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

Sara Daniela de Sousa Reis CHARACTERIZATION OF THE EFFECTS OF PROTON PUMP INHIBITORS ON BONE METABOLISM: IN VITRO STUDY IN COCULTURES OF OSTEOCLASTS AND BREAST CANCER CELLS AND IN CULTURES OF OSTEOBLASTS 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 2 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 3 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. 5 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 7 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 8 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 9 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. 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.) 17 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 HER2-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 luminalB breast cancer has been its poor outcome compared with the luminalA 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 breastconserving surgery. Radiation therapy is generally given over a 5or 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 estrogenresponsive 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 signaltransduction 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 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 benzimidazolebased 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 sideeffect 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 acidrelated 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-, gastrinor acetylcholinestimulated 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, 5dimethylthiazol-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. AntiVNR 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 Absorbance (550nm) 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 * * * * * * * * * 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 nmol/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 TRAP+ multinucleated cells 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 10-7M 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 % Resorption Area 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 antiosteoclastogenic 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 nmol/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 nmol/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 Absorbance (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 nmol/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 106 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 Absorvância (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 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 nmol/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) 64 Following the assessment of the osteoclastogenic effects of the different PPI in cocultures 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 References 1. Boyle, W.J., Simonet, W.S., and Lacey, D.L. (2003). Osteoclast differentiation and activation. Nature. 423: 337-342; 2. Datta, H.K.,Ng, W.F., Walker, J.A., Tuck, S.P., and Varanasi, S.S. (2008). The cell biology of bone metabolism. J. Clin. Pathol. 61: 577-587; 3. Marks, S.C., and Hermey, D.C. (1996). The structure and development of bone. In Principles of Bone biology. Bilezikian, J.P., Raisz, L.G., Rodan, G.A., ed. pp. 3-14; 4. Newton, A.C. (1995). Protein Kinase C: Structure, function and regulation. J. Biol. Chem. 270: 28495-28498; 5. Blair, H.C. (2006). Osteoclastic differentiation and function regulated by old and new pathways. Rev. Endocr. Metab. Disord. 7: 23-32; 6. Zhao, Q., Shao, J., Chen, W., and Li, Y. (2007). Osteoclast differentiation and gene regulation. Frontiers in Bioscience. 12: 2519-2529; 7. Mansky, K. C., Sankar, U., Han, J., and Ostrowsky, C. (2002). Microphthalmia transcription factor is a target of the p38 MAPK pathway in response to receptor activator of NF-kB ligand signaling. J. Biol. Chem. 277: 11077-11083; 8. Aeschlimann, D., and Evans, B. A. J. (2004). The vital osteoclast: how is it regulated? Cell Death and differentiation. 11: S5-S7; 9. Karsdal, M. A. et al. (2005). Acidification of the Osteoclastic Resorption Compartment Provides Insight into the Coupling of Bone Formation to Bone Resorption. American Journal of Pathology. Vol. 166. 