The antineoplastic role of bisphosphonates: from basic research to clinical evidence

D. Santini1,+, U. Vespasiani Gentilucci2, B. Vincenzi1, A. Picardi2, F. Vasaturo3, A. La Cesa1, N. Onori4, S. Scarpa3 and G. Tonini1

Interdisciplinary Center for Biomedical Research (CIR), 1 Oncology, 2 Laboratory of Internal Medicine and Hepathology and 4 Clinical Pharmacology, University Campus Bio-Medico, Rome; 3 Department of Experimental Medicine and Pathology, ‘La Sapienza’ University, Rome, Italy

Received 19 December 2002; revised 7 March 2003; accepted 14 April 2003

Abstract

Bisphosphonates are now well established as successful agents for the prevention and treatment of postmenopausal osteoporosis, corticosteroid-induced bone loss and Paget’s disease. Bisphosphonates have also recently become important in the management of cancer-induced bone disease, and they now have a widely recognized role for patients with multiple myeloma and bone metastases secondary to breast cancer and prostate cancer. Recent studies suggest that, besides the strong antiosteoclastic activity, the efficacy of such compounds in the oncological setting could also be due also to direct antitumor effect, exerted at different levels. Here, after a brief analysis of the chemical structure, we will review the antineoplastic and biological properties of bisphosphonates. We will start from well estabilished mechanisms of action and go on to discuss the latest evidence and hypotheses. In particular, we will review the antiresorptive properties in malignant osteolysis and the recent evidence of a direct antitumor effect. Furthermore, this review will analyze the influence of bisphosphonates on cancer growth factor release, their effect on cancer cell adhesion, invasion and viability, the proapoptotic potential on cancer cells, the antiangiogenic effect, and, finally, the immunomodulating properties of bisphosphonates on the {gamma}{delta} T cell population.

Key words: angiogenesis, antineoplastic, apoptosis, bisphosphonates, clinical and preclinical evidence

Introduction

Bisphosphonates are analogs of endogenous pyrophosphates in which a carbon atom replaces the central oxygen atom. Their potential for strong inhibition of osteoclastic bone resorption has progressively extended the field of their clinical indications. Bisphosphonates are now well established as successful agents for the prevention and treatment of postmenopausal osteoporosis, since they have been shown to increase bone mass and diminish by half fracture rates at the spine, hip and other sites in postmenopausal osteoporosis [1, 2]. They are also utilized in corticosteroid-induced bone loss [3]. In Paget’s disease, bisphosphonates can alleviate bone pain in the short term and prevent bone, joint, and neurological complications in the long term [4, 5]. Bisphosphonates have also recently become important in the management of cancer-induced bone disease, and they have now a widely recognized role for patients with multiple myeloma and bone metastases secondary to breast cancer [611]. Clinical studies have shown that, independent of the method of administration (intravenous or oral), bisphosphonates can reduce the overall amount of skeletal events in patients with myeloma and breast cancer by ~50% [7, 8]. Increased osteoclastic bone resorption is the central mechanism underlying hypercalcemia of malignancy, and bisphosphonates have been shown to be extremely effective in the management of this disorder. In fact, bisphosphonates are the treatment of choice for this disorder [12]. Recent studies have suggested that, besides the strong antiosteoclastic activity, the efficacy of such compounds in the oncological setting could also be due to a direct antitumor effect. Here, after a brief analysis of the chemical structure, we will review the antineoplastic and biological properties of bisphosphonates, starting from well estabilished mechanisms of action and finishing with the latest evidence and hypotheses.

