Sara 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

Dissertação de Candidatura ao grau de Mestre em Oncologia Molecular

submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade

do Porto, realizada sob orientação do Professor Doutor João Miguel Silva e Costa

Rodrigues, professor auxiliar convidado, e co-orientação da Professora Doutora

Maria Helena Raposo Fernandes, professora catedrática da Faculdade de

Medicina Dentária da Universidade do Porto

Porto, 2013

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Agradecimentos

Agradeço aos meus orientadores, Professor Doutor João Miguel da Costa Rodrigues e

Professora Doutora Maria Helena Raposo Fernandes por todos os ensinamentos, toda a

ajuda e disponibilidade permanente ao longo da elaboração deste trabalho.

Ao Professor Doutor Manuel Teixeira (Instituto Português de Oncologia), pela cedência

das linhas celulares de cancro da mama.

À Doutora Paula Sampaio (Instituto de Biologia Molecular e Celular), pela

disponibilidade na aquisição de imagens de microscopia confocal.

À Técnica Mónica Garcia (Faculdade de Medicina Dentária da Universidade do Porto),

pela disponibilidade e ajuda laboratorial.

Aos meus colegas e amigos por todo o apoio e companheirismo.

Aos meus familiares e namorado por todo o apoio e paciência

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Resumo

Os inibidores de bombas de protões são uma classe de fármacos amplamente utilizada

em várias condições patológicas, como refluxo gastroesofágico, dispepsia e úlceras

pépticas. Apesar de existirem diversas moléculas diferentes pertencentes a esta classe,

sabe-se que estas partilham várias propriedades funcionais e estruturais, bem como

algumas características farmacocinéticas. O seu mecanismo de ação baseia-se na

inibição de H+/K+ ATPases, responsáveis pela secreção de ácido gástrico. Neste

contexto, uma vez que no processo de reabsorção os osteoclastos solubilizam a fase

óssea mineral através da secreção de ácido para a lacuna de reabsorção por bombas de

protões do tipo vacuolar, V-ATPases, os inibidores de bombas de protões surgem como

potenciais modeladores farmacológicos da atividade osteoclástica, particularmente em

condições patológicas caracterizadas pela hiperactivação de osteoclastos, tal como

acontece nas metástases osteolíticas associadas ao cancro da mama.

Neste trabalho avaliaram-se os efeitos celulares e moleculares de três inibidores de

bombas de protões, nomeadamente, omeprazole, esomeprazole e lansoprazole, em

culturas de células do cancro da mama, em co-culturas de osteoclastos e células do

cancro da mama e, ainda, em culturas de osteoblastos. As culturas foram mantidas na

presença de diferentes concentrações de cada inibidor durante 21 dias. As células foram

caracterizadas aos dias 7, 14 e 21 através da proliferação/viabilidade celular, conteúdo

proteico, atividade e coloração histoquímica da fosfatase alcalina e da fosfatase ácida

resistente ao tartarato, presença de células multinucleadas com anéis de actina e

expressão de recetores de vitronectina e calcitonina, apoptose e capacidade de

reabsorção. Posteriormente, as culturas foram também tratadas com inibidores das vias

MAPK/ERK, NFkB, PKC e JNK para ser avaliado o envolvimento destas vias de

sinalização intracelulares na resposta celular. Observou-se que os inibidores de bombas

de protões inibem a osteoclastogénese em co-culturas de osteoclastos e células do

cancro da mama, de forma dependente de dose e fármaco testado, envolvendo

alterações significativas nas diversas vias de sinalização intracelulares testadas.

Verificou-se, ainda, que estes compostos têm, também, a capacidade de inibir o

crescimento de culturas de células do cancro da mama e a osteoblastogénese. Os

inibidores de bombas de protões surgem, assim, como potenciais modeladores do

metabolismo ósseo no contexto da metastização óssea.

4

Abstract

Proton pump inhibitors are a class of drugs widely used in many pathological

conditions, such as gastroesophageal reflux, dyspepsia and peptic ulcers. Although there

are several different molecules belonging to this class, it is well known that they share

many functional and structural properties as well as some pharmacokinetic

characteristics. The mechanism of action relies in the inhibition of H+/K+ ATPases,

responsible for gastric acid secretion. In this context, since in the resorption process

osteoclasts solubilize the bone mineral phase through the secretion of acid to the

resorption lacuna by proton pumps of the vacuolar type, V-ATPases, proton pump

inhibitors appear as potentially pharmacological modulators of osteoclastic activity,

particularly in pathological situations characterized by hiperactivation of osteoclasts, such

as osteolytic metastases associated with breast cancer.

In this study were assessed the cellular and molecular effects of three proton pump

inhibitors, namely, omeprazole, esomeprazole and lansoprazole, in cultures of breast

cancer cells, in co-cultures of osteoclasts and breast cancer cells and in osteoblasts

cultures. The cultures were maintained in the presence of different concentrations of each

inhibitor for 21 days. Cells were characterized at day 7, 14 and 21 for cellular

proliferation/viability, total protein content, alkaline phosphatase and tartarate resistant

acid phosphatase activity and histochemical staining, presence of multinucleated cells

with actin rings and expression of vitronectin and calcitonin receptors, apoptosis and

resorbing ability. Later, the cultures were treated with inhibitors of the MAPK/ERK, NFkB,

PKC and JNK pathways to assess the involvement of these intracellular signaling

pathways in the cellular response. Results revealed that proton pump inhibitors inhibit

osteoclastogenesis in co-cultures of osteoclasts and breast cancer cells, depending on

the identity and dose of the tested molecule and involving significant changes in the

several intracellular signaling pathways tested. Moreover, it was seen that these

compounds also have the ability to inhibit cell growth in breast cancer cell cultures and

osteoblastogenesis. Thus, proton pump inhibitors appear as potential modulators of bone

metabolism in the context of bone metastasis.

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Index

List of abbreviations……………………………………………………………………………….7

1. Introduction………………………………………………………………………………………9

1.1 Bone tissue…………………………………………………………………………………..9

1.1.1 Structural and functional characteristics…………………………………………...9

1.1.2 Bone cells……………………………………………………………………………10

1.1.3 Osteoclastogenesis…………………………………………………………………12

1.1.4 Hormonal control……………………………………………………………………13

1.1.5 Signaling pathways…………………………………………………………………14

1.2 Breast cancer………………………………………………………………………………17

1.2.1 Epidemiology and pathology………………………………………………………17

1.2.2 Risk factors…………………………………………………………………………..18

1.2.3 Molecular types of breast cancer………………………………………………….18

1.2.4 Therapies…………………………………………………………………………….20

1.3 Bone metastasis in breast cancer……………………………………………………….24

1.3.1 Bone metastasis…………………………………………………………………….24

1.3.2 Types of bone metastases…………………………………………………………26

1.3.3 Molecular types of breast cancer and metastatic behavior…………………….28

1.3.4 Treatment……………………………………………………………………………29

1.4 Proton pump inhibitors…………………………………………………………………….30

1.4.1 Mechanism of action………………………………………………………………..30

1.4.2 Adverse effects……………………………………………………………………...32

1.4.3 PPIs versus H2RAs…………………………………………………………………33

1.4.4 Secondary effects of PPI in bone metabolism…………………………………...34

2. Materials and methods………………………………………………………………………..36

2.1 Cell cultures………………………………………………………………………………..36

2.1.1 Isolation of peripheral blood mononuclear cells (PBMC)……………………….36

2.1.2 Breast cancer cell lines cultures (T47D and SK-BR-3)…………………………36

2.1.3 Co-cultures of SK-BR-3 and T47D and PBMC…………………………………..36

2.1.4 Human fetal osteoblastic cell line (hFOB) culture……………………………….37

2.2 Characterization of cell cultures………………………………………………………….38

2.2.1 Total protein quantification…………………………………………………………38

2.2.2 TRAP activity quantification………………………………………………………..38

2.2.3 Histochemical staining of TRAP…………………………………………………..38

2.2.4 ALP activity quantification………………………………………………………….39

2.2.5 Histochemical staining of ALP……………………………………………………..39

6

2.2.6 Cellular proliferation/viability……………………………………………………….39

2.2.7 Immunofluorescence staining and visualization by CLSM……………………..39

2.2.8 Apoptosis quantification……………………………………………………………40

2.2.9 Calcium phosphate resorbing ability……………………………………………...40

2.2.10 Statistical analysis…………………………………………………………………40

3. Results………………………………………………………………………………………….41

3.1 Assessment of the effects of different PPI in cultures of SK-BR-3 and T47D………41

3.1.1 Characterization of SK-BR-3 cultures…………………………………………….41

3.1.2 Characterization of T47D cultures ………………………………………………..42

3.2 Assessment of the osteoclastogenic effects of different PPI in co-cultures of PBMC

and breast cancer cell lines……………………………………………………………………..44

3.2.1 Characterization of PBMC + SK-BR-3 co-cultures………………………………44

3.2.2 Characterization of PBMC + T47D co-cultures………………………………….48

3.3 Assessment of the effects of different PPI in cultures of hFOB………………………52

3.3.1 Characterization of hFOB cultures (-DEX)……………………………………….52

3.3.2 Characterization of hFOB cultures (+DEX)………………………………………55

3.3.3 Characterization of the intracellular effects involved in cellular response…....57

4. Discussion……………………………………………………………………………………...62

5. Conclusion……………………………………………………………………………………..66

References………………………………………………………………………………………..67

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List of abbreviations

α-MEM - α-minimal essential medium

ALP – alkaline phosphatase

BMP – bone morphogenetic protein

BP - bisphosphonate

CA2 – carbonic anhydrase II

cAMP – cyclic adenosine monophosphate

CaSR – calcium-sensing receptor

CATK – cathepsin K

CLSM – confocal laser scanning microscopy

CSF – colony-stimulating factor

CTR – calcitonin receptor

DMEM/F12 - Dulbecco’s Modified Eagle’s Medium/HAM’s Nutrient Mixture F12

ECM – extracellular matrix

EGF – epidermal growth factor

ER – estrogen receptor

ERK - extracellular signal regulated kinase

FGF - fibroblasts growth factor

GMCSF - granulocyte macrophage colony-stimulating factor

HER2/erB2 - human epidermal growth factor receptor 2

hFOB – human fetal osteoblastic cell line

HR - hormonal receptor

H2RA - histamine 2 receptor antagonist

IGF - insulin-like growth factor

IL - interleukin

JNK - C-jun N-terminal kinase

MAPK - mitogen activated protein kinases

M-CSF – macrophage colony stimulating factor

MITF - microphthalmia associated transcription factor

MSC - mesenchymal stem cells

NF-kB - Nuclear factor kappa B

OPG – osteoprotegerin

PBMC – pheripheral blood mononuclear cells

PDGF - platelet-derived growth factor

PGE2 – prostaglandin E2

PKC – protein kinase C

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pNPP - para-nitrophenilphosphate

PPI – proton pump inhibitor

PR – progesterone receptor

PTH - parathyroid hormone

PTHrP - parathyroid hormone related protein

RANK – receptor activator of nuclear factor kappa B

RANKL - receptor activator of nuclear factor kB ligand

TGF-β - transforming growth factor-betas

TNF – tumor necrosis factor

TRAF – tumor necrosis factor receptor associated factor

TRAP - tartarate resistant acid phosphatase

VEGF - vascular endothelial growth factor

VNR – vitronectin receptor

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1. Introduction

1.1 Bone tissue

1.1.1 Structural and functional characteristics

Bone is a specialized connective tissue that is constantly remodeling and repairing

itself. With a microstructure developed to enable and support a maximum strength

associated with a minimal mass, bone tissue can be divided in two components: an

organic component, composed essentially by bone cells and an extracellular matrix

constituted by type I collagen (90% of total protein), proteoglycans and other proteins,

which confers flexibility; and an inorganic, or mineralized, component, composed mainly

by calcium and phosphate salts in the form of hydroxyapatite (Ca10(P04)6(OH)2),

representing about 60% of bone weight, and also small amounts of silicon, sodium and

magnesium, which confers rigidity [1, 3, 18].

In addition to presenting a series of mechanical functions such as, providing form,

protection and support, the bone also acts as a reservoir, storing and releasing ions in

order to maintain a constant concentration in body fluids, and absorbing and accumulating

toxins and metals preventing its adverse effects in other tissues [2, 20].

Its multicellular base comprises essentially three distinct types of cells: osteoblasts

(cells responsible for the formation of new bone), osteoclasts (cells specialized in bone

resorption), and osteocytes, which are osteoblast completely embedded in bone matrix

that are thought to play a key role as mechanosensors (Fig. 1) [2, 20].

Figure 1. Bone remodeling process. Osteoclasts are activated and the resorption process

begins. Resorption phase takes approximately 10 days. Then, osteoblasts precursors are recruited,

proliferate and differentiate. Osteoblasts produce bone mineralized matrix in a much longer

process that can take several months.

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(http://www.york.ac.uk/res/btr/Image%20Library/Bone%20remodelling.jpg)

Once formed, bone performs its remodeling process balancing these actions of bone

resorption and synthesis in order to ensure the renewal of tissue with minimal temporary

loss. Unbalances between these actions can result in some pathological conditions. Most

adult skeletal diseases are caused by an excess of osteoclastic activity, such as

osteoporosis and rheumatoid arthritis [1].

1.1.2 Bone cells

1.1.2.1 Osteoblasts

Osteoblasts arise from mesenchymal stem cells (MSC) and are the bone-forming cells.

The process of differentiation of osteoblasts is less well understood than the osteoclastic

one.