2: 467-476; 10. Edwards, J. R. and Mundy, G. R. (2011). Advances in osteoclast biology: old findings and new insights from mouse models. Nature Reviews Rheumatology. 7: 235-243; 11. Tolar, J. Teitelbaum, SL. and Orchard, PJ. (2004). Osteopetrosis. N Engl J Med. 351:2839-2840; 12. Yamaguchi, A. et al. (2000). Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs and Cbfal. Endocrine Reviews. 21(4): 393-411; 13. Komori, T. (2006). Regulation of Osteoblast Differentiation by Transcription Factors. Journal of Cellular Biochemistry. 99: 1233-1239; 14. Stein, G. S. and Lian, J. B. (1993) Molecular mechanisms mediating developmental and hormone-regulated expression of genes in osteoblasts: an integrated relationship of cell growth and differentiation. In: Noda M, editor. Cellular and molecular biology of bone. Tokyo: Academic Press: 47–95; 15. Kasperk. C. et al. (1995). Human bone cell phenotypes differ depending on their skeletal site of origin. J. Clin. Endocrinol. Metab. 80(8): 2511-2517; 68 16. Neve, A. Corrado, A. and Cantatore, F. P. (2010). Osteoblast physiology in normal and pathological conditions. Cell Tissue Res.; 17. Aubin, J. E. Lian, J. B. Stein, G. S. (2006). Bone formation: maturation and functional activities of osteoblast lineage cells. Primer on the metabolic bone diseases and disorders of mineral metabolism. Washington, D.C.: The American society for bone and mineral research; 18. Adebisi, S. S. (2009). Current trends in human bone development and growth. J. Life Sci. 1: 9-14; 19. Costa-Rodrigues, J. Teixeira, C.A. Sampaio, P. and Fernandes, M.H. (2010). Characterization of the osteoclastogenic potencial of human osteoblastic and fibroblastic conditioned media. J. Cell. Biochem. 109: 205-216; 20. Junqueira, L.C., and Carneiro, J. (2008). Tecido Ósseo. In Histologia Básica (Guanabara Koogan) pp. 135-152; 21. Blair, H. C. (1998). How the osteoclast degrades bone. BioEssays. 20: 837-846; 22. Ross, F. P. et al. (1993). Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin avb3 potentiate bone resorption. J Biol Chem. 268:9901-9907; 23. Kierszenbaum, A. L. (2002). Histology and cell biology: an introduction to pathology. Elsevier Health Sciences Edition. 24. Chitu, V., and Stanley, E.R. (2006). Colony-stimulating factor-1 in immunity and inflammation. Curr. Opin. Immunol. 18: 39-48; 25. Fixe, P., Rougier, F., Ostyn, E., Gachard, N., Faucher, J. L., Praloran, V., and Denizot, Y. (1997) Spontaneous and inducible production of macrophage colony-stimulating factor by human bone marrow stromal cells. Eur. Cytokine Netw. 8: 91-95; 26. Pixley, F.J., and Stanley, E. R. (2004) CSF-1 regulation of the wandering macrophage: complexity in action. Trends Cell Biol. 14: 628-638; 27. Lacey, D. L., Timms, E., Tan, H. L., Kelley, M. J., Dunstan, C. R., Burgess, T., Elliott, R., Colombero, A., Elliot, G., Scully, S., et al. (1998). Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 93: 165-176; 28. Yasuda, H., Shima, N., Nakagawa, N., Mochizuki, S. I., Yano, K., Fujise, N., Sato, Y., Goto, M., Yamaguchi, K., Kuriyama, M., et al. (1998) Identify of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibitis osteoclastogenesis in vitro. Endocrinology. 139: 1329-1337; 29. Wong, B. R., Rho, J., Arron, J., Robinson, E., Orlinick, J., Chao, M., Kalachikov, S., Cayani, E., Bartlett, F. S., Lee, S. Y., et al. (1997). TRACE is a novel ligand of the tumor necrosis factor family that activates c-Jun N-terminal kinase in T cells. J. Biol. Chem. 272: 25190-25190-25194; 69 30. Wada, T., Nakashima, T., Hiroshi, N., and Penninger, J. M. (2006). RANKL-RANK signaling in osteoclastogenesis and bone disease. Trends Mol. Med. 12: 17-25; 31. Horton, M. A. (1997). The alpha v beta 3 integrin “vitronectin receptor”. Int. J. Biochem. Cell Biol. 29: 721-725; 32. Uy, H. L. et al. (1995). Effects of PTHrP and PTH on osteoclasts and osteoclast precursors in vivo. Endocrinology. 36:3207; 33. McSheehy, P. M. J. and Chambers, T. J. (1986). Osteoblastic cells mediate osteoclastic responsiveness to parathyroid hormone. Endocrinology. 118:824-828; 34. Abu-Amer, Y. (2005). Advances in Osteoclast Differentiation and Function. Immune, Endocrine & Metabolic Disorders. 