3. Ma ks, S.C., and He mey, D.C. (1996). 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