First, mesenchymal stem cells are committed to the osteoblast lineage. These cells,

called osteoprogenitors, proliferate and differentiate into pre-osteoblasts. Pre-osteoblasts

in turn start to produce extracellular matrix (ECM) and differentiate into mature

osteoblasts. Mature osteoblasts ensure ECM synthesis and mineralization. However, only

a fraction of the mature osteoblasts will be incorporated into newly formed bone matrix

and remain as osteocytes. The remaining osteoblasts will die or become lining cells (Fig.

2) [17].

Figure 2. Stages of osteoblasts differentiation. Osteoblasts undergo a complex differentiation

process that essentially comprises: cell proliferation, matrix maturation and matrix mineralization.

(Adapted from Aubin, J. E. et al.)

During the active proliferation phase, committed progenitor cells express genes that

support proliferation and several genes encoding for extracellular matrix proteins, such as

type I collagen and fibronectin. The accumulation of matrix proteins will contribute, in part,

to the cessation of cell proliferation. During the post-proliferative phase, characterized by

the high synthesis of alkaline phosphatase (ALP), the extracellular matrix progresses to

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the mineralization phase. Here, osteoblasts synthesize several proteins that are

associated with the mineralized matrix, including sialoprotein, osteopontin and osteocalcin

[14, 15, 16].

It is believed that each of the differentiation steps during osteoblastogenesis is

regulated by different kinds of morphogens, hormones, growth factors, cytokines and

ECM proteins [12, 13].

1.1.2.2 Osteoclasts

Osteoclasts are mobile multinucleated cells that arise from precursor cells in the CD14+

monocyte-macrophage lineage [1, 23]. The osteoclast precursor cells reach the bone

through the blood circulation and fuse into multinucleated cells to form mature osteoclasts

in a process called osteoclastogenesis. Inactive osteoclasts adhere to bone surface

through specialized adhesion structures called podosomes, forming a sealing zone sealed

by actin filaments together with αvβ3 integrin, the vitronectin receptor (VNR) [20-22].

Once formed, the resorption lacuna, or Howship’s lacuna, is acidified by the export of

hydrogen ions mediated by H+-ATPase located in osteoclasts plasma membrane. These

protons are derived from the carbonic acid formed by solubilization of CO2 by carbonic

anhydrase II (CA2). Thus, mineral phase is dissolved by H+ action, producing and

releasing into the resorption lacuna calcium, water and phosphate, according to the

following equation [9, 20, 21, 75].

[Ca3(PO4)2]3Ca(OH)2 + 8 H+ → 10 Ca2+ + 6 HPO4+ 2 H2O

Hydroxyapatite (solid) (solution)

The process continues with the degradation of the organic phase, being carried out

through the action of lytic enzymes such as tartarate resistant acid phosphatase (TRAP)

and cathepsin K (CATK), both secreted by osteoclasts (Fig. 3) [9, 20, 21].

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Figure 3. Functional elements of the mature osteoclast. Osteoclasts adhere to bone via

podosomes containing αvβ3 integrin. Protons generated by action of CA2 are transported into the

resorption lacuna by the vacuolar-type H+-ATPase. CATK is an important enzyme for the removal

of the organic components of bone in the resorption lacuna. (Adapted from Tolar, J. et al.)

1.1.3 Osteoclastogenesis

Osteoclastogenesis is a complex process regulated by different cells, such as

osteoblasts and bone marrow stromal cells, responsible for the production of many

modulators of the process, including the two key protein factors that shape osteoclast

differentiation and activation, namely macrophage colony stimulating factor (M-CSF) and

receptor activator of nuclear factor kB ligand (RANKL) [1, 2, 23].

M-CSF is a homodimeric glycoprotein produced by monocytes, granulocytes,

endothelial cells, osteoblasts, fibroblasts, lymphocytes B and T, and some tumor cell lines.

M-CSF promotes the formation of granulocytes and macrophages colonies and acts on

osteoclastogenesis at an early stage in the fusion and further differentiation of monocytes

[19, 24-26]. RANKL is a homotrimeric protein member of the tumor necrosis factor (TNF)

family that stimulates differentiation and activation of osteoclasts by linkage to RANK

receptor on the surface of precursor cells. RANKL is produced by osteoblasts but it can

also be synthesized by fibroblasts, lymphocytes T and bone marrow cells.

The osteoclastogenic process may, however, be negatively regulated by

osteoprotegerin (OPG) which, acting as a soluble receptor to RANKL, prevents RANKL-

RANK binding, decreasing its bioavailability [1, 19, 27-30].

M-CSF and RANKL are, by themselves, enough to induce the expression of several

osteoclastic features, such as the reorganization of the cytoskeleton with formation of

actin rings that establish the contact with the resorption lacuna, production of TRAP,

enzyme used as a marker of osteoclastic activity, synthesis of VNR, responsible for the

adhesion process, and calcitonin receptor (CTR) of which the ligand is an inhibitory

hormone of the osteoclastic activity, preserving bone mass (Fig.4) [1, 31].

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Figure 4. Osteoclastogenesis. M-CSF and RANKL act in crucial phases of osteoclastogenesis,

being able to induce the expression of several osteoclastic characteristics. (Adapted from Feng, X.)

Upon resorption, bone-stored growth factors, such as bone morphogenetic proteins

(BMPs), insulin-like growth factors (IGFs), and transforming growth factor-betas (TGF-β),

are released and activated. These local factors, along with the systemic factors

parathyroid hormone (PTH), estrogens, and prostaglandins, are constantly released in the

bone marrow cavity by osteoclastic bone resorption, and recruit new osteoblasts to fill the

gap created by osteoclasts [47].

1.1.4 Hormonal control

PTH (parathyroid hormone) is a peptide produced by the parathyroid glands that is

important in the maintenance of normal calcium homeostasis due to its stimulatory effects

on osteoclastic bone resorptive activity and renal reabsorption of calcium [32].

Parathyroid hormone related protein (PTHrP) binds PTH receptor and activates adenyl

cyclase to produce cyclic adenosine monophosphate (cAMP). The effects of PTH and

PTHrP on osteoclasts and precursors were studied in vivo. These studies demonstrated

that neither PTH nor PTHrP had an effect on early osteoclast precursors, but increased

the number of committed mononuclear osteoclast progenitors as well as mature

osteoclasts [32].

However, the primary target cell for PTH appears to be the osteoblast. PTH binds to

osteoblasts stimulating osteoblasts to increase their expression of RANKL and to inhibit

their expression of OPG. Isolated osteoclasts seem to not resorb bone in response to

PTH and only do so when osteoblasts or osteoblastic cell lines are added to the cultures.

Furthermore, PTH induces RANKL expression by marrow stromal cells. On the other

14

hand, PTHrP stimulates bone formation by enhancing osteoblast differentiation and

reducing osteoblast apoptosis (Fig. 5) [33].

Figure 5. Regulation of Bone Resorption. Both systemic factors and locally acting factors induce

the formation and activity of osteoclasts. Systemic hormones such as parathyroid hormone, 1,25-

dihydroxyvitamin D 3, and thyroxine (T 4) stimulate the formation of osteoclasts by inducing the

expression of RANKL on marrow stromal cells and osteoblasts. In addition, osteoblasts produce

interleukin-6 (IL-6), interleukin-1 (IL-1), prostaglandins, and colony-stimulating factors (CSFs),

which induce the formation of osteoclasts. (Adapted from Roodman, G. D.)

1.1.5 Signaling pathways

Osteoclastogenesis is an extremely complex process dependent on the action of

several hormones, growth factors, and others, therefore involving multiple intracellular

signaling pathways.

Several signaling pathways are activated by RANKL mediated protein kinase signaling.

Some of them mediate directly osteoclastogenesis and others osteoclast activation. The

intracellular domain of RANK contains three binding domains for TNF receptor-associated

factors (TRAF). TRAF2, -5 and -6 have been shown to bind to RANK and studies revealed

that TRAF6 is indeed an essential adaptor required for RANK-associated signaling (Fig. 6)

[10, 34].

Among the major types of signal transduction pathways in eukaryotic cells are protein

kinase cascades which culminate in the activation of a family of protein kinases known as

15

mitogen activated protein kinases, or MAP kinases. Although they vary in their substrate

specificities and responses to extracellular stimuli, MAP kinases are highly conserved in

their primary structure and mode of activation. All MAP kinases are serine/threonine

kinases that are activated in response to their phosphorylation on invariant threonine and

tyrosine residues [36]. The mitogen activated protein kinase and extracellular signal

regulated kinase (MAPK/ERK) pathway triggers intracellular responses through binding of

growth factors to surface receptors promoting osteoclastic proliferation and differentiation

[35].

Recently, Miyazaki et al. reported that ERK is involved in survival of osteoclasts by

showing that inhibition of ERK activity induced the apoptosis of osteoclast-like cells while

ERK activation remarkably lengthened their survival by preventing spontaneous

apoptosis. In short, results show that ERK is localized in the clear zones of osteoclasts. It

seems that both the prevention of ruffled border formation and the acceleration of

apoptosis in osteoclasts are associated with a decrease in phosphorylated ERK. Results

suggest that ERK in osteoclasts may be involved in their survival, motility, and cell polarity

[35].

Nuclear factor kappa B (NF-kB) is a family of five transcription factors that are

expressed in most cell types. NF-κB dimers are normally inactive in the cytosol. Activation

of this transcription factor is a consequence of phosphorylation of its inhibitory protein, IκB

proteins (IκBa, IκBb, IκBe, IκBg, Bcl-3), by an upstream kinase complex termed IκB kinase

(IKK), resulting in the release and translocation of NF-κB to the nuclear compartment.

Once in the nucleus, they attach to κB binding sites in the promoter region of a large

number of genes including IL-1, IL-6, TNFα-, and granulocyte macrophage colony-

stimulating factor (GMCSF) and, after stimuli-induced IκB protein degradation in response

to extracellular signals, induce their transcription [37]. Thus, NF-kB pathway regulates the

expression of various genes essential to inflammatory and immune responses, by

regulating the expression of a variety of pro inflammatory cytokines and other

inflammatory mediators, cellular survival and proliferation. It is crucial for

osteoclastogenesis in terms of differentiation of monocytic precursors into osteoclasts and

further activation such as the C-jun N-terminal kinase (JNK) pathway, which acts

mobilizing a series of osteoclastogenic transcription factors [5, 6, 8]. JNK is a subgroup of

the MAPK family and is also known to be activated by cell stressors such UV radiation

[36].

Other relevant signaling pathways are protein kinase C (PKC) and stress-activated

protein kinase (p38). PKC pathway when activated induces cellular proliferation, apoptosis

16

and actin remodeling. Stimulation of the stress-activated protein kinase, p38, results in the

downstream activation of the microphthalmia associated transcription factor (MITF), which

controls the expression of the genes encoding TRAP and CATK, required for osteoclastic

function [4, 7].

Figure 6. Signaling pathways involved on osteoclastogenic differentiation. Binding of RANKL

to its receptor RANK induces various intracellular signaling cascades, such as MAPK (JNK, ERK,

MAPK 14) and NF-κB. Several transcription factors have been found crucial for osteoclast

differentiation downstream of RANKL/RANK signaling. (Adapted from Edwards, J. R. and Mundy,

G. R.)

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1.2 Breast cancer

1.2.1 Epidemiology and pathology

Breast cancer is by far the most frequent cancer among women (small occurrence in

men) with an estimated 1.38 million new cancer cases diagnosed in 2008 (23% of all

cancers), and ranks second overall (10.9% of all cancers). It is now the most common

cancer both in developed and developing regions and it is second only to lung cancer as a

cause of cancer mortality.

It is estimated that in the year of 2008, the incidence rate of breast cancer in Portugal

was 60 per 100,000 and the mortality rate of 13.5 per 100,000 [GLOBOCAN 2008 IARC].

Ninety-five percent of breast cancers are carcinomas. Carcinomas can be divided into:

in situ carcinoma and invasive or infiltrating carcinoma, and these may arise either in

ducts or lobules. Most carcinomas show ductal differentiation (90%). Other malignant

lesions are associated with mesenchymal neoplasms, such as cystosarcoma phyllodes,

angiosarcoma, among others [38, 39].

Figure 7. The female breast. The anatomic structures and lesions of the female breast.

(http://medicinembbs.blogspot.pt/2010/12/breast-anatomy.html)

Benign lesions include hyperplasia, cystic changes, adenomas, papillomas and

fibroadenomas, the most common benign tumor of the breast. Pathologies of the adipose

tissue can also occur, called traumatic fat necrosis (Fig. 7) [38, 39].

18

1.2.2 Risk factors

There are several risk factors considered for the development of breast cancer.

Obviously, the age and the occurrence of a prior breast cancer or benign lesion are two of

them, but there is also a number of lifestyle, environmental and hormonal factors that are

probably involved (unbalanced diet, lack of exercise, early menarche, hormone

replacement therapy, nulliparity, …) [38, 39,40].

Women with a family history of breast cancer should obtain as much information as

possible about those relatives, including age at onset and type of cancer. The risk of

breast cancer development related to family history increases with the number of affected

relatives, specific lineage and age at diagnosis [38, 39, 40].

About 5-10% of breast cancer is thought to be linked to changes in certain genes. The

most common are those of the BRCA1 and BRCA2 genes. Women with mutations in

these genes have a higher risk of developing breast and ovarian cancer during their lives

[38, 39, 40].

Despite advances, 20-30% of patients with early breast cancers experience relapse

with distant metastatic disease. Risk of recurrence is influenced by stage at initial

presentation and the underlying biology of the tumor. Tumor size, nodal involvement,

grade, lymphovascular invasion, and estrogen receptor (ER) and human epidermal growth

factor receptor 2 (HER2/erB2) status are all independent risk factors for relapse [38, 39,

59].