5:347-355; 35. Nakamura, H. et al. (2003). Role of osteoclast extracellular signal-regulated kinase (ERK) in cell survival and maintenance of cell polarity. J. Bone. Miner. Res. 18: 1198-1205; 36. Minden, A. and Karin, M. (1997). Regulation and function of the JNK subgroup of MAP kinases. Biochim. Biophys. Acta. 1333: 85-104; 37. Miyazaki, T. et al. (2000). Reciprocal role of ERK and NFkB pathways in survival and activation of osteoclasts. J. Cell. Biol. Vol. 148. 2:333-342; 38. DeVita, V. T., Lawrence, T. S., Rosenberg, S. A., Weinberg, R. A. and DePinho, R. A. (2008). DeVita, Hellman, and Rosenberg’s cancer: principles & practice of oncology. 8th Edition. Lippincott Williams & Wilkins. 39. Richie, R. C. and Swanson, J. O. (2003). Breast cancer: a review of the literature. J. Insur. Med. 35: 101; 40. Kalogerakos, K. Sofoudis, C. and Baltayiannis, N. (2008). Early breast cancer: A review. Cancer Therapy. Vol 6: 463-476; 41. Pusztai, L. (2009). Gene expression profiling of breast cancer. Breast Cancer Research. 11 (Suppl. 3); 42. Sotiriou, C. and Pusztai, L. (2009). Gene expression signatures in breast cancer. N. Engl. J. Med. 360: 790-800; 43. Tran, B. and Bedard, P. L. (2011). Luminal B breast cancer and novel therapeutic. Breast Cancer Research. 13: 221; 44. Ring, A. and Dowsett, M. (2004). Mechanisms of tamoxifen resistance. Endocrine Related Cancer. 11: 643-658 45. Johnston, S. R. D. and Dowsett, M. (2003). Aromatase inhibitors for breast cancer: lessons from the laboratory. Nat. Rev. Canc. 3: 821-831; 46. Mundy, G.R. (2002). Metastasis to bone: causes, consequences and therapeutic opportunities. Nature Reviews. Volume 2; 47. Roodman, G.D. (2004). Mechanisms of bone metastasis. N. Engl. J. Med. 350: 16551664; 70 48. Feng, X. (2005). Regulatory roles and molecular signaling of TNF family members in osteoclasts. Gene. Vol. 350. 1: 1-13; 49. Maki, D. D., Grossman, R. I. (2000). Patterns of Disease Spread in Metastatic Breast Carcinoma: Influence of Estrogen and Progesterone Receptor Status. Am. J. Neuroradiol. 21: 1064-1066; 50. Kingsley, L. A. et al. (2007). Molecular biology of bone metastasis. Mol. Cancer Ther. 6: 2609-2617; 51. Kakonen, S. M., Mundy, G. R. (2003). Mechanisms of osteolytic bone metastases in breast carcinoma. Cancer Supplement. 97(3): 834-839; 52. James, J. J. et al. (2003). Bone metastases from breast carcinoma: histopathological – radiological correlations and prognostic features. B. J. Cancer. 89(4): 660-665; 53. Costa-Rodrigues, J., Moniz, K.A., Teixeira, M.R., and Fernandes M.H. (2012). Variability of the paracrine-induced osteoclastogenesis by human breast cancer lines. J. Cell. Biochem. 113: 1069-1079; 54. Foumier, P.G. et al. (2010). How do biphosphonates inhibit bone metastasis in vivo? Neoplasia. Vol. 12, 17: 571-578; 55. Fromigue, O., Kheddoumi, N., and Body, J.J. (2003). Bisphosphonates antagonise bone growth factors’ effects on human breast cancer cells survival. B. J. Cancer. 89: 178-184; 56. Gall, C. et al. (2007). A Cathepsin K Inhibitor Reduces Breast Cancer–Induced Osteolysis and Skeletal Tumor Burden. Cancer Res. 67 (20): 9894-99902; 57. Schlotter, C. M., Vogt, U. and Brandt. B. (2008). Molecular targeted therapies for breast cancer treatment – review. Breast Cancer Research. Vol. 10. 4: 211; 58. Higgins, M. J. and Baselga. J. (2011). Targeted therapies for breast cancer. The Journal of Clinical Investigation. Vol. 121. 10: 3797-3803; 59. Kennecke, H. et al. (2010). Metastatic behavior of breast cancer subtypes. J. Clin. Oncol. 28: 3271-3277; 60. Thomas, C. and Gustafsson, J.A. (2011). A CUE hints at tumor resisitance. Nature Medicine. 17: 658-660; 61. Hudis, C. A. (2007). Transtuzumab – Mechanism of action and use in clinical practice. N. Engl. J. Med. 357: 39-51; 62. Rogers, T. L. and Holen, I. (2011). Tumour macrophages as potential targets of bisphosphonates. Journal of Translational Medicine. 9: 177; 63. Guise, T. A. et al. (2006). Basic mechanism responsible for osteolytic and osteoblastic bone metastases. Clin. Cancer Res. 12: 6213-6216; 64. Pederson, L. Winding, B. and Foged, N. T. (1999). Identification of breats cancer cell linederived paracrine factors that stimulate osteoclast activity. Cancer Res. 59: 5849-5855; 71 65. Sachs, G., Shin, J.M., and Howden, C.M. (2006). Review article: the clinical pharmacology of proton pump inhibitors. Aliment. Pharmacol. Ther. 23 (suppl. 2) 2-8; 66. Yang, Y.X. (2008). Proton pump inhibitor therapy and osteoporosis. Current Drug Safety. 