Chemical structure

Bisphosphonates are compounds with a chemical structure that closely resembles that of inorganic pyrophosphate (PPi). Whereas the two phosphate groups in PPi are linked by phosphoanidride bonds, which are extremely unstable, the two phosphonate groups of bisphosphonates are linked to the central carbon atom by highly hydrolysis-resistant phosphoether bonds. The central carbon atom can form two additional covalent bonds, and the resulting side chains are usually indicated as R1 and R2 (Figure 1). The P-C-P moiety of bisphosphonates is responsible for their strong affinity for divalent metal ions, such as calcium ions, and for the skeleton. Furthermore, when the R1 side chain is a hydroxyl group, such compounds are able to chelate calcium ions more effectively, by tridentate rather than bidentate binding (Figure 1). Etidronate, the first bisphosphonate used to treat a human disease, was synthesized exactly 100 years ago [13]. It consists in a simple chemical structure in which the R1 side chain is a methyl group (-CH3) and the R2 side chain is a hydroxyl (-OH). When the the length of the R2 side chain was increased from a simple methyl group to longer alkyl chains, significantly more potent compounds were obtained [14]. An up to 1000-fold increase in potency was achieved by the introduction of a primary amino group (-NH2) at the extremity of the R2 alkyl chain, to form the amino-bisphosphonates (e.g. alendronate, pamidronate and neridronate) [15]. Amino-bisphosphonates with a secondary amino group (e.g. incadronate) and a tertiary amino group (e.g. olpadronate) are even more effective, and potency reaches the peak when the tertiary nitrogen is included within a ring structure in the R2 side chain (as in risedronate and zoledronic acid) [16, 17] (Figure 1).



View larger version (26K):
[in this window]
[in a new window]
 
Figure 1. Bisphosphonate chemical structure. The two phosphonate groups are linked to the central carbon atom by phosphoeter bonds. The carbon atom forms two additional covalent bonds, and the resulting side chains are referred to as R1 and R2.

 
Antiresorptive properties in malignant osteolysis

Numerous clinical studies have shown that bisphosphonates can reduce the occurrence of pathological fractures, bone pain, hypercalcemic episodes, and the need for radiation therapy and surgery in patients with osteolytic bone metastases [611]. Such compounds are particularly indicated in the palliative setting, for the reduced side-effects and the improvement in quality of life deriving from the reduction of skeletal events. Bisphosphonates have been clearly demonstrated to reduce tumor osteolysis and bone destruction and to prolong survival in animal tumor models [1820]. Such efficacy is first of all related to the inhibitory activity on osteoclast resorption. Bisphosphonates affect osteoclast-mediated bone resorption in a variety of ways, which include effects on osteoclast formation, resorptive activity and viability [2123]. Osteoclasts are the bone cells most likely to be exposed to high concentrations of drug; experimental studies support the hypothesis that they are able to internalize bisphosphonates by endocytosis [24]. After cellular uptake, bisphosphonate-treated osteoclasts show important changes in morphology, both in vitro [23, 25] and in vivo [2628]. These include the lack of the ruffled border [29], the disruption of cytoskeleton and the loss of actin rings [30, 31]. These structural alterations lead to a decreased osteoclast function and are by themselves sufficient to prevent bone resorption [32]. Furthermore, it has been reported that bisphosphonates can induce osteoclast apoptosis, both in vitro and in vivo [22, 33]. Another mechanism by which bisphosphonates exert their antiresorptive activity is the inhibition of osteoclast differentiation. In fact, such compounds have been shown to inhibit the formation of osteoclast-like cells in a dose-dependent manner in long-term cultures of human bone marrow [21]. These results suggest that the antiproliferative activity on osteoclasts could play a major role in bisphosphonate efficacy. Finally, it has been shown that bisphosphonates may also act through the modulation of the osteoclast–osteoblast interrelations. When pure osteoblastic cell populations were pretreated with alendronate and ibandronate and then co-cultured with osteoclasts, a decrease in osteoclast-mediated resorption was observed [34]. It was subsequently shown that bisphosphonate-treated osteoblasts produce an inhibitor of osteoclast resorption, which has not yet been well characterized but is known to be a labile factor of ~3–4 kDa [35].