1.2.3 Molecular types of breast cancer

The heterogeneity of breast cancers makes them both a fascinating and challenging

solid tumor to diagnose and treat.

Until very recently, personalized cancer medicine in breast cancer relied on only two

predictive markers, ER and HER2/erB2. Breast cancer is now recognized not as a single

disease with variable morphology, but as at least four molecularly distinct neoplastic

disorders: luminal-A breast cancer (low proliferative ER positive), luminal-B breast cancer

(high proliferative ER positive), HER2-positive breast cancer (amplification and high

expression of the ERBB2 gene and several other genes of the ERBB2 amplicon) and

triple-negative or basal-like breast cancer (ER/progesterone receptor negative and HER-

2-negative) (Fig. 8) [41, 42].

19

Although the immediate additional clinical value of this molecular classification is limited

by its close correlation to traditional methods of testing for ER and HER2, the identification

of genetic aberrations that underlie molecularly distinct subtypes of breast cancer has

revealed new therapeutic targets and has reshaped breast cancer clinical trial design [41,

42].

Figure 8. Molecular types of breast cancer. To distinguish the molecular type of cancer, first

HER2 membrane receptor must be assessed and only after test for nuclear hormone receptor. Ki

67 denotes nuclear antigen ki-67, a marker of cell proliferation. (Adapted from Sotiriou, C. and

Pusztai, L.)

Estrogen receptor/progesterone receptor positive (ER/PR+) tumors have a better

prognosis and a better response to treatment than receptor-negative tumors. Two thirds of

women with diagnosed breast cancer have ER/PR+ tumors. These tumors are highly

responsive to anti-estrogen therapeutic strategies. However, despite the widespread use

of hormonal adjuvant therapy, a quarter of women with ER+ disease will relapse. Since

the earliest studies of the intrinsic molecular subtypes in breast cancer, the defining

feature of luminal- B breast cancer has been its poor outcome compared with the luminal-

A subtype. Several studies have suggested that luminal-B breast cancer is relatively

insensitive to endocrine therapy compared with luminal-A [43, 49].

HER2 is over expressed in 15-20% of breast cancer, and it is associated with a highly

aggressive behavior. HER family is constituted by four members, HER1, HER2, HER3

and HER4. An intracellular tyrosine kinase domain exists for HER1, HER2 and HER4 and

phosphorylation of these domains by homodimerization or heterodimerization induces

both cell proliferation and survival signaling [61]. HER2 is the preferred dimerization

Breast Cancer

HER2 Negative

ER/PR Negative

Basal like

ER/PR positive

Good differentiation low Ki 67

Luminal A

Poor differentiation high Ki 67

Luminal B

HER2 Positive

ER positive or ER negative

HER2 like

20

partner for the other HER family members. One of the other downstream effects of these

reactions is the production of vascular endothelial growth factor (VEGF) supporting

angiogenesis [58].

The availability of the anti-HER2 monoclonal antibody therapy has significantly improved

the prognosis of patients with HER2 positive breast cancer both in early and advanced

disease stages.

Triple-negative breast cancers in particular are difficult to define. This tumor subgroup

does not respond to hormonal therapies or Her2-targeted therapies due to the lack of

expression of these targets. These tumors are associated with a poor prognosis.

The subtypes most in need of therapeutic advances are triple negative breast cancer

and luminal-B breast cancer, where therapeutic resistance is common and where

advances in molecular profiling have identified promising new therapeutic targets [43, 49].

1.2.4 Therapies

Breast cancer surgery has changed dramatically over the past 20 years. With the

emergence of breast conserving therapy, many women now have the option of preserving

a cosmetically acceptable breast without sacrificing survival. Conserving therapy refers to

surgical removal of the tumor without removing excessive amounts of normal breast

tissue. The aims of this therapy are to provide a cancer operation equivalent to

mastectomy and a cosmetically acceptable breast, with a low rate of recurrence in the

treated breast. The main obstacle to widespread acceptance and utilization of conserving

therapy is the risk of in-breast recurrence. Depending of several factors, such as tumor

size, multifocality and possibility of radiotherapy used, mastectomy may be the method of

choice. Regardless of the method used, axillary lymph node dissection is always

mandatory for evaluation [39, 40].

Radiation adjuvant therapy is known to substantially reduce the risk of recurrence and

decrease breast cancer mortality, both when given after mastectomy and after breast-

conserving surgery. Radiation therapy is generally given over a 5- or 6-week time span,

with care taken to try to avoid damage to the heart or lungs. The only usual changes with

breast radiation are skin erythema and possibly some transient lymphedema [39].

Adjuvant systemic therapy refers to the administration of chemotherapy, hormone

therapy and/or monoclonal antibodies used following primary surgery for breast cancer.

The purpose is to eliminate or delay the subsequent appearance of clinically occult

21

micrometastases. The original regimen of chemotherapy used was cyclophosphamide,

methotrexate, 5-fluorouracil (CMF). Thereafter, many other regimes have been used. A

major determinant of the choice of adjuvant therapy is whether an individual breast cancer

expresses hormonal receptors (HR). Hormone therapy benefits patients with hormone

receptor-positive breast cancer, but not those with hormone receptor negative disease

[39].

Targeting the ER is the oldest molecular targeted therapy approach, and the

widespread use of the selective ER modulator tamoxifen in breast cancer is responsible

for major improvements in cure rates, quality of life and disease prevention for the past 25

years [39, 40].

Tamoxifen, a selective estrogen receptor modulator (SERM), inhibits the growth of

breast cancer cells by competitive antagonism of estrogen at the ER, inhibiting both

translocation and nuclear binding of the ER. Tamoxifen and its metabolites bind to the ER,

and subsequently occurs translocation of this complex to the nucleus and binding to the

estrogen-response element. This binding prevents transcriptional activation of estrogen-

responsive genes (Fig. 8). However, its actions are complex and it has also a partial

estrogen agonist activity. Additionally, there are marked differences between the anti

proliferative properties of tamoxifen and its metabolites [40, 57].

Their long-term efficacy, however, is limited by relapse of disease and development of

resistance. Laboratory and clinical data have demonstrated that ER-positive breast

cancers that overexpress HER2 are associated with resistance to tamoxifen and to

hormonal therapy in general. Despite continuous expression of ER at relapse in either

locally recurrent or secondary metastatic tumors, up to half of patients with HR-positive

primary breast cancer who develop metastatic disease do not respond to first-line

endocrine treatment (de novo resistance), and the remainder will eventually relapse

despite an initial response (acquired resistance) [40].

Several factors may contribute to tamoxifen resistance in breast cancer, including

variable expression of estrogen receptor alpha and beta isoforms, interference with

binding of co-activators and co-repressors, alternatively spliced ER mRNA variants and

ER modulators expression, such as epidermal growth factor (EGF) and its receptor

(EGFR1, also called HER1), as well as the type 2 EGFR, also called HER2. Emerging

studies also suggest that relative resistance to tamoxifen may be related to inheritance of

22

certain drug metabolizing CYP2D6 genotypes that are associated with a reduced

activation of tamoxifen to its active metabolite endoxifen [40, 44, 60].

Other hormonal therapeutic agents include aromatase inhibitors that interfere with the

enzyme aromatase, which plays a critical role in the production of estrogen in

postmenopausal women. Aromatase is an enzyme that naturally converts estrogen from

androgen. In premenopausal women, most of the estrogen is produced in the ovaries, but

in postmenopausal women, most estrogen is synthesized in peripheral tissue from

conversion of androgens. In contrast to tamoxifen, these compounds lack partial agonist

activity (Fig. 9). Examples of this class include anastrozole, letrozole and exemestane [40,

45].

Figure 9. Comparison of the mechanisms of action of estradiol, tamoxifen and aromatase inhibitors.

Estradiol binds to ER, leading to dimerization, conformational change and binding to estrogen response

elements (ERE) upstream of estrogen responsive genes including those responsible for proliferation.

Tamoxifen competes with estradiol for ER binding whereas Aromatase inhibitors reduce the synthesis of

estrogen from androgenic precursors. (Adapted from Johnston, S. R. D. and Dowsett, M.)

Approximately 25-30% of breast cancer tumours display amplification of the HER2

gene or overexpression of its protein product, and this is associated with an adverse

prognosis. Trastuzumab is a humanized monoclonal antibody directed against the

external domain of the receptor with clinical activity as a single agent in patients whose

cancers overexpress HER2 (Fig. 10). Overexpression of the receptor is associated with

increased disease recurrence and worse prognosis. Targeting both HER2 with

23

trastuzumab and VEGF with bevacizumab in combination with chemotherapy has become

a further milestone of molecular targeted therapy. However, intrinsic and acquired

resistance to endocrine and/or cytostatic treatments is still a common feature that limits

the benefits of these novel therapeutic strategies. In tumors that are HER2 and ER

positive, HER2 signaling is dominant. This resistance can be partially overcome by

combining anti-estrogen and anti-HER2 therapies [40, 57, 61].

Figure 10. Potencial mechanisms of action of transtuzumab. Cleavage of the extracellular

domain of HER2 leaves a membrane-bound phosphorylated p95, which can activate signal-

transduction pathways. Binding of trastuzumab to a juxtamembrane domain of HER2 reduces the

release of the extracellular domain, thereby reducing p95. Trastuzumab may reduce HER2

signaling by physically inhibit homodimerization or heterodimerization. Trastuzumab may also

recruit immune effector cells and other components of antibody-dependent cell-mediated

cytotoxicity, leading to tumor-cell death. Other mechanisms such as receptor down-regulation

through endocytosis have been suggested. (Adapted from Hudis, C. A.)

24

1.3 Bone metastasis in breast cancer

1.3.1 Bone metastasis

Most patients with cancer die not because of the primary tumour, but rather because it

has spread to other sites. It is difficult to determine, precisely, how frequently different

tumours metastasize to bone. Patients with advanced breast and prostate cancers almost

always develop bone metastases. Moreover, the chances are high that, in patients who

are originally diagnosed with breast or prostate cancers, the bulk of the tumour burden at

the time of death will be in bone [46]. Metastasis to bone occurs in the late stages of

tumor progression, in a multistep process [50, 51].

Because tumor metastasis is a complex process involving individual discrete steps,

interruption of one or more of these steps can inhibit the metastatic process. Each of

these steps represents cellular interactions caused by specific determinants of both the

tumor and the tissue. The steps involved in the shedding of the tumor cells from the

primary site involve detachment of tumor cells from adjacent cells, followed by invasion of

adjacent tissue in the primary organs. The cells then enter the tumor capillaries

(stimulated by specific angiogenesis factors produced by the tumor) and via these

capillaries reach the general circulation. The steps involved in entering the tumor blood

vessels at the primary site are similar to those involved in exiting the vasculature in the

bone marrow cavity. These steps include the attachment of the tumor cells to basement

membrane, the secretion of proteolytic enzymes that enable tumor cells to disrupt that

membrane, and the migration of the tumor cells through it. Tumor cells that metastasize to

the skeleton adhere to the endosteal surface and colonize bone (Fig. 11) [50, 51].

Figure 11. The steps involved in tumour-cell metastasis from a primary site to the skeleton.

The primary malignant neoplasm promotes new blood-vessel formation, and these blood vessels

25

carry the cancer cells to capillary beds in bone. Aggregates of tumour cells and other blood cells

eventually form embolisms that arrest in distant capillaries in bone. These cancer cells can then

adhere to the vascular endothelial cells to escape the blood vessels. As they enter the bone, they

are exposed to factors of the microenvironment that support growth of metastases. (Adapted from

Mundy, G. R.)

Although the distribution of metastases in distant organs can be predicted by the

anatomic distribution of blood flow from the primary site in 30% of cases, specific

properties of the tumor cell and features at the metastatic site determine where the

metastasis occurs in the majority of cases [51].

The metastasis of tumor cells to specific sites in the skeleton is not a simple and

random event determined solely by blood flow. Rather, it is a complex process that is

dependent on specific properties of the tumor cells and on factors in the bone

microenvironment that favor metastasis. Tumor cells most frequently affect the heavily

vascularized areas of the skeleton, particularly the red bone marrow of the axial skeleton

and the proximal ends of the long bones, the ribs, and the vertebral column [51].

Physical factors within the bone microenvironment, including low oxygen levels, acidic

pH, and high extracellular calcium concentrations, may also enhance tumor growth.

Hypoxia is a major contributor to tumor metastasis, regulating secreted products that

drive tumor-cell proliferation and spread. Hypoxia also contributes to resistance to

radiation and chemotherapy in primary tumors. Solid tumors are particularly prone to

hypoxia because they proliferate rapidly, outgrowing the malformed tumor vasculature,

which is unable to meet the increasing metabolic demands of the expanding tumor.

Hypoxia regulates normal marrow hematopoiesis and chondrocyte differentiation. Cancer

cells capable of surviving at low oxygen levels can thrive in the hypoxic bone

microenvironment and participate in the vicious cycle of bone metastasis [50].

Acidosis of the bone microenvironment also potentiates the vicious cycle of bone

metastasis. Extracellular pH is tightly regulated within bone and has significant effects on

osteoblast and osteoclast function with osteoclasts being maximally stimulated at pH

levels of <6.9. Osteoblast mineralization and bone formation is significantly affected by

acid. Tumor metastasis leads to localized regions of acidosis within the skeleton.