3: 204-209; 67. Cheng, H. C., Sheu, B. S. (2011). Intravenous proton pump inhibitors for peptic ulcer bleeding: Clinical benefits and limits. World J. Gastrointest. Endosc. 3(3): 49-56; 68. Thomson, A. B. et al. (2010). Safety of long-term use of proton pump inhibitors. World J. Gastroenterol. 16(19): 2323-2330; 69. Robinson, M. (2004). Review article: the pharmacodynamics and pharmacokinetics of proton pump inhibitors – overview and clinical implications. Aliment. Pharmacol. Ther. 20(6): 1-10; 70. McCarthy, D. M. (2010). Adverse effects of proton pump inhibitor drugs: clues and conclusions. Curr. Opin. Gastroenterol. 26: 624-631; 71. Olbe, L. Carlsson, E. and Lindberg, P. (2003). A proton pump inhibitors expedition: the case histories of omeprazole and esomeprazole. Nature Reviews Drug Discovery. 2: 132139; 72. Insogna, K. L. (2009). The effect of proton pump inhibiting drugs on mineral metabolism. Am. J. Gastroenterol. 104: S2-S4; 73. Ali, T. Roberts, D. N. and Tierney, W. M. (2009). Long term safety concerns with proton pump inhibitors. Am. J. Med. 122: 896-903; 74. Eom, C. S. et al. (2011). Use of acid suppressive drugs and risk of fracture: a metaanalysis of observational studies. Ann. Fam. Med. 9: 257-267; 75. Vaananen, H. K. et al. (1990). Evidence for the presence of a proton pump of the vacuolar H*-ATPase type in the ruffled borders of osteoclasts. J. Cell. Biol. 111: 1305-1311; 76. Luciani, F. et al. (2004). Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs. J. National Cancer Institute. Vol. 96, No.22; 77. Fais, S. et al. (2007). Targeting vacuolar H+-ATPase as a new strategy against cancer. Cancer Res.67: 10627-10630; 78. Sennoune, S. R. et al. (2003). Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity. Am. J. Physiol. Cell Physiol. 286: 1443-1452; 79. Zojaji, H. and Bonehy, S. M. (2008). Effect of omeprazole on bone mineral density and frequency of osteopenia and osteoporosis. Gastroenterology and Hepatology from Bed to Bench. 1(3): 123-126; 80. Targownik. L. E. et al. (2008). Use of proton pump inhibitors and risk of osteoporosisrelated fractures. C.M.A.J. 179(4): 319-326; 72 81. Mizunashi, K. et al. (1993). Effect of omeprazole, an inhibitor of H+/K+-ATPase, on bone resorption in humans. Calcif. Tissue. Int. 53: 21-25; 82. Tuukkanen, J. and Vaananen, H. K. (1986). Omeprazole, a specific inhibitor of H+/K+- ATPase, inhibits bone resorption in vitro. Calcif. Tissue. Int. 38: 123-125; 83. Morita, T. Nagaki, T. Fukuda I. and Okumura, K. (1992). Clastogenicity of low pH to various cultured mammalian cells. Mutant. Res. 268: 297-305; 84. Martinez-Zaguilan, R. et al. (1996). Acidic pH enhances the invasive behavior of human melanoma cells. Clin. Exp. Metastasis. 14: 176-186; 85. Hall, T. J. (1994). Cytotoxicity of vacuolar H+-ATPase inhibitors to UMR-106 rat osteoblasts: an effect on iron uptake into cells? Cell. Biol. Int. Vol. 18, No. 3, 189-193; 86. Francis, M. J. O. et al. (2002). ATPase pumps in osteoclasts and osteoblasts. Int. J. Biochem. Cell. Biol. 24: 459-476; 87. Anderson, T. et al. (1998). Pharmacokinetics and effect on caffeine metabolism of the proton pump inhibitors, omeprazole, lansoprazole, and pantoprazole. Br. J. Clin. Pharmacol. 45(4): 369-375; 88. Junghard, O. Hassan-Alin, M. and Hasselgren, G. (2002). The effect of the area under the plasma concentration vs time curve and the maximum plasma concentration of esomeprazole on intragastric pH. Eur. J. Clin. Pharmacol. 58(7): 453-458; 89. Ogston, N. et al. (2002). Dexamethasone and retinoic acid differentially regulate growth and differentiation in an immortalized human clonal bone marrow stromal cell line with osteoblastic characteristics. Steroids. Vol. 67. 11: 895-906; 90. Cheng, S. L. et al (1994). Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology. Vol. 134. 1: 277-286; 91. Cheng, S. L. Zhang, S. F. and Avioli, L. (1998). Expression of bone matrix proteins during dexamethasone-induced mineralization of human bone marrow stromal cells. J. Cell. Biochem. Vol. 61. 2: 182-193; 92. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72: 248254; 93. Yin, J. J. et al. (2003). A causal role for endothelin-1 in the pathogenesis of osteoblastic bone metastases. Proc. Natl. Acad. Sci. USA.100: 10954-10959.