Molecular level of action
The molecular events by which bisphosphonates lead to osteoclast inactivation and diminish osteoclast maturation are not fully understood. To date, there is no evidence of active receptors or binding sites on the osteoclast membrane. However, it is well known that bisphosphonates can be divided in two different classes (amino-bisphosphonates and non-amino-bisphosphonates), not only because of the molecular structures (as discussed above), but also the intracellular mechanism of action. Early studies suggested that non-amino-bisphosphonates, such as etidronate and clodronate, can affect a large number of metabolic processes, including glycolysis, fatty acid oxidation and lactate production [36, 37]. Non-amino-bisphosphonates can be metabolized to methylene-containing analogs of ATP, which are extremely resistant to hydrolysis [38, 39]. These metabolic reactions are likely to be catalyzed by members of the family of type 2 class of aminoacyl-tRNA synthetases [40], which play a pivotal role in protein synthesis. Since osteoclasts are able to internalize bisphosphonates, consequently formed non-hydrolyzable ATP analogs can accumulate intracellularly and inhibit metabolic enzymes, such as phosphatases and pyrophosphatases [41, 42], which play important roles in cellular growth, differentiation and activity. On the other hand, the more potent amino-bisphosphonates (such as alendronate, risedronate, pamidronate, ibandronate and zoledronic acid) are not metabolized to ATP analogs, and show a completely different mechanism of action. These compounds are able to inhibit the mevalonate pathway [43, 44] (Figure 2), a biosynthetic pathway on which depends the synthesis of cholesterol and isoprenoid lipids, such as isopentenyldiphosphate (IPP), farnesyl diphosphate (FPP) and geranylgeranyldiphosphate (GGPP). FPP and GGPP are required for the prenylation of small GTPases, such as Ras, Rho and Rac [45], which are signaling proteins that regulate a variety of cellular processes. The lack of GTPase prenylation, derived from inhibition of the mavelonate pathway, is responsible for their inadequate function [46]. The function of these GTPases has been shown to be important for osteoclast morphology and activity [47]. Therefore, inhibiting farnesyl pyrophosphate synthase or other enzymes of the mevalonate pathway [43, 44], amino-bisphosphonates can deprive osteoclasts of important regulators of intracellular dynamics, leading to poor cell functioning or programmed cell death. This mechanism of action is confirmed by the ability of geranylgeraniol (a cell-permeable form of GGPP) and, to a lesser extent, farnesol (a cell-permeable form of FPP) to protect osteoclasts from the inhibitory properties of amino-bisphosphonates [48, 49].



View larger version (16K):
[in this window]
[in a new window]
 
Figure 2. Bisphosphonate level of action on the mevalonate pathway. Bisphosphonates inhibit the formation of farnesyl diphosphate (PP) and geranylgeranyl-PP, which are required for the prenylation of small GTPases that regulate a variety of important cell processes. HMG-Co A, 3-hydroxy-3-methylglutaryl-Co A; GTPases, guanosine triphosphatases.

 
Direct antitumor effects

Bisphosphonate efficacy for the treatment of bone metastases was initially thought to depend only on the antiosteoclast activity of such compounds. Antiresorptive properties were considered to be sufficent by themselves to explain their ability to reduce skeletal morbidity in patients with lytic bone disease. Bisphosphonates were shown to inhibit establishment and growth of osteoblastic bone metastases from prostate cancer; the efficacy was attributed to the observation that abnormal osteoblastic bone formation within metastases is preceded by osteoclastic activation [50]. However, several lines of evidence suggesting a direct antitumor effect of bisphosphonates have progressively accumulated. Sasaki et al. [51] demonstrated a reduced tumor burden in nude mice pretreated with risedronate and then injected with human breast carcinoma cells. In the same animal model, risedronate was shown to also reduce the number, extent and size of bone metastases when given after tumor cell inoculation [52]. In humans, trials with adjuvant clodronate in primary breast carcinoma reported conflicting results. Diel et al. [9] described a reduction in bone and, surprisingly, visceral metastases, and an improvement in survival in patients with bone marrow disseminated tumor cells. On the other hand, Saarto et al. [53] reported an increase in the incidence of bone and visceral metastases and a poorer survival in node-positive breast cancer patients. Even though further investigation of bisphosphonates in the adjuvant setting is still required, such observations led to the suggestion that bisphosphonates might have some kind of direct effect on cancer cells. Currently, several studies are being published with the aim of clarifying the mechanisms by which this effect is achieved.

Effect on cancer growth factor release
It was postulated previously that bisphosphonates could have an antitumor activity by altering the release of growth factors in the bone microenvironment. Although calcified bone matrix shows relatively low cellularity and metabolic activity, it stores many different osteoblast-derived growth factors [54]. These growth factors, such as transforming growth factor-ß (TGF-ß) and insulin-like growth factor-I (IGF-I), are released into the bone marrow as a consequence of osteoblast resorption, and represent the essential nutrients for cancer cells localized in bone [55]. Furthermore, although still controversial, it has been shown that cancer cells are unable to degrade bone matrix by themselves [56], meaning that the release of such essential nutrients is strictly osteoclast resorption related. Moreover, cultured human breast cancer cells respond to TGF-ß by releasing PTHrP, a major stimulator of osteoclast activity in breast cancer osteolysis [57, 58]. Thus, there is a vicious circle, where osteoclasts and cancer cells interact through the mediation of several soluble factors in the bone microenvironment. Bisphosphonates may interrupt this cycle by decreasing osteoclast activity, thereby inhibiting the release of TGF-ß, IGF-I and other peptides from bone matrix. Cancer cells result deprived of essential nutrients and reduce the release of osteoclast stimulating factors.