Increased glycolysis and lactic acid production by proliferating cancer cells (due to the

hypoxic conditions) and decreased buffering capacity of the interstitial fluid contribute to

26

the acidic microenvironment within primary tumors. The acid-mediated tumor invasion

hypothesis states that altered glucose metabolism in cancer cells stimulates cancer cell

proliferation resulting in a more invasive tumor phenotype. Acidosis alters the cellular

dynamics at the interface between the tumor and normal tissue, promoting apoptosis in

adjacent normal cells and facilitating extracellular matrix degradation through the release

of proteolytic enzymes. Unlike normal cells, cancer cells have compensatory mechanisms

to allow proliferation and metastasis even at low extracellular pH, being resistant to acid-

induced apoptosis [50].

Calcium released from the mineralized bone matrix contributes to the vicious cycle of

metastasis by several mechanisms. Calcium is the primary inorganic component of the

bone matrix and, in the bone microenvironment, levels are maintained within a narrow

physiologic range. Active osteoclastic bone resorption causes extracellular calcium levels

to rise up. The effects of Ca2+ are mediated through the extracellular calcium-sensing

receptor (CaSR), a G protein–coupled receptor, which, in the presence of high Ca2+,

inhibits cyclic AMP and activates phospholipase C. The CaSR is expressed in normal

tissues and is overexpressed in several types of cancer, including breast and prostate

cancer and it regulates the secretion of PTHrP [50].

Finally, estimates of the frequency of metastasis depend also on the sensitivity of the

diagnostic technique utilized to detect them. The most commonly used techniques are

bone scans, X-rays, and histologic evaluation of autopsy specimens. Both radiologic and

histologic assessments are limited by the extent of the sample evaluated. Bone scans

survey the whole body. However, because isotope accumulation depends on osteoblast

activity, lesions with very little osteoblastic component cannot be detected [51].

1.3.2 Types of bone metastases

There are different patterns of bone effects in patients with cancer, ranging from purely

or mostly destructive, designated by osteolytic metastases, to mostly bone-forming or

osteoblastic metastases [46]. However, patients can have both osteolytic and osteoblastic

metastasis or mixed lesions containing both elements. Both osteoblastic and osteolytic

bone metastases lead to numerous skeletal complications, including bone pain,

hypercalcemia, pathologic fractures, and spinal cord and nerve compression syndromes.

Such complications increase morbidity and diminish the quality of life in these patients [47,

63].

27

Breast cancer is one of the most frequent tumours that originate bone metastasis, being

the most common the osteolytic ones. However, 15 to 20 percent of the patients have

predominantly osteoblastic lesions. Moreover, secondary formation of bone occurs in

response to bone destruction. This reactive process makes it possible to detect the

osteolytic lesions by scanning techniques, identifying the sites of active bone formation

[47]. This increase in bone formation in patients with osteolytic lesions can be also

reflected by the increase of serum ALP levels, a marker of osteoblasts activity. In contrast,

the lesions in prostate cancer are predominantly osteoblastic.

In breast cancer, osteolytic bone metastasis results from a hiperactivation of

osteoclasts induced directly or indirectly by several factors produced by cancer cells.

Osteoblastic metastases, conversely, are believed to be caused by cancer-cell production

of factors that stimulate osteoblast proliferation, differentiation and bone formation [46,

63].

1.3.2.1 Osteoblastic metastases

The mechanisms of osteoblastic metastasis and the factors involved are unknown.

Endothelin-1 has been implicated in osteoblastic metastasis from breast cancer. This

molecule is one of the most well studied mediators that stimulates bone formation and

osteoblast proliferation in bone organ cultures. Endothelin-1 levels are increased in the

circulation of patients with osteoblastic metastases and prostate cancer, being also

expressed by breast cancer cell lines that cause osteoblastic metastases. Osteoblast

proliferation and bone metastasis have both been shown to be inhibited in vivo by

endothelin-1A-receptor antagonists. In addition to endothelin-1, platelet-derived growth

factor (PDGF), a polypeptide produced by osteoblasts in the bone microenvironment,

urokinase, and prostate- specific antigen may also be involved [47, 93].

1.3.2.2 Osteolytic metastases

Breast cancer cells produce factors that direct or indirectly induce osteoclasts

formation. In turn, bone resorption carried out by osteoclasts release growth factors from

bone matrix which stimulate tumor growth and bone destruction. This reciprocal

interaction between breast-cancer cells and the bone microenvironment results in a

vicious cycle that increases both bone destruction and the tumor burden [47, 64].

Bone is an abundant source of inactive growth factors which are activated during the

resorption process, including TGF-β, IGFs I and II, fibroblasts growth factors (FGFs),

28

PDGFs, BMPs and calcium. These factors are then released providing a fertile ground in

which tumour cells can grow. On the other hand, breast cancer cells and many of other

solid tumours are thought to stimulate the formation of osteoclasts by the production of

PTHrP. This molecule, in association with other factors produced by tumor cells, such as

IL-6, prostaglandin E2 (PGE2), TNF-α (tumour necrosis factor α) and M-CSF, increase the

expression of RANKL mainly by osteoblasts, which directly acts on osteoclast precursors

promoting osteoclastogenesis and bone resorption (Fig. 12) [46, 47, 63, 64].

Figure 12. The Vicious Cycle of Osteolytic Metastasis. Breast-cancer cells, secrete PTHrP as

the primary stimulator of osteoclastogenesis. In addition, tumor cells produce other factors that

increase the formation of osteoclasts (IL-6, PGE2, TNF-α, and M-CSF). All these factors increase

the expression of RANKL. The process of bone resorption releases factors such as TGF-b,IGFs,

FGFs, PDGF, and BMPs, which increase the production of PTHrP by tumor cells as well as growth

factors that increase tumor growth. This symbiotic relationship between bone destruction and tumor

growth further increases bone destruction and tumor growth. (Adapted from Roodman, G. D.)

Two pieces of evidence support the view that osteoclastic bone resorption is the

predominant mechanism of bone destruction in bone metastasis: histological analysis and

scanning electron microscopy studies invariably detect tumor cells adjacent to osteoclasts

resorbing bone, and the use of bisphosphonates, potent inhibitors of osteoclastic bone

resorption, in women with breast cancer metastases to bone results in reduced skeletal

morbidity [51].

1.3.3 Molecular types of breast cancer and metastatic behavior

It is known that estrogen-responsive breast cancer cells have a more aggressive bone

metastatic behavior than their estrogen receptor-negative counterparts which metastasize

more aggressively to visceral organs (liver, lung, pleura…). There are differences in the

29

anatomic sites of relapse according to molecular subtypes. The increased incidence of

brain metastases in HER2-positive and basal like breast cancer is well recognized.

Luminal breast cancers appear to have a predilection for metastasis to bone and pleura.

In a small study of patients with metastatic breast cancer, no differences in sites of

metastasis were observed between luminal-B and luminal-A breast cancers.

1.3.4 Treatment

Bisphosphonates (BPs) are a class of pyrophosphate analogues that bind with high

affinity to mineralized bone surfaces and inhibit osteoclastic bone resorption. Common

examples of these drugs are pamidronate (Aredia), alendronate (Fosamax), zoledronate

(Zometa), risedronate (Actonel) and clodronate (Bonefos) [46]. Bisphosphonates are used

extensively to treat patients with diseases associated with bone loss, such as

osteoporosis, Paget’s disease and osteolytic metastasis [54, 55].

Bisphosphonates inhibit both bone lesions and tumor cell burden in bone in

experimental models of breast carcinomas. They act by decreasing the recruitment,

proliferation and differentiation of pre-osteoclasts, their adhesion to the mineralized matrix

and, most importantly, the resorptive activity of mature osteoclasts. They also reduce the

release of bone derived tumor growth factors and shorten the osteoclast lifespan by

induction of programmed cell death (Fig. 13) [54, 55].

Figure 13. Inhibitory action of bisphosphonates in the vicious cycle of osteolytic metastasis.

(Adapted from Rogers, T. L.)

In addition, there is extensive evidence that these agents exert direct antitumor effects in

vitro, inhibiting tumor cell adhesion, invasion, proliferation and angiogenesis, and inducing

apoptosis.

30

Because BPs home to bone very quickly due to their high affinity for hydroxyapatite,

they are present in the skeleton for prolonged periods.

However, BPs therapy is only for palliative treatment and does not provide a life

prolonging benefit in the majority of the patients. New therapeutics targeting osteoclast

activities are therefore required. They could be used alone or in combination with BPs to

treat more efficiently breast cancer patients with bone (osteolytic) metastases [56].

Newer bone resorption inhibitors are currently in clinical development. These new

therapeutics include OPG, anti-PTHrP antibodies, vitamin D analogues (to decrease

PTHrP production) and RANK-Fc, a hybrid chimeric molecule that acts in an identical way

to OPG [46, 56].

The use of CATK inhibitors has been investigated as well. CATK inhibitors are

successfully used in the treatment of bone loss associated with osteoporosis. This

protease is also expressed by human breast cancer cells in skeletal metastases,

suggesting that CATK inhibitors could target not only osteoclasts but also tumour cells.

Preclinical studies show some evidence that these inhibitors, alone or in combination with

bisphosphonates, reduce breast cancer–induced osteolysis and skeletal tumor burden in

animals [46, 56].

31

1.4 Proton pump inhibitors

Proton pump inhibitors (PPI) and histamine 2 receptor antagonists (H2RAs) are the

most popular acid-suppressive drugs (ASDs) available, and millions of individuals

currently take these medications on a continuous or long-term basis. These potent drugs

are used to treat various disorders, and indications for long-term maintenance therapy

with this drug class continue to expand [74]. First, the H2RAs were introduced, offering

patients the first single-agent therapy that effectively reduced gastric acid secretion. Then,

PPI became widely available in the early 1990s, and they generally appeared to be

superior to the H2RAs in their acid-suppressing activity, symptom control and healing [69].

1.4.1 Mechanism of action

PPIs are a class of drugs widely used in many pathological conditions, such as

gastroesophageal reflux, dyspepsia and peptic ulcers. Although there are several different

molecules belonging to this class, it is known that they share many functional and

structural properties as well as some pharmacokinetic characteristics. The mechanism of

action of PPIs relies on their chemical activation in a significant acidic environment and

consequent inhibition of H+/K+ATPases. Nevertheless, like any other medication, they

also display several side effects [65, 67].

.

Omeprazole was the first PPI used in clinical practice in the late 1980s. Other

analogues such as lansoprazole, rabeprazole and esomeprazole have been developed

since that (Fig. 14) [67].

Figure 14. Structures of PPIs. Timoprazole, omeprazole, lansoprazole and rabeprazole are

benzimidazole-based PPIs. Esomeprazole is an optical isomer of omeprazole (S-omeprazole).

(Adapted from Sachs, G. et al.)

32

PPIs are lipophilic and are inactive in the neutral environment of the bloodstream. After

absorption, PPIs cross the plasma membrane, enter and accumulate in the secretory

canaliculi of parietal cells, where they are protonated by acid and then converted into their

active form, sulfenamide. The activated sulfenamide reacts covalently with the cysteine

residues on the extracellular surface of the proton pump, inhibiting gastric acid secretion

(Fig. 15) [67].

Figure 15. PPIs activation. PPIs such as omeprazole concentrate in the acidic secretory

canaliculi of the parietal cell, where they are activated and generate a sulphenamide. The

sulphenamide interacts covalently with the sulphydryl goups of cysteine residues in the

extracellular domain of the ATPase thereby inhibiting its activity. (Adapted from Olbe, L. et al)

PPIs are very specific to the inhibition of gastric proton pumps because they are

activated only in the acidic environment of the stomach, whereas they are not activated by

the similar enzyme found in the colon and the kidney [67]. Differences in cysteine binding

properties among the PPIs may, at least partly, underlie the differences among them in

the duration of the inhibition of gastric acid secretion. Currently available benzimidazole-

based PPIs have similar half-lives of 1–2 h. As these agents are usually prescribed once

daily, this means that there is effectively no circulating PPI present at the end of the

dosing interval. A PPI with an extended half-life might have a prolonged anti-secretory

effect with consequent therapeutic advantages [65].

1.4.2 Adverse effects

As mentioned above, there are common adverse events that occur in 1-4% of patients.

The most common are headache, abdominal pain, gastrointestinal alterations (nausea

and diarrhea, for example), fatigue, community-acquired pneumonia, acute interstitial

nephritis and allergy-related problems. However, the knowledge of the adverse events of

newly marketed drugs is limited, and it is only after widespread clinical use that the side-

effect profile of a drug is performed more assertively. The presence of side effects on

33

proton pump inhibitors is greatest for omeprazole, since this drug has been on the market

for a longer period [68, 70, 73].

PPIs reduce gastric acidity, which is necessary to activate pepsinogen into pepsin,

which, by its turn is important for the beginning of the digestion of dietary proteins and to

release vitamin B12 from food. PPIs used for a short-term may minimally reduce the

absorption of protein-bound vitamin B12. Elderly patients who already have gastric

atrophy may present lower levels of vitamin B12 in serum with long term PPIs used.

However, studies have shown that PPIs used for a long-term in younger patients do not

reduce serum vitamin B12 concentrations. In patients long-term treated with high dose of

PPIs duodenal absorption of organic and non-organic iron may be reduced. Nevertheless,

this effect is small, and PPIs are not associated with an increased risk of latent iron

deficiency or iron deficiency [68, 70, 73].

For the proton pump inhibitors omeprazole, lansoprazole, pantoprazole and

esomeprazole, certain hepatic isoenzyme pathways, notably cytochrome P450

(CYP2C19), have an important role in the drug metabolism. In particular, among patients

who are genotypically rapid, or extensive, metabolizers, proton pump inhibitors with

significant CYP2C19 metabolism tend to yield lower plasma levels and, thus, have low

efficacy. The contribution of the CYP2C19 pathway varies in the metabolism of the

different proton pump inhibitors [69].

1.4.3 PPI’s versus H2RAs

Despite the efficiency of the action of the H2RAs, there were patients whose acid-

related disorders failed to respond to or required very high doses of these drugs.

Furthermore, the phenomenon of H2RA tolerance was recognized, with implications on

possible failures of chronic/maintenance H2RA therapy. PPIs were subsequently

developed, and, in most cases, have been found to be superior to H2RAs in their acid

suppressing ability. They also relieve oesophagitis symptoms and heal erosions more

effectively than H2RAs. In contrast to H2RAs or anticholinergic agents, which only

partially inhibit histamine-, gastrin- or acetylcholine- stimulated acid secretion, PPIs inhibit

acid secretion in response to all stimulatory agents (Fig. 16). Consequently, PPIs are

exceptionally effective for the control of acid production. Moreover, PPIs elevate pH above

3-4 4 for longer time periods than do H2RAs[69].

34

Figure 16. Gastric acid secretion by parietal cell. Gastric acid secretion is a process regulated

by three types of receptors on the parietal cell (histamine, gastrin and acetylcholine). Activation of

these receptors leads to activation of the gastric acid proton pump, H+K+-ATPase, which regulates

acid transport and is the final common pathway to acid secretion for these three receptors. In

contrast to H2RAs which only partially block these receptors, PPIs directly block the action of

H+K+-ATPase, suppressing gastric acid secretion, regardless of the stimulus. (Adapted from Olbe,

L. et al.)

1.4.4 Secondary effects of PPI in bone metabolism

Long-term PPI therapy and its attendant potent acid suppressive properties have

generated great concern regarding to the potential effects on calcium absorption and bone

metabolism. Several epidemiological studies have suggested an association between PPI

use and the risk of fractures of the hip, or other sites, including spine, wrists and forearms.

Some potential mechanisms by which PPI therapy may lead to fractures have been

identified. First, the small intestine’s ability to absorb ingested calcium depends on pH.

Without an acidic environment in the gastrointestinal tract, calcium may be retained in the

food matrix preventing absorption. Second, impaired calcium absorption may lead to

compensatory secondary hyperparathyroidism. Secondary hyperparathyroidism refers to

the increase in circulating levels of parathyroid hormone, which, in an attempt to rectify the

deficit in calcium absorption, increase the rate of osteoclastic activity [66, 72]. Finally, PPI

may directly interfere with osteoclasts activity [74]. Studies have shown that omeprazole

inhibits bone resorption in vitro and in vivo in humans by inhibiting osteoclastic vacuolar

H+ ATPase activity. A reduced bone resorption should lead to increases in bone density,

however, bone resorption is necessary for the normal bone development, replacing old

bone and repairing microfractures. Thus, a decrease in osteoclast activity can affect bone

structure and, thus, predispose patients to fractures [66, 70, 72, 74].

35

Taken together, these studies are limited by a low magnitude of association and

inability to assess potential confounding factors [79]. Despite all these reservations, it

seems likely that PPI may affect significantly bone metabolism, particularly in some

vulnerable individuals, such as postmenopausal women, where PPI use contributes to an

increased risk of fractures at various sites, in a manner related to the dose and duration of

exposure to the drug [66, 70, 74, 80].

In this context, since in the resorption process osteoclasts solubilize the bone mineral

phase through the secretion of acid to the resorption lacuna by proton pumps of the

vacuolar type, V-ATPases, PPIs appear as potentially pharmacological modulators of

osteoclastic activity, particularly in pathological situations characterized by hiperactivation

of osteoclasts such as osteolytic metastases associated with breast cancer.

36

2. Materials and methods

2.1 Cell Cultures

2.1.1 Isolation of Peripheral blood mononuclear cells (PBMC)

The cells were isolated from blood of male healthy donors with ages between 25 and

35 years old. The blood was diluted with PBS (phosphatase buffer solution) supplemented

with 2 mM EDTA (1:2) and applied on top of 4ml of Ficoll-PaqueTM PREMIUM (GE

Healthcare Bioscience). After centrifugation at 400 g for 30 minutes at room temperature,

PBMC were collected and washed twice with PBS supplemented with 2 mM EDTA with

centrifugations at 300 g for 10 minutes at 4ºC in each. PBMC were seeded at

1.5x106cells/cm2.

2.1.2 Breast cancer cell lines culture (T47D and SK-BR-3)

Human breast cancer cell lines, estrogen-receptor positive T47D and estrogen-negative

SK-BR-3, were maintained in α-Minimal Essential Medium (α-MEM) supplemented with

10% (v/v) fetal bovine serum, 100 IU/mL penicillin, 2.5 µg/mL streptomycin, 2.5 µg/mL

amphotericin B and 50 µg/mL ascorbic acid. Cultures were performed at 37ºC in a 5%

CO2 humidified atmosphere. At about 70-80% confluence, cells were detached with 0.05%

trypsin and 0.5 mM EDTA, and separately seeded at 104cells/cm2.

Cell cultures were maintained in α-MEM supplemented with 10% (v/v) fetal bovine

serum, 100 IU/mL penicillin, 2.5 µg/mL streptomycin, 2.5 µg/mL amphotericin B and 50

µg/mL ascorbic acid. Cultures were treated with different PPI (omeprazole, esomeprazole

and lansoprazole), at a concentration range of 10-9-10-3 M. Culture medium was changed

once a week and PPI were renewed at each medium change. Cell cultures were

maintained for 21 days in a 5% CO2 humidified atmosphere at 37ºC.

Cultures were assessed for cellular viability/ proliferation and apoptosis.

2.1.3 Co-culture of SK-BR-3 and T47D and PBMC

SK-BR-3 and T47D cell lines were separately seeded at 102 cells/cm2 and 104cells/cm2,

respectively. Cultures were incubated for 24h at 37ºC in α-MEM supplemented with 10%

(v/v) fetal bovine serum, 100 IU/mL penicillin, 2.5 µg/mL streptomycin, 2.5 µg/mL

37

amphotericin B and 50 µg/mL ascorbic acid. After that time, PMBC were added at

1.5x106cells/cm2.

Cell cultures were maintained in α-MEM supplemented with 30% (v/v) human serum

(from the same donor where PBMC were obtained), 100 IU/mL penicillin, 2.5 µg/mL

streptomycin, 2.5 µg/mL amphotericin B and 2 mM L-glutamine. Cultures were treated

with different PPI (omeprazole, esomeprazole and lansoprazole), at a concentration range

of 10-7-10-3 M. Culture medium was changed once a week and PPI were renewed at each

medium change. Cell cultures were maintained for 21 days in a 5% CO2 humidified

atmosphere at 37ºC.

Cultures were assessed for total protein content, TRAP activity, number of TRAP

positive cells, presence of multinucleated cells with actin rings and expressing VNR and

CTR by confocal laser scanning microscopy (CLSM) and calcium phosphate resorbing

ability. In order to assess the intracellular mechanisms involved, when indicated, the

medium was also supplemented with several signalling pathway inhibitors, namely, 1µM

U0126 (MAPK/ERK pathway inhibitor), 10µM PDTC (NF-kB pathway inhibitor), 5µM GO

6983 (PKC pathway inhibitor) and 10µM SP (JNK pathway inhibitor).

2.1.4 Human fetal osteoblastic cell line (hFOB) culture

The hFOB cells were cultured in Dulbecco’s Modified Eagle’s Medium/HAM’s Nutrient

Mixture F12 (DMEM/F12) supplemented with 10% (v/v) fetal bovine serum, 100 IU/mL

penicillin, 2.5 µg/mL streptomycin, 2.5 µg/mL amphotericin B, 50 µg/mL ascorbic acid and

0,6% gentamicin. Cultures were performed at 37ºC in a 5% CO2 humidified atmosphere.

At about 70-80% confluence, cells were detached with 0.05% trypsin and 0.5 mM EDTA,

and seeded at 104cells/cm2.

Cell cultures were maintained in DMEM/F12 supplemented with 10% (v/v) fetal bovine

serum, 100 IU/mL penicillin, 2.5 µg/mL streptomycin, 2.5 µg/mL amphotericin B, 50 µg/mL

ascorbic acid and 0,6% gentamicin, in absence and presence of 1% dexamethasone.

Cultures were treated with different PPI at a concentration range of 10-7-10-3 M. Culture

medium was changed once a week and PPI were renewed at each medium change. Cell

cultures were maintained for 21 days in a 5% CO2 humidified atmosphere at 37ºC.

Cultures were assessed for cellular viability/ proliferation, total protein content, ALP

activity and histochemical staining and visualized by CLSM. It was also carried out a

38

characterization of the intracellular mechanisms involved, for that, cells were seeded at

104cells/cm2 and maintained in DMEM/F12 as described above. When indicated, the

medium was also supplemented with several signalling pathway inhibitors, namely, 1µM

U0126 (MAPK/ERK pathway inhibitor), 10µM PDTC (NF-kB pathway inhibitor), 5µM GO

6983 (PKC pathway inhibitor) and 10µM SP (JNK pathway inhibitor).

2.2 Characterization of cell cultures

2.2.1 Total protein quantification

Cellular protein content was determined by Bradford’s method. Cell cultures were

washed twice with PBS and solubilized with 0.1 M NaOH. Samples were treated with

Coomassie® Protein Assay Reagent (Fluka) and incubated for 2 minutes at room

temperature. The absorbance was quantified at 595 nm in an ELISA plate reader (Sinergy

HT, Bioteck). Results were expressed as mg/mL.

2.2.2 TRAP activity quantification

TRAP activity was assessed by para-nitrophenilphosphate (pNPP) hydrolysis method.

Cell cultures were washed twice with PBS and solubilized with 0.1% (V/V) Triton X-100.

Samples were incubated for 1 hour at 37ºC with 10 mM pNPP prepared in 0.04 M tartaric

acid and 0.09 M citrate at pH 4.8. After incubation, the reaction was stopped with 5 M

NaOH and the absorbance was measured at 400 nm in an ELISA plate reader (Synergy

HT, Bioteck). Results were normalized with total protein content and expressed as

nmol/min/µgprotein.

2.2.3 Histochemical staining of TRAP

At days 14 and 21, cell cultures were washed twice with PBS and fixed with 3.7% (v/v)

formaldehyde for 15 minutes at room temperature. After new wash with PBS, cells were

stained for TRAP with Acid Phosphatase Leukocyte (TRAP) kit (SIGMA), according to

manufacturer’s instructions. Shortly, cells were incubated with naphtol AS-BI 0.12 mg/mL,

in the presence of 6.76 nM tartrate and 0.14 mg/mL Fast Garnet GBC, for 1 hour at 37ºC

in the dark. After incubation, cell layers were washed, stained with hematoxilin and

visualized by light microscopy. TRAP positive multinucleated cells (purple/dark red) were

counted.

39

2.2.4 ALP activity quantification

ALP activity was also determined by the pNPP hydrolysis method used to TRAP activity

quantification. Here, after solubilization with 0.1% (V/V) Triton X-100, samples were

incubated with 10 mM pNPP in a 0.15 M bicarbonate buffer in 2.5 mM MgCl2 at pH 10.

The procedure was the same as described for TRAP activity.

2.2.5 Histochemical staining of ALP

Cell cultures were washed twice with PBS and fixed with 1.5% glutaraldehyde in

cacodylate buffer, for 10 minutes at room temperature. After fixation, cells were incubated

with 30 mg/mL phosphatase naphthyl in 0.1 M Tris buffer at pH 10, in the presence of 30

mg/mL Fast Blue RR Salt, for 1 hour at 37ºC, in the dark. After incubation, cell layers were

washed and visualized by light microscopy.

2.2.6 Cellular proliferation/viability

The celular proliferation/viability assessment was performed by MTT (3-(4, 5-

dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Cell cultures were

incubated with 5 mg/mL MTT, for 4 hours at 37ºC. After incubation, the medium was

removed and 100 µL dimethyl sulfoxide (DMSO) was added. The absorbance was

quantified at 550nm in an ELISA plate reader (Synergy HT, Bioteck).

2.2.7 Immunofluorescence staining and visualization by CLSM

Cell cultures were washed twice with PBS and fixed with 3.7% (v/v) paraformaldehyde,

for 15 minutes at room temperature. After fixation, cells were washed again with PBS and

permeabilized with 0.1% (V/V) Triton X-100 for 5 minutes. Cultures were then stained for

F-actin with 5 U/mL Alexa Fluor® 647-Phalloidin (Invitrogen), for nucleus with 500 nM

propidium iodide, and for vitronectin receptors (VNR) and calcitonin receptors (CTR) with

50 µg/mL mouse IgGs anti-VNR and IgGs anti-CTR (R&D Systems), respectively. Anti-

VNR and anti-CTR detection was performed with 2 µg/mL Alexa Fluor® 488 anti-mouse

IgGs (Invitrogen). Cultures were visualized by CLSM.

40

2.2.8 Apoptosis quantification

Apoptosis was analyzed through caspase 3 activity quantification. For that, breast

cancer cell cultures were washed twice with PBS and assessed for caspase 3 activity with

EnzCheck® Caspase 3 Assay Kit # 2 (Molecular Probes), according to manufacturer’s

instructions. The fluorescence was measured at 486/520 nm (excitation/emission) in an

ELISA plate reader (Synergy HT, Bioteck).

2.2.9 Calcium phosphate resorbing ability

Co-cultures of SK-BR-3 and T47D and PBMC were performed on calcium phosphate

coated culture plates (BD BioCoat™ Osteologic™ Bone Cell Culture Plates, BD

Biosciences), for 21 days. After that period, cells were bleached with 6% NaOCl and 5.2%

NaCl, and the remaining calcium phosphate layers were visualized by phase contrast light

microscopy (Nikon TMS phase contrast microscope). Resorption lacunae were identified

and total resorbed area was quantified with ImageJ 1.41 software. Results were

presented as a % of resorbed area.

2.2.10 Statistical analysis

Data presented in this work are the means of separate experiments. Three replicas of

each condition were made for each experiment and data are expressed as the mean ±

standard deviation. Data were evaluated using a two-way analysis of variance (ANOVA)

and no significant differences in the pattern of the cell behavior were observed. Statistical

differences found between control and experimental conditions were determined by

Bonferroni’s method. For values of p ≤ 0.05, differences were considered statistical

significant.

41

3. Results

3.1 Assessment of the effects of different PPI in cultures of SK-BR-3 and T47D

In the first part of this work, it was pretended to evaluate the effects of PPI in cultures of

SK-BR-3 and T47D. Each culture was treated with different concentrations of omeprazole,

esomeprazole and lansoprazole, and maintained for 21 days. Samples were assessed at

days 7, 14 and 21.

3.1.1 Characterization of SK-BR-3 cultures

3.1.1.1 Cellular proliferation/viability

Figure 17. Cellular proliferation/viability of SK-BR-3 cultures treated with different concentrations of

omeprazole, esomeprazole and lansoprazole. * Significantly different from the control.

In general, SK-BR-3 cell cultures maintained a cell proliferation/viability identical to

negative control for the lowest concentrations of each PPI. However, with the increase of

PPI concentrations, cell proliferation/viability started to decrease. This dose-dependent

decrease was seen for the three PPI starting from the concentration 10-5 M, with a

decrease of about 18.79%, 21.13% and 3.64% for omeprazole, esomeprazole and

lansoprazole, respectively.

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

7 14 21

Ab

sorb

ance

(55

0nm

)

Days

Omeprazole

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

7 14 21

Days

Esomeprazole

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

7 14 21

Days

Lansoprazole

Negative Control

10-9M

10-8M

10-7M

10-6M

10-5M

10-4M

10-3M

* * *

* * *

* * *

42

3.1.1.2 Apoptosis quantification

Figure 18. Caspase-3 activity of SK-BR-3 cultures treated with different concentrations of

omeprazole, esomeprazole and lansoprazole. * Significantly different from the control.

Concentrations up to 10-5 M did not cause any significant effect on caspase-3 activity of

SK-BR-3 cultures. However, cellular response increased significantly with the highest

tested concentration of omeprazole and esomeprazole. Compared to the control, at day

21 the increase was about 85.20% and 58%, in the case of omeprazole and

esomeprazole, respectively. In the presence of lansoprazole, caspase activity was not

significantly affected by the tested concentrations.

3.1.2 Characterization of T47D cultures

3.1.2.1 Cellular proliferation/viability

0

0,5

1

1,5

2

2,5

3

3,5

4

7 14 21

flu

ore

sce

nce

x10

4

Days

Omeprazole

0

0,5

1

1,5

2

2,5

3

3,5

4

7 14 21

Days

Esomeprazole

0

0,5

1

1,5

2

2,5

3

3,5

4

7 14 21 Days

Lansoprazole

Negative Control

10-9M

10-8M

10-7M

10-6M

10-5M

0

0,5

1

1,5

2

2,5

3

7 14 21

Ab

sorb

ance

(550

nm

)

Days

Omeprazole

0

0,5

1

1,5

2

2,5

3

7 14 21

Days

Esomeprazole

*

*

*

* *

*

*

*

*

43

Figure 19. Cellular proliferation/viability of T47D cultures treated with different concentrations of

omeprazole, esomeprazole and lansoprazole. * Significantly different from the control.

Cell proliferation/viability was higher for T47D cultures than for SK-BR-3 cultures. In

general, T47D cultures maintained a cellular proliferation/viability identical to negative

control for the lowest concentrations of each PPI. However, proliferation/viability started to

decrease with the increase of PPI concentration starting from 10-4 M omeprazole and

esomeprazole, and 10-5 M lansoprazole. The decrease was about 53.80%, 30.75% and

27.36%, respectively.

3.1.2.2 Apoptosis quantification

Figure 20. Caspase-3 activity of T47D cultures treated with different concentrations of omeprazole,

esomeprazole and lansoprazole. * Significantly different from the control.

0

0,5

1

1,5

2

2,5

3

7 14 21

Days

Lansoprazole

Negative Control

10-9M

10-8M

10-7M

10-6M

10-5M

10-4M

10-3M

0

0,5

1

1,5

2

2,5

3

3,5

4

7 14 21

Flu

ore

scen

ce x

104

Days

Omeprazole

0

0,5

1

1,5

2

2,5

3

3,5

4

7 14 21 Days

Esomeprazole

0

0,5

1

1,5

2

2,5

3

3,5

4

7 14 21

Days

Lansoprazole

Negative Control

10-9M

10-8M

10-7M

10-6M

10-5M

* *

*

*

*

44

T47D cultures presented lower caspase-3 activity values than those obtained in SK-BR-

3 cultures. In general, caspase-3 activity was not affected by the presence of neither

omeprazole nor esomeprazole, presenting similar values to the negative control at all

tested concentrations. However, caspase activity increased in the presence of the highest

concentration of lansoprazole (10-5 M), with an increase of about 50.80% at day 21.

3.2 Assessment of the osteoclastogenic effects of different PPI in co-cultures of

PBMC and breast cancer cell lines

In the second part of this work, it was pretended to evaluate the effects of PPI in co-

cultures of PBMC and SK-BR-3 or T47D. SK-BR-3 and T47D cells were seeded at

different densities, 102 cells/cm2 and 104 cells/cm2, respectively, based on previous

studies (data not shown). Cultures were treated with different concentrations of

omeprazole, esomeprazole and lansoprazole, and maintained for 21 days. Samples were

assessed at days 7, 14 and 21.

3.2.1 Characterization of PBMC + SK-BR-3 co-cultures

3.2.1.1 TRAP activity

Figure 21. TRAP activity on PBMC + SK-BR-3 co-cultures treated with different concentrations of

omeprazole, esomeprazole and lansoprazole. * Significantly different from the control.

0

5

10

15

20

25

30

7 14 21

nm

ol/

min

.µg-1

Days

Omeprazole

0

5

10

15

20

25

30

7 14 21 Days

Esomeprazole

0

5

10

15

20

25

30

7 14 21

Days

Lansoprazole

Negative control

10-7M

10-6M

10-5M

10-4M

10-3M

* * * *

* * *

* * *

45

As observed in Figure 21, TRAP activity of PBMC + SK-BR-3 co-cultures decreased

with the increase of each PPI concentration. Among the three tested PPI, the one that

induced a more significant dose-dependent decrease in TRAP activity was omeprazole,

whereas esomeprazole was the one that affected the least. Compared to the control, the

effects became statistically significant for the concentration of 10-7 M of omeprazole

(~15.55%), and 10-6 M of esomeprazole (~24.40%) and lansoprazole (~34.04%). Co-

cultures displayed null levels of activity for the highest tested concentrations of

omeprazole and lansoprazole.

3.2.1.2 Histochemical staining of TRAP

Figure 22. Presence of TRAP+ multinucleated cells on PBMC + SK-BR-3 co-cultures treated with

different concentrations of omeprazole, esomeprazole and lansoprazole. *

Significantly different

from the control.

The results of histochemical staining of TRAP (Fig. 22) were consistent with those

obtained for TRAP activity. The number of positive TRAP multinucleated cells decreased

with the increase of each PPI concentration, particularly in the case of omeprazole and

lansoprazole.

0

20

40

60

80

100

120

140

14 21

TRA

P+

mu

ltin

ucl

eate

d c

ells

Days

Omeprazole

0

20

40

60

80

100

120

140

14 21 Days

Esomeprazole

0

20

40

60

80

100

120

140

14 21 Days

Lansoprazole

Negative control

10-7M

10-6M

10-5M

10-4M

10-3M

* *

* *

*

*

* *

* * *

* *

*

* * *

* *

*

* *

* *

* *

46

3.2.1.3 Calcium phosphatase resorbing ability

Figure 23. Visualization of calcium phosphatase resorbed area of PBMC + SK-BR-3 co-cultures

treated with 10-6

M omeprazole, esomeprazole and lansoprazole, by light microscopy. White bars

represent 600 µm.

Figure 24. Calcium phosphatase resorbed area of PBMC + SK-BR-3 co-cultures treated with 10-6

M omeprazole, esomeprazole and lansoprazole. *Significantly different from the control.

Visualization of calcium phosphatase resorbed area by light microscopy showed a

decrease in calcium phosphatase resorbing ability in the presence of the three PPI. The

co-culture that presented the lowest area of resorption was the one that was treated with

10-6 M omeprazole, with a maximal inhibition of 41% compared to control. In the presence

of esomeprazole and lansoprazole the inhibition was about 27% and 34%, respectively.

3.2.1.5 Characterization of the intracellular mechanisms involved in the cellular

response

Following the assessment of the osteoclastogenic effects of PPI in co-cultures of PBMC

and SK-BR-3, it was also carried out a characterization of the intracellular mechanisms

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

% R

eso

rpti

on

Are

a

PBMC + SK-BR-3

Control Omeprazole

Esomeprazole Lansoprazole

Negative control Omeprazole (10-6

M)

Esomeprazole (10-6

M) Lansoprazole (10-6

M)

* * *

Esomeprazole (10-6

M) Lansoprazole (10-6

M)

47

involved in this process. For that, co-cultures were maintained for 21 days in the absence

or presence of 10-6 M (the lowest common concentration that elicited a significant anti-

osteoclastogenic effect either in TRAP activity and staining) of each PPI and treated with

intracellular signaling pathway inhibitors: 1µM U0126 (MAPK/ERK inhibitor); 10µM PDTC

(NF-kB inhibitor); 5µM GO 6983 (PKC inhibitor) and 10µM SP 600125 (JNK inhibitor).

Samples were assessed at days 7, 14 and 21.

3.2.1.5.1 TRAP activity

Figure 25. TRAP activity on PBMC + SK-BR-3 co-cultures treated with 10-6

M omeprazole, 10-6

M

esomeprazole and 10-6

M lansoprazole and supplemented with different intracellular signalling

pathway inhibitors. * Significantly different from the control.

TRAP activity of untreated PBMC + SK-BR-3 co-cultures decreased in the presence of

all the inhibitors tested, with the one exception being GO6983, which did not significantly

affect the cellular behavior. Co-cultures treated with omeprazole displayed a similar

pattern of cell response. In the presence of esomeprazole, only PDTC negatively affected

TRAP activity, while the remaining inhibitors did not affect cellular response. Finally, in the

presence of lansoprazole, U0126 and PDTC inhibited TRAP activity similarly to the

control, whereas co-cultures supplemented with GO6983 displayed a significantly higher

decrease on cell response. On the other hand, in the presence of SP600125 TRAP

activity was less affected.

0

5

10

15

20

25

30

7 14 21

nm

ol/

min

.µg-1

Days

Control

0

5

10

15

20

25

30

7 14 21

Days

Omeprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

0

5

10

15

20

25

30

7 14 21

nm

ol/

min

.µg-1

Days

Esomeprazole

0

5

10

15

20

25

30

7 14 21 Days

Lansoprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

* * *

*

**

**

48

The results of histochemical staining of TRAP were consistent with those obtained for

TRAP activity, showing more evidently that GO6983 was the inhibitor that less affected

the cultures, except for the cultures treated with lansoprazole (data not shown).

3.2.2 Characterization of PBMC + T47D co-cultures

3.2.2.1 TRAP activity

Figure 26. TRAP activity of PBMC + T47D co-cultures treated with different concentrations of

omeprazole, esomeprazole and lansoprazole. * Significantly different from the control.

PBMC + T47D co-cultures presented levels of TRAP activity higher than those obtained

in PBMC + SK-BR-3 co-cultures. TRAP activity decreased with the increase of each PPI

concentration. This dose-dependent inhibition was higher in the presence of omeprazole

and lansoprazole. Compared to the control, the effects became statistically significant for

the concentration of 10-7 M omeprazole (30.93%) 10-5 M of esomeprazole (16.87%) and

10-7 M of lansoprazole (34%). Again, co-cultures treated with the highest concentrations of

omeprazole and lansoprazole displayed null levels of TRAP activity.

0

10

20

30

40

7 14 21

nm

ol/

min

.µg-1

Days

Omeprazole

0

10

20

30

40

7 14 21 Days

Esomeprazole

0

10

20

30

40

7 14 21

Days

Lansoprazole

Negative control

10-7M

10-6M

10-5M

10-4M

10-3M

* * * *

* *

* * *

49

3.2.2.2 Histochemical staining of TRAP

Figure 27. Presence of TRAP+ multinucleated cells on PBMC + T47D co-cultures treated with

different concentrations of omeprazole, esomeprazole and lansoprazole. *Significantly different

from the control.

PBMC + T47D co-cultures showed a higher presence of TRAP multinucleated cells

than PBMC + SK-BR-3 co-cultures. As observed for TRAP activity, the number of TRAP+

multinucleated cells decreased with the increase of each PPI concentration, particularly in

the presence of omeprazole and lansoprazole.

3.2.2.3 Calcium phosphatase resorbing ability

0

20

40

60

80

100

120

140

14 21

TRA

P+

mu

ltin

ucl

eate

d c

ells

Days

Omeprazole

0

20

40

60

80

100

120

140

14 21 Days

Esomeprazole

0

20

40

60

80

100

120

140

14 21 Days

Lansoprazole

Negative control

10-7M

10-6M

10-5M

10-4M

10-3M

Negative control Omeprazole (10-7

M)

Esomeprazole (10-5

M) Lansoprazole (10-7

M)

* *

* *

*

* *

*

*

* * * *

* * *

* *

* *

*

* *

50

Figure 28. Visualization of calcium phosphatase resorbed area of PBMC + T47D co-cultures

treated with 10-7

M omeprazole, 10-5

M esomeprazole and 10-7

M lansoprazole, by light microscopy.