Effect on cancer cell adhesion, invasion and viability
As reviewed elsewhere [59], the process leading to bone metastases involves cancer cell migration, adhesion to cortical bone and finally invasion of extracellular bone matrix. It has been shown previously that extracellular matrix-bound bisphosphonates may inhibit the adhesion of osteoclast precursors and inhibit their subsequent differentiation into mature resorbing osteoclasts [60, 61]. Other studies have suggested that such compounds may also inhibit the attachment of mature osteoclasts to cortical bone slices and to specific extracellular matrix proteins, such as bone sialoprotein-derived peptide [62]. Recently, a similar effect was observed on cancer cell lines. Bisphosphonate pretreatment of prostate and breast carcinoma cell lines has been shown to inhibit tumor cell adhesion to unmineralized and mineralized bone extracellular matrices in a dose-dependent way [63]. Van der Pluijm et al. [64] showed that pretreatment of bovine cortical bone slices and cryostat sections of trabecular bone from neonatal mouse tail with amino-bisphosphonates (pamidronate, olpadronate, alendronate and ibandronate) can inhibit the adhesion of breast cancer cells in a dose-dependent manner. In contrast, no effect was obtained with the non-amino-bisphosphonates clodronate and etidronate [64]. The order of potency of the four amino-bisphosphonates corresponded to their ranking in bone resorption assays. Moreover, Boissier et al. [65] demonstrated that bisphosphonate pretreatment inhibits breast and prostate carcinoma cell invasion in a dose-dependent manner. Although no activity was observed on cancer cell matrix metalloproteinases (MMPs) production, bisphosphonates were shown to inhibit MMP activity through zinc chelation [65]. Senaratne et al. [66] investigated the in vitro effects of bisphosphonates zoledronic acid, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines. All four bisphosphonates significantly reduced cell viability in all three cell lines. In addition, in different murine models bisphosphonate markedly inhibited the progression of established osteolytic lesions and the expansion of breast and prostate cancer cells within bone [67]. Finally, Magnetto et al. [68] reported the ability of ibandronate to enhance the antitumor activity of taxoids against breast cancer cell invasion and adhesion to bone.