White bars represent 600µm.

Figure 29. Resorbed area of PBMC + T47D co-cultures treated with 10-7

M omeprazole, 10-5

M

esomeprazole and 10-7

M lansoprazole. *Significantly different from the control.

Visualization of calcium phosphatase resorbed area by light microscopy showed a

decrease in calcium phosphatase resorbing ability in the presence of the three PPI. The

co-culture that presented the lowest area of resorption was the one that was treated with

10-7 M omeprazole, with a maximal inhibition of 44% compared to the control. In the

presence of esomeprazole and lansoprazole the inhibition was about 23% and 35%,

respectively. Globally, PBMC + T47D co-cultures presented a higher resorbing ability

than PBMC + SK-BR-3 co-cultures (53% versus 30%).

3.2.2.4 Visualization of cells with actin rings and expression of VNR and CTR

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

% R

eso

rpti

on

Are

a

T47D + PBMC

Control Omeprazole

Esomeprazole Lansoprazole

Negative control Omeprazole (10-6

M)

Negative control Omeprazole (10-6

M)

V

N

R

C

T

R

* *

*

51

Figure 30. Visualization of cells with actin rings and expression of VNR and CTR of PBMC + T47D

co-cultures in absence and presence of 10-6

M omeprazole, by CLSM. Cells were stained blue for

actin and green for VNR and CTR. White bars represent 150µm.

Visualization of PBMC + T47D co-cultures by CSLM revealed the presence of

osteoclastic cells in all analysed conditions. The results were somehow in line with the

data obtained from the previous analysis. That means, co-cultures treated with 10-6 M

omeprazole showed a decrease in osteoclastic cells compared to the negative control.

3.2.2.5 Characterization of the intracellular mechanisms involved in the cellular

response

Characterization of the intracellular mechanisms involved in osteoclastogenesis was

also performed in PBMC + T47D co-cultures. For that, co-cultures were maintained for 21

days in the absence and presence of 10-7 M omeprazole, 10-5 M esomeprazole and 10-7 M

lansoprazole (the lowest concentration of each PPI that elicited a significant anti-

osteoclastogenic effect) and treated with intracellular signalling pathway inhibitors: 1µM

U0126 (MAPK/ERK inhibitor); 10µM PDTC (NF-kB inhibitor); 5µM GO 6983 (PKC

inhibitor) and 10µM SP 600125 (JNK inhibitor). Samples were assessed at days 7, 14 and

21.

3.2.2.5.1 TRAP activity

0

5

10

15

20

25

30

35

40

7 14 21

nm

ol/

min

.µg-1

Days

Control

0

5

10

15

20

25

30

35

40

7 14 21 Days

Omeprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

0

5

10

15

20

25

30

35

40

7 14 21

nm

ol/

min

.µg-1

Days

Esomeprazole

0

5

10

15

20

25

30

35

40

7 14 21 Days

Lansoprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

**

* *

* *

*

* **

52

Figure 31. TRAP activity on PBMC + T47D co-cultures treated with 10-7

M omeprazole, 10-5

M

esomeprazole and 10-7

M lansoprazole and supplemented with different intracellular signalling

pathway inhibitors. * Significantly different from the control.

TRAP activity of untreated and treated PBMC + T47D co-cultures decreased in the

presence of all the tested inhibitors. In co-cultures treated with omeprazole and

esomeprazole, with exception of U0126 that equally affected TRAP activity, the presence

of all the inhibitors induced a less significant inhibition than in untreated cultures. In the

presence of lansoprazole, TRAP activity decreases were similar to those seen on

untreated cultures for those supplemented with U0126, PDTC and GO6983, whereas

SP600125 supplemented cultures were less affected.

The results of histochemical staining of TRAP were consistent with those obtained for

TRAP activity. (data not shown)

3.3 Assessment of the effects of different PPI in cultures of hFOB

Finally, in the last part of this work, the aim was to evaluate the effects of PPI in

cultures of hFOB. Cultures were performed in the absence and presence of the

osteogenic inducer dexamethasone (DEX) and supplemented with different

concentrations of omeprazole, esomeprazole and lansoprazole. Cell cultures were

maintained for 21 days and samples were assessed at days 7, 14 and 21.

3.3.1 Characterization of hFOB cultures (-DEX)

3.3.1.1 Cellular proliferation/viability

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1

7 14 21

Ab

sorb

ance

(550

)

Days

Omeprazole

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1

7 14 21 Days

Esomeprazole

*

*

*

** * *

*

*

*

53

Figure 32. Cellular proliferation/viability of hFOB cultures treated with different concentrations of

omeprazole, esomeprazole and lansoprazole, in absence of DEX. * Significantly different from the

control.

Cellular proliferation/viability of hFOB cultures, maintained in the absence of DEX,

decreased with the increase of each PPI concentration. This dose-dependent inhibition

was higher in the presence of omeprazole and lansoprazole, with null levels of cellular

proliferation/viability for the highest concentration of lansoprazole (10-3 M). The inhibition

became statistically significant in the presence of 10-6 M omeprazole and lansoprazole

and 10-5 M esomeprazole, with a corresponding inhibition of about 17.78%, 31.30% and

16.69%.

3.3.1.2 ALP activity

Figure 33. ALP activity of hFOB cultures treated with different concentrations of omeprazole,

esomeprazole and lansoprazole, in absence of DEX. * Significantly different from the control.

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1

7 14 21 Days

Lansoprazole

Negative control

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

nm

ol/

min

.µg-

1

Days

Omeprazole

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

14 21 Days

Esomeprazole

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

14 21 Days

Lansoprazole

Negative control

10-7M

10-6M

10-5M

10-4M

10-3M

**

**

*

*

*

** *

* * **

*

**

*

*

*

*

*

54

As observed for cellular proliferation/viability, ALP activity of hFOB cultures, maintained

in the absence of DEX, decreased with the increase of PPI concentration, particularly in

the presence of omeprazole and lansoprazole, with null levels of activity for the highest

concentration (10-3 M). The inhibition became statistically significant in the presence of 10-

6 M omeprazole (39.46%), 10-6 M esomeprazole (8.16%) and 10-6 M lansoprazole

(25.85%).

3.3.1.3 Histochemical staining of ALP

Figure 34. Histochemical staining of ALP in hFOB cultures treated with 10-6

M omeprazole,

esomeprazole and lansoprazole, in absence of DEX. White bars represent 200µm.

Visualization of histochemical staining of ALP revealed a cellular behavior consistent

with the data obtained from ALP activity analysis. Cultures treated with 10-6 M omeprazole

and lansoprazole displayed a significant decrease in ALP expression, while the same

concentration of esomeprazole did not have a so sharp effect in the cellular response.

Negative Control Omeprazole (10-6

M)

Esomeprazole (10-6

M) Lansoprazole (10-6

M)

55

3.3.2 Characterization of hFOB cultures (+DEX)

3.3.2.1 Cellular proliferation/viability

Figure 35. Cellular proliferation/viability of hFOB cultures treated with different concentrations of

omeprazole, esomeprazole and lansoprazole, in the presence of DEX. * Significantly different from

the control.

Cellular proliferation/viability of hFOB cultures, in presence of DEX, decreased with the

increase of each PPI concentration. This dose-dependent inhibition was higher in the

presence of omeprazole and lansoprazole. The inhibition became statistically significant in

the presence of 10-7 M omeprazole, 10-5 M esomeprazole and 10-4 M lansoprazole, with a

corresponding inhibition of about 13.92%, 12.66% and 31.65%.

3.3.2.2 ALP activity

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1

7 14 21

Ab

sorv

ânci

a (5

50)

Days

Omeprazole

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1

7 14 21 Days

Esomeprazole

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1

7 14 21 Days

Lansoprazole

Negative control

10-7M

10-6M

10-5M

10-4M

10-3M

0

0,5

1

1,5

2

2,5

3

3,5

14 21

nm

ol/

min

.µg-

1

Days

Omeprazole

0

0,5

1

1,5

2

2,5

3

3,5

14 21 Days

Esomeprazole

** *

*

**

*

*

*

*

*

** *

* *

56

Figure 36. ALP activity of hFOB cultures treated with different concentrations of omeprazole,

esomeprazole and lansoprazole, in the presence of DEX. * Significantly different from the control.

ALP activity of hFOB cultures, in the presence of DEX, decreased with the increase of

each PPI concentration, particularly in the presence of omeprazole and lansoprazole. The

inhibition became statistically significant starting from the concentration 10-6 M, with a

decrease of about 21.07%, 10.98% and 13.95% for omeprazole, esomeprazole and

lansoprazole, respectively. Once again, co-cultures treated with the highest

concentrations of omeprazole and lansoprazole displayed null levels of TRAP activity.

3.3.2.3 Histochemical staining of ALP

Figure 37. Histochemical staining of ALP in hFOB cultures treated with 10-6

M omeprazole,

esomeprazole and lansoprazole, in the presence of DEX. White bars represent 200µm.

The results of histochemical staining of ALP were consistent with those obtained in ALP

activity analysis, showing that cultures treated with omeprazole and lansoprazole had less

expression of ALP.

0

0,5

1

1,5

2

2,5

3

3,5

14 21 Days

Lansoprazole

Negative control

10-7M

10-6M

10-5M

10-4M

10-3M

Negative Control Omeprazole (10-6

M)

Esomeprazole (10-6

M) Lansoprazole (10-6

M)

*

*

*

57

3.3.2.4 Visualization of hFOB cultures by CLSM

Figure 38. Visualization of hFOB cultures treated with 10-6

M omeprazole, esomeprazole and

lansoprazole, in the presence of DEX, by CLSM. Cells were stained green for actin and red for

nuclei. White bars represent 60µm.

Visualization of hFOB cultures by CLSM revealed the presence of osteoblastic cells in

all tested conditions. Results were consistent with the data obtained from the previous

analysis, that is, cultures treated with omeprazole and lansoprazole showed a more

significant decrease in the amount of osteoblastic cells compared to the negative control.

3.3.3 Characterization of the intracellular effects involved in the observed cellular

response

Following the assessment of the effects of PPI in cultures of hFOB, it was also carried

out a characterization of the intracellular mechanisms involved in the observed cellular

behavior. For that, cultures were maintained for 21 days in the absence and presence of

10-6 M of each PPI and treated with intracellular signalling pathway inhibitors: 1µM U0126

(MAPK/ERK inhibitor); 10µM PDTC (NF-kB inhibitor); 5µM GO 6983 (PKC inhibitor) and

10µM SP 600125 (JNK inhibitor). Samples were assessed at days 7, 14 and 21.

Negative control Omeprazole (10-6

M)

Esomeprazole (10-6

M) Lansoprazole (10-6

M)

58

3.3.3.1 Cellular proliferation/viability of hFOB cultures (-DEX)

Figure 39. Cellular proliferation/viability of hFOB cultures treated with 10-6

M omeprazole, 10-6

M

esomeprazole and 10-6

M lansoprazole, in absence of DEX, and supplemented with different

intracellular signalling pathway inhibitors. *Significantly different from the control.

Cellular proliferation/viability of untreated hFOB cultures decreased in the presence of

all the inhibitors. In cultures treated with omeprazole, U0126 and PDTC induced a less

significant inhibition of cellular proliferation/viability than in control, whereas cultures

supplemented with GO6983 and SP600125 displayed a higher decrease. In the presence

of esomeprazole, with exception of U0126 that did not affect cultures, all the inhibitors

induced similar changes to those seen for cultures treated with omeprazole. Finally, in the

presence of lansoprazole, cultures supplemented with U0126 and GO6983 displayed

cellular proliferation/viability decreases similar to those obtained in control, while PDTC

induced a higher decrease. On the other hand, in the presence of SP600125 cultures

were less affected.

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1

7 14 21 Ab

sorb

ance

(550

nm

)

Days

Control

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1

7 14 21 Days

Omeprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1

7 14 21 Ab

sro

ban

ce (5

50n

m)

Days

Esomeprazole

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

1

7 14 21 Days

Lansoprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

***

**

* * *

***

***

59

3.3.3.2 ALP activity of hFOB cultures (-DEX)

Figure 40. ALP activity of hFOB cultures treated with 10-6

M omeprazole, 10-6

M esomeprazole and

10-6

M lansoprazole, in absence of DEX, and supplemented with different intracellular signalling

pathway inhibitors. *Significantly different from the control.

ALP activity of control cultures decreased in the presence of all the inhibitors. In the

presence of omeprazole, U0126 and PDTC did not affect ALP activity, while cultures

supplemented with GO6983 showed a lower decrease compared to the control. The

presence of SP600125 induced a higher decrease. Cultures treated with esomeprazole

displayed a less significant decrease for inhibitors U0126 and PDTC. On the other hand,

the presence of GO6983 and SP600125 induced a higher decrease in ALP activity. In

contrast, in the presence of lansoprazole, GO6983 and SP600125 induced an increase in

ALP activity. Cultures supplemented with U0126 showed a higher decrease compared to

the control and those supplemented with PDTC a lower one.

3.3.3.3 Cellular proliferation/viability of hFOB cultures (+DEX)

0

0,25

0,5

0,75

1

1,25

1,5

1,75

2

7 14 21

nm

ol/

min

.µg-1

Days

Control

0

0,25

0,5

0,75

1

1,25

1,5

1,75

2

7 14 21 Days

Omeprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

0

0,25

0,5

0,75

1

1,25

1,5

1,75

2

7 14 21

nm

ol/

min

.µg-1

Days

Esomeprazole

0

0,25

0,5

0,75

1

1,25

1,5

1,75

2

7 14 21 Days

Lansoprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

0

0,25

0,5

0,75

1

1,25

1,5

1,75

2

7 14 21 Ab

sorb

ance

(550

nm

)

Days

Control

0

0,25

0,5

0,75

1

1,25

1,5

1,75

2

7 14 21 Days

Omeprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

*

***

* *

*

***

*

*

60

Figure 41. Cellular proliferation/viability of hFOB cultures treated with 10-6

M omeprazole, 10-6

M

esomeprazole and 10-6

M lansoprazole, in presence of DEX, and supplemented with different

intracellular signalling pathway inhibitors. *Significantly different from the control.

Cellular proliferation/viability of untreated hFOB cultures decreased in the presence of

all the inhibitors tested with exception of U0126 which did not affect the cultures. In

cultures treated with omeprazole, only GO6983 and SP600125 negatively affected cellular

proliferation/viability, inducing a higher decrease compared to the control. In the presence

of esomeprazole, cultures supplemented with U0126 were negatively affected. The

presence of PDTC induced a lower decrease of cellular proliferation/viability to that on

control cultures, whereas GO6983 and SP600125 induced similar changes. Finally, in the

presence of lansoprazole, PDTC and SP600125 induced similar changes to those seen

on control, while GO6983 induced a higher decrease. Again, cultures supplemented with

U0126 were negatively affected.

3.3.3.4 ALP activity of hFOB cultures (+DEX)

0

0,25

0,5

0,75

1

1,25

1,5

1,75

2

7 14 21 Ab

sorb

ance

(550

nm

)

Days

Esomeprazole

0

0,25

0,5

0,75

1

1,25

1,5

1,75

2

7 14 21 Days

Lansoprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

0

0,5

1

1,5

2

2,5

3

3,5

4

7 14 21

nm

ol/

min

.µg-

1

Days

Control

0

0,5

1

1,5

2

2,5

3

3,5

4

7 14 21 Days

Omeprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

* * *

*

*

*

***

61

Figure 42. ALP activity of hFOB cultures treated with 10-6

M omeprazole, 10-6

M esomeprazole and

10-6

M lansoprazole, in presence of DEX, supplemented with different intracellular signalling

pathway inhibitors. *Significantly different from the control.

ALP activity of untreated hFOB cultures decreased in the presence of all the inhibitors.

In cultures treated with omeprazole and esomeprazole, U0126 and GO6983 induced a

lower decrease in ALP activity compared to the control, while SP600125 induced a higher

inhibition. The presence of PDTC did not affect the enzyme activity on cultures treated

with omeprazole but did negatively affect ALP activity on cultures treated with

esomeprazole, inducing a higher decrease compared to the control. In the presence of

lansoprazole, cultures supplemented with U0126 and SP600125 displayed activity

decreases inferior to those seen in the control. On the other hand, PDTC and GO6983

induced a higher inhibition.

0

0,5

1

1,5

2

2,5

3

3,5

4

7 14 21

nm

ol/

min

.µg-1

Days

Esomeprazole

0

0,5

1

1,5

2

2,5

3

3,5

4

7 14 21 Days

Lansoprazole

No inhibitor

U0126

PDTC

GO6983

SP 600125

*

*

** *

62

4. Discussion

PPI are a class of drugs widely used in many pathological conditions, such as

gastroesophageal reflux, dyspepsia and peptic ulcers. The mechanism of action relies in

the inhibition of H+/K+ ATPases, responsible for gastric acid secretion. Osteoclasts are

specialized cells that solubilize extracellular bone matrix through the production of acid, by

proton pumps that belong to the same family as the gastric ones [75]. Thus, PPI appear

as potential candidates to modulate osteoclast resorption activity, particularly in conditions

associated with hyperactivation of these cells, such as bone osteolytic metastasis. Breast

cancer is one of the most frequent tumours that originate bone metastasis, being the most

common the osteolytic ones [47]. In this context, it seems relevant to assess the effects of

PPI on human osteoclastogenesis in co-cultures of human osteoclasts and breast cancer

cell lines.

The range of PPI concentrations used in this study was chosen according to

concentrations found in human serum during therapeutic use of these molecules. It was

seen that after a week of treatment with a daily dose of 20 mg omeprazole, the plasma

maximum concentration was of about 1.1-3.2 µM, depending on the metabolizing capacity

of the individual [87]. For esomeprazole, daily doses of 20 mg or 40 mg elicited a

maximum concentration of 2.1 and 4.7 µM, respectively [88]. As for lansoprazole, after

treatments with daily doses of 30 mg, plasma maximum concentration ranged between

2.2 and 4.8 µM [87]. Thus, the observed values fall completely within the concentration

range used in this study (10-9 – 10-3 M). However, it is important to notice that these are

just mean values and that PPI pharmacokinetics depends on various factors.

Regarding the breast cancer cells, the tested cell lines are known to be associated with

the development of bone metastasis. These lines were also selected by their different

ability to express estrogen receptors, a factor known to be related with bone metastatic

behavior of breast cancer cells [52, 53].

In the first part of the work it was observed that the presence of the tested PPI under

certain experimental conditions inhibits the proliferation/viability of breast cancer cell

cultures. Thereby, indicating that those drugs have the ability to act directly on these cells.

More precisely, in SK-BR-3 cultures, cellular proliferation/viability inhibition occurred for

concentrations higher than 10-5 M of each PPI. In the presence of 10-5 M omeprazole and

esomeprazole, the cultures also displayed an increase in caspase-3 activity, suggesting

that for those PPI, cellular proliferation/viability decrease may be due to apoptosis

63

mechanisms. In general, T47D line was less affected by PPI treatment. In this case,

cellular growth inhibition occurred at concentrations slightly higher and particularly in the

presence of lansoprazole. In general, these cultures exhibited lower caspase-3 activity

values compared to SK-BR-3 cultures. Nevertheless, caspase-3 activity was only affected

by the presence of the highest concentration (10-5 M) of lansoprazole.

In addition to the possible effects on the apoptosis mechanisms, the results observed

on cultures of breast cancer cell lines might also be explained by the presence of vacuolar

H+/ATPases in these cells. Acidity has been shown to have an important role in

proliferation and metastatic behavior of cancer cells [83, 84]. Studies conducted on this

matter revealed the presence of these proton pumps in the plasma membrane of human

breast cancer cells with metastatic potential. However, these studies did not include SK-

BR-3 or T47D lines [78]. Following these studies, others have been performed in order to

assess the potential of PPI to reverse the intrinsic resistance of human breast cancer cells

to chemotherapy with promising results [76, 77].

In the second part of the work it was performed an analysis of the effects of the different

PPI tested in osteoclastogenesis, in co-cultures of PBMC and breast cancer cell lines.

Globally, the presence of breast cancer cells, particularly T47D cells, greatly induced

osteoclastogenesis in the co-cultures with PBMC. T47D line exhibited the highest

osteoclastogenic potential and elicited the highest resorption ability. These results of

osteoclastic stimulation are consistent with previous studies on the action of different

breast cancer cell lines on bone cells, both on animal and human osteoclast cultures [53].

It was observed that the presence of the three PPI at certain concentrations inhibited

osteoclastogenesis in both co-cultures, particularly in the presence of omeprazole.

Generally, the effects observed presented a dose-dependent inhibition. Although the

highest concentrations seemed to be toxic for osteoclastic cells (10-4 M and 10-3 M), the

inhibition observed at lower concentrations appeared to be due to specific effects on the

osteoclastogenic process, rather than to a significant decrease on the cellular viability.

Previous studies have reported that omeprazole could inhibit bone resorption at

concentrations similar to those found in blood during therapeutic use of these molecules,

likely because of inhibition of vacuolar H+/ATPases. In vitro, the presence of omeprazole

inhibited bone resorption on rat cell cultures, and in vivo, treatment with omeprazole

reduced the urinary excretion of calcium in patients with a history of gastric disease [81,

82]. Up to our knowledge, there is no information regarding the ability of PPIs to modulate

osteoclastogenesis promoted by breast cancer cells.

64

Following the assessment of the osteoclastogenic effects of the different PPI in co-

cultures of PBMC and breast cancer cell lines, it was also carried out a characterization of

the intracellular mechanisms involved in this process. For that, it was used the lowest

common concentration of each PPI that elicited a significant anti-osteoclastogenic effect in

each co-culture. In SK-BR-3 co-cultures it was used 10-6 M of the three PPI whereas in

T47D co-cultures were used the following concentrations: 10-7 M omeprazole, 10-5 M

esomeprazole and 10-7 M lansoprazole.

In control co-cultures of PBMC and SK-BR-3, the osteoclastogenic process was highly

dependent on NFkB pathway and significantly dependent on MEK and JNK pathways. It

was seen that the presence of omeprazole seemed not to affect the signaling pathways of

co-cultures, compared to control. In the presence of esomeprazole, osteoclastogenesis

appeared to be significantly dependent on NFkB pathway, while in the presence of

lansoprazole this process appeared to be significantly dependent on PKC pathway.

In control co-cultures of PBMC and T47D, osteoclast differentiation was highly

dependent on NFkB pathway and significantly dependent on PKC and JNK pathway. It

was observed that in the presence of omeprazole and esomeprazole all the tested

pathways seemed to become less important in osteoclastogenesis, compared to control.

On the other hand, the presence of lansoprazole only affected the JNK pathway, making

this pathway less important to the observed osteoclastic response.

It was also observed that the PPI tested are capable of directly act on osteoblastic

hFOB cells. The presence of dexamethasone, an osteogenic enhancer, greatly induced

ALP activity, as reported previously [89, 90, 91]. Cellular proliferation/viability of hFOB

cultures maintained in the absence and presence of DEX decreased in the presence of

the three PPI starting from the concentration 10-6 M. This inhibition was also seen for ALP

activity, however, the percentage of inhibition was higher for ALP activity compared to that

related to cell growth. This suggests that PPI effects are more likely to be due to specific

changes on the cellular differentiation rather than to decreases on cellular viability due to

toxic mechanisms. The inhibitory effects were more pronounced in the presence of

omeprazole and lansoprazole. As observed in the previous culture systems, the highest

tested concentrations of PPI appeared to be toxic for the cells.

In comparison with osteoclasts, significantly less attention has been paid to ATPases in

osteoblasts. Osteoblastic cells have been shown to express some types of ATPases,

namely, Na+/K+-ATPase and Ca+-ATPase, however, there is only indirect evidence of the

65

presence of vacuolar H+-ATPase in osteoblasts [86]. In vitro studies demonstrated that

bafilomycin A1 and concanamycin A, which are potent and specific inhibitors of H+-

ATPases, dose-dependently inhibited the growth and changed the morphology of rat

osteoblast cell lines [85, 86]. The present results are in agreement with this latter study,

although the molecules do not have the same mechanism of action.

It was also carried out a characterization of the intracellular mechanisms involved in the

osteoblastic response.

In control hFOB cultures maintained in the absence of DEX, cellular growth was highly

dependent on NFkB pathway. The PKC and JNK pathways seemed to be more important

for cellular growth in the presence of omeprazole and esomeprazole, whereas in the

presence of lansoprazole, cellular proliferation/viability appeared to be significantly

dependent on NFkB pathway.

In control cultures, ALP activity was highly dependent on NFkB and significantly

dependent on MEK and PKC pathways. In the presence of omeprazole the enzyme

activity appeared to be significantly dependent on JNK pathway. In the presence of

esomeprazole PKC and JNK pathways are the most important, while the MEK pathway

seemed to stand out in the presence of lansoprazole.

In control hFOB cultures maintained in presence of DEX, proliferation/viability was

highly dependent on NFkB pathway. The pathways that appeared to be more important

for cellular proliferation/viability in the presence of omeprazole were the PKC and JNK. In

the presence of esomeprazole the MEK pathway was more important, while in the

presence of lansoprazole cellular growth seemed significantly dependent on PKC

pathway.

In control cultures, ALP activity was highly dependent on MEK and PKC pathways and

significantly dependent on NFkB and JNK pathways. As observed in the cultures

maintained in the absence of DEX, the ALP activity appeared to be significantly

dependent on JNK pathway in the presence of omeprazole and esomeprazole. The NFkB

pathway was also important in the presence of esomeprazole. The enzyme activity was

significantly dependent on NFkB and PKC pathways.

66

5. Conclusion

The PPI, omeprazole, esomeprazole and lansoprazole, have the ability to negatively

affect the osteoclastogenic process on co-cultures of osteoclasts and breast cancer cells,

affecting not only osteoclastic cells differentiation but also their resorbing ability. This

inhibition was depending on the identity and concentration of the tested molecule, and

involved significant changes in different intracellular signaling pathways. PPI also inhibits

cellular proliferation and viability of breast cancer cell cultures. This inhibition appeared to

be, at least partially, due to an increase on apoptosis. Similarly, these compounds induced

a decrease on osteoblastogenesis, an effect that might contribute to eventual disruption of

normal bone cellular metabolic activities and consequently, lead to deleterious effects of

these drugs in the bone tissue. Globally, these effects were observed at concentrations

identical to those found in human serum during therapeutic use of these molecules.

This study contributed to a better understanding of the role of PPI in bone metabolism

in the context of breast cancer bone metastases. In conclusion, PPI appeared to have the

potential to control the destructive effects of osteolytic metastases, which might open

doors to new therapeutic approaches regarding this pathological condition.

67

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