Proapoptotic effect on cancer cells
Recently, numerous studies have shown that bisphosphonates can induce apoptosis in in vitro models. These results are consistent with earlier reports that bisphosphonates can inhibit cell proliferation and induce apoptosis in osteoclasts [33] and in J774 macrophage-like cells [69]. In the work previously mentioned by Senaratne et al. [66], the investigators demonstrated that all the bisphosphonates studied decreased breast cancer cell number and viability, but also induced apoptosis in a dose-dependent manner. This study has shown that there is no equivalence between bisphosphonate potency in bone resorption and in the induction of apoptosis: zoledronic acid was the most effective compound. The proapoptotic efficacy of zoledronic acid on breast cancer cells was confirmed in two subsequent studies [70, 71]. In the first, Jadgev et al. [70] showed that acute exposure to zoledronic acid (2, 6 and 12 h), more accurately reflecting the in vivo condition than long-term exposure (72 h), was sufficient to determine an antitumor effect in breast cancer cells. A synergic action with paclitaxel was also observed. Moreover, zoledronic acid-induced apoptosis was inhibited by the addition of intermediates of the mevalonate pathway (completely inhibited by geranylgeraniol and partially by farnesol), suggesting that amino-bisphosphonate activity on breast cancer cells is strictly related to the inhibition of enzymes of the same pathway [70]. The second study aimed to identify the signaling pathways involved in zoledronic acid-induced apoptosis [71]. Zoledronate treatment was shown to induce the failure of Ras protein membrane localization, the release of mitochondrial cytochrome c into the cytosol and the subsequent activation of caspase-3 proteases [71]. Such events were inhibited by the addition of farnesol, and by forced expression of bcl-2. The authors suggested that the reduced Ras protein prenylation, with impaired membrane localization and functioning, represents the initial event inducing apoptosis [71]. This apoptosis is associated with the release of cythocrome c into the cytosol and the subsequent activation of the caspase cascade. Fromigue et al. [72] demonstrated that the apoptosis of breast cancer cells induced by clodronate, pamidronate, ibandronate and zoledronic acid was almost completely reversed by the z-VAD-fmk caspase inhibitor. This suggests a role of caspase activation in bisphosphonate-induced apoptosis. When analyzing non-amino-bisphosphonate-induced apoptosis, mechanisms other than the lack of Ras-protein prenylation need to be considered. In fact, caspase activation should be induced by toxic ATP analogs accumulating intracellularly. Bisphosphonate activity was also observed in other cancer cell lines. Shipman et al. [73] demonstrated that the amino-bisphosphonate incadronate may induce apoptosis of human multiple myeloma cells in vitro. This effect is mediated by the inhibition of the mevalonate pathway [74], and is completely abrogated by forced expression of bcl-2 [75]. Moreover, pamidronate and clodronate were shown to inhibit cell proliferation and to induce apoptosis in the UMR 106-01 clonal rat osteosarcoma cell line in a dose- and time-dependent fashion [76]. Recently, Riebeling et al. [77] reported that pamidronate can induce apoptosis and inhibit proliferation of human melanoma cells in vitro in a dose-dependent manner. In contrast, clodronate did not show any effect on the same melanoma cell line. Apoptosis was associated with caspase-3 activation and was strongly reduced by the addition of geranylgeraniol to the culture medium. On the other hand, p53 or bcl-2 overexpression did not abolish pamidronate-induced apoptosis [77]. Finally, Lee et al. [78] reported that pamidronate and zoledronic acid significantly reduce the growth of prostate cancer cell lines in vitro. Pamidronate treatment was shown to induce significant amounts of cell death, while only zoledronic acid exerted a dramatic effect on cell proliferation [78].

Antiangiogenic effect
Recent evidence suggests that part of the antitumor activity of bisphosphonates may be attributed to an antiangiogenic effect. The studies by Wood et al. [79] with the amino-bisphosphonate zoledronate are the most important in describing this effect. The authors reported that the cell proliferation induced by fetal calf serum, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) is inhibited by zoledronic acid on human umbilical vein endothelial cells (HUVEC) in vitro. Zoledronic acid inhibition on HUVEC migration was dependent on the dose administered. Zoledronic acid was also shown by the same investigators to reduce vessel sprouting in cultured aortic rings and in the chicken egg chorioallantoic membrane assay. Moreover, in a subcutaneous growth factor implant model in mice, zoledronate treatment strongly inhibited the angiogenic response induced by bFGF and VEGF [80]. The INSERM research group has recently demonstrated that zoledronic acid clearly inhibits the angiogenesis both in bone and in prostate tissues in a murine model [81]. Finally, for the first time in humans, our research group showed a significant decrease of circulating levels of VEGF in bone metastatic cancer patients receiving a single dose of pamidronate [82]. A significant decrease of VEGF was already evident the first day after single pamidronate infusion (90 mg), and the effect was still pesent on day 7. A shorter-lasting increase of interferon (IFN)-{gamma} circulating levels was also observed, while no significant modifications in interleukin (IL)-8 levels were found. With these encouraging results, further investigations on the antiangiogenic effects of bisphosphonates both in vitro and in vivo are urgently needed.

Effect on {gamma}{delta} T cell activation and proliferation
{gamma}{delta} T cells represent a minor subset of human peripheral T cells (1–10%), differing from {alpha}ß T cells in that they have a limited combinatorial diversity of T-cell receptor (TCR) and an human leukocyte antigen-unrestricted antigen recognition site. In adults, most of these {gamma}{delta} T cells present a V{gamma}9/V{delta}2 TCR [83], and are able to recognize a broad spectrum of non-peptide compounds of low molecular weight (100–600 Da) with an essential phosphate residue. {gamma}{delta} T cells are suggested to play a surveillance role for infected and transformed cells [84], and they have already been shown to recognize and lyze certain hematopoietic tumor cells (such as the Burkitt’s lymphoma cell line Daudi, and myeloma cell lines PPMI 8226 and U266) in vitro [8587]. Kunzmann et al. [87, 88] demonstrated for the first time that amino-bisphosphonates are potent activators of human {gamma}{delta} T cells both in vitro and in vivo. In the first study, the amino-bisphosphonates alendronate, ibandronate and pamidronate have been shown to induce a dose-dependent activation and expansion of {gamma}{delta} T cells in primary peripheral blood mononuclear cell cultures of healthy donors at clinically relevant concentrations, while non-amino-bisphosphonates clodronate and etidronate were demonstrated to be inactive [87]. The activation of {gamma}{delta} T cells was associated with CD25 and CD69 expression and increased secretion of IFN-{gamma}, while {gamma}{delta} T cell proliferation was obtained only when low doses of exogenous IL-2 were added to the culture medium. In the same study, pamidronate-treated bone marrow cultures of patients with multiple myeloma showed a reduced survival of plasma cells [87]. Such reduction in survival was observed particularly when the bone marrow {gamma}{delta} T cells were activated, suggesting that pamidronate can induce a {gamma}{delta} T cell activity against the plasma cells through a direct cell contact-dependent lysis or by the secretion of inhibitory cytokines such as IFN-{gamma} [89]. In vivo, pamidronate treatment has been shown to significantly increase the number of peripheral blood {gamma}{delta} T cells [90]. Amino-bisphosphonate activity on {gamma}{delta} T cells (V{gamma}9/V{delta}2 subset) has been demonstrated to be strictly dependent on the presence of monocyte lineage cells [90]. It was suggested that monocytes act as antigen presenting cells, presenting amino-bisphosphonates to {gamma}{delta} T cells, and then determining their activation [90]. In conclusion, the amino-bisphosphonates activate the {gamma}{delta} T cell population, which shows potential cytotoxic activity toward a broad spectrum of tumors [8991]. This represents another intriguing aspect of the antitumor activity of such compounds, which deserves further investigation.

Conclusions

The literature clearly shows the potential and impressive antineoplastic properties of bisphosphonates (Tables 1 and 2), and sheds new light on the biological applications of such compounds in the clinical setting. Bisphosphonates interact with osteoclasts, osteoblasts, tumor cells, cytokine and growth-factor production, leading to the interruption of bone destruction. For these reasons they may represent a new class of drug with antitumor power.


View this table:
[in this window]
[in a new window]
 
Table 1. Experimental evidence of bisphosphonate’s direct antitumor activity
 

View this table:
[in this window]
[in a new window]
 
Table 2. Evidence of bisphosphonate antitumor activity in cancer patients
 
Several ongoing preclinical trials are evaluating the possibility of associating zoledronic acid with: (i) tyrosine kinase inhibitors, such as imatinib; (ii) aromatase inhibitors, such as letrozole; and (iii) antineoplastic agents, such as paclitaxel or docetaxel, with the aim of obtaining a synergic effect in experimental models.

Other fields of research concern the immunomodulating properties of bisphosphonates on {gamma}{delta} T cells, their proapoptotic and antiangiogenic potentials and their use as radiation sensitizers. All these findings indicate that it is possible to extend the potential use of bisphosphonates, and in particular of zoledronic acid, to other diseases that involve angiogenesis or an immunogical component.

Footnotes

+ Correspondence to: Dr D. Santini, University Campus Bio-Medico, Via Emilio Longoni 83, 00155 Rome, Italy. Tel: +39-06-22541739; Fax: +39-06-22541445; E-mail: d.santini{at}unicampus.it Back

References

1. Watts NB, Harris ST, Genant HK et al. Intermittent cyclical etidronate treatment of postmenopausal osteoporosis. N Engl J Med 1990; 323: 73–79.[Abstract]

2. Harris ST, Watts NB, Jackson RD et al. Four-year study of intermittent cyclic etidronate treatment of postmenopausal osteoporosis: three years of blinded therapy followed by one year of open therapy. Am J Med 1993; 95: 557–567.[ISI][Medline]

3. Reid IR, King AR, Alexander CJ, Ibbertson HK. Prevention of steroid-induced osteoporosis with (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD). Lancet 1988; 1: 143–146.[Medline]

4. Roux C, Gennari C, Farrerons J et al. Comparative prospective, double-blind, multicenter study of the efficacy of tiludronate and etidronate in the treatment of Paget’s disease of bone. Arthritis Rheum 1995; 38: 851–858.[Medline]

5. Miller PD, Brown JP, Siris ES et al. A randomized, double blind comparison of risedronate and etidronate in the treatment of Paget’s disease of bone. Paget’s Risedronate/Etidronate Study Group. Am J Med 1999; 106: 513–520.[CrossRef][ISI][Medline]

6. Paterson AHG, Powles TJ, Kanis JA et al. Double-blind controlled trial of oral clodronate in patients with bone metastases from breast cancer. J Clin Oncol 1993; 11: 59–65.[Abstract]

7. Berenson J, Lichtenstein A, Porter L et al. Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma. Myeloma Aredia Study Group. N Engl J Med 1996; 334: 488–493.[Abstract/Free Full Text]

8. Hortobagyi GN, Theriault RL, Porter L et al. Efficacy of pamidronate in reducing skeletal complications in patients with breast cancer and lytic bone metastases. Protocol 19 Aredia Breast Cancer Study Group. N Engl J Med 1996; 335: 1785–1791.[Abstract/Free Full Text]

9. Diel IJ, Solomayer EF, Costa SD et al. Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N Engl J Med 1998; 339: 357–363.[Abstract/Free Full Text]

10. McCloskey EV, MacLennan CM, Drayson MT et al. A randomized trial of the effect of clodronate on skeletal morbidity in multiple myeloma. MRC Working Party on Leukaemia in Adults. Br J Haematol 1998; 100: 317–325.[CrossRef][ISI][Medline]

11. Theriault RL, Lipton A, Hortobagyi GN et al. Pamidronate reduces skeletal morbidity in women with advanced breast cancer and lytic bone lesions: a randomized, placebo-controlled trial. Protocol 18 Aredia Breast Cancer Study Group. J Clin Oncol 1999; 17: 846–854.[Abstract/Free Full Text]

12. Fleisch H. Bisphosphonates. Pharmacology and use in the treatment of tumor-induced hypercalcemic and metastatic bone disease. Drugs 1991; 42: 919–944.[ISI][Medline]

13. Von Baeyer H, Hofmann KA. Acetodiphosphorige Saure. Beitr Dtsch Chem Ges 1897; 30: 1973–1978.

14. Shinoda H, Adamek G, Felix R et al. Structure–activity relationship of various bisphosphonates. Calcif Tissue Int 1983; 35: 87–99.[ISI][Medline]

15. Schenk R, Eggli P, Fleisch H, Rosini S. Quantitative morphometric evaluation of the inhibitory activity of new amino-bisphosphonates on bone resorption in the rat. Calcif Tissue Int 1986; 38: 342–349.[ISI][Medline]

16. Sietsema WK, Ebetino FH, Salvagno AM, Bevan JA. Antiresorptive dose–response relationship across three generations of bisphosphonates. Drugs Exp Clin Res 1989; 15: 389–396.[ISI][Medline]

17. Green JR, Muller K, Jaeggi KA. Preclinical pharmacology of CGP 42'446, a new, potent, heterocyclic bisphosphonate compound. J Bone Miner Res 1994; 9: 745–751.[ISI][Medline]

18. Johnson KY, Wesseler MA, Olson HM et al. The effects of diphosphonates on tumor-induced hypercalcemia and osteolysis in Walker carcinosarcoma 256 (W-256) of rats. In Donath A, Courvoisier B (eds): Diphosphonates and Bone. Geneva, Switzerland: Editions Medecine et Hygiene 1982; 386–389.

19. Jung A, Bornand J, Mermillod B et al. Inhibition by diphosphonates of bone resorption induced by the Walker tumor of the rat. Cancer Res 1984; 44: 3007–3011.[Abstract]

20. Martodam RR, Thornton KS, Sica DA et al. The effects of dichloromethylene diphosphonate on hypercalcemia and other parameters of the humoral hypercalcemia of malignancy in the rat Leydig cell tumor. Calcif Tissue Int 1983; 35: 512–519.[ISI][Medline]

21. Hughes DE, Mac Donald B, Russell RGG, Gowen M. Inhibition of osteoclast-like cell formation by bisphosphonates in long term cultures of human bone marrow. J Clin Invest 1989; 83: 1930–1935.[ISI][Medline]

22. Hughes DE, Wright KR, Uy HL et al. Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res 1995; 10: 1478–1487.[ISI][Medline]

23. Sato M, Grasser W. Effects of bisphosphonates on isolated rat osteoclasts as examined by reflected light microscopy. J Bone Miner Res 1990; 5: 31–40.[ISI][Medline]

24. Fisher JE, Rogers MJ, Halasy JM et al. Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption and kinase activation in vitro. Proc Natl Acad Sci USA 1999; 96: 133–138.[Abstract/Free Full Text]

25. Endo Y, Nakamura M, Kikuchi T et al. Aminoalkylbisphosphonates, potent inhibitors of bone resorption, induce a prolonged stimulation of histamine synthesis and increase macrophages, granulocytes, and osteoclasts in vivo. Calcif Tissue Int 1993; 52: 248–254.[ISI][Medline]

26. Schenk R, Merz WA, Muhlbauer R et al. Effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl2[scap]mDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats. Calcif Tissue Res 1973; 11: 196–214.[ISI][Medline]

27. Plasmans CM, Jap PHK, Kuijpers W, Slooff TJ. Influence of a diphosphonate on the cellular aspect of young bone tissue. Calcif Tissue Int 1980; 32: 247–266.[ISI][Medline]

28. Miller SC, Jee WS. The effect of dichloromethylenediphosphonate, a pyrophosphate analog, on bone and bone cell structure in the growing rat. Anat Rec 1979; 193: 439–462.[ISI][Medline]

29. Flanagan AM, Chambers TJ. Inhibition of bone resorption by bisphosphonates: interactions between bisphosphonates, osteoclasts, and bone. Calcif Tissue Int 1991; 49: 407–415.[ISI][Medline]

30. Murakami H, Takahashi N, Sasaki T et al. A possible mechanism of the specific action of bisphosphonates on osteoclasts: tiludronate preferentially affects polarized osteoclasts having ruffled borders. Bone 1995; 17: 137–144.[CrossRef][ISI][Medline]

31. Selander K, Lehenkari P, Vaananen HK. The effects of bisphosphonates on the resorption cycle of isolated osteoclasts. Calcif Tissue Int 1994; 55: 368–375.[ISI][Medline]

32. Halasy-Nagy JM, Rodan GA, Reszka AA. Inhibition of bone resorption by alendronate and risedronate does not require osteoclast apoptosis. Bone 2001; 29: 553–559.[CrossRef][ISI][Medline]

33. Ito M, Amizuka N, Nakajima T, Ozawa H. Ultrastructural and cytochemical studies on cell death of osteoclasts induced by bisphosphonate treatment. Bone 1999; 25: 447–452.[CrossRef][ISI][Medline]

34. Sahni M, Guenther HL, Fleisch H et al. Bisphosphonates act on rat bone resorption through the mediation of osteoblasts. J Clin Invest 1993; 91: 2004–2011.[ISI][Medline]

35. Vittè C, Fleisch H, Guenther HL. Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption. Endocrinology 1996; 137: 2324–2333.[Abstract]

36. Fast DK, Felix R, Dowse C et al. The effects of diphosphonates on the growth and glycolysis of connective-tissue cells in culture. Biochem J 1978; 172: 97–107.[ISI][Medline]

37. Felix R, Fleisch H. Increase in fatty acid oxidation in calvaria cells cultured with diphosphonates. Biochem J 1981; 196: 237–245.[ISI][Medline]

38. Rogers MJ, Ji X, Russell RG et al. Incorporation of bisphosphonates into adenine nucleotides by amoebae of the cellular slime mould Dictyostelium discoideum. Biochem J 1994; 303–311.

39. Pelorgeas S, Martin JB, Satre M. Cytotoxicity of dichloromethane diphosphonate and of 1-hydroxyethane-1,1-diphosphonate in the amoebae of the slime mould Dictyostelium discoideum. A 31P NMR study. Biochem Pharmacol 1992; 44: 2157–2163.[CrossRef][ISI][Medline]

40. Rogers MJ, Brown RJ, Hodkin V et al. Bisphosphonates are incorporated into adenine nucleotides by human aminoacyl-tRNA synthetase enzymes. Biochem Biophys Res Commun 1996; 224: 863–869.[CrossRef][ISI][Medline]

41. Felix R, Graham R, Russell RGG, Fleisch H. The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes. Biochim Biophys Acta 1976; 429: 429–438.[ISI][Medline]

42. Shimdt A, Rutledge SJ, Endo N et al. Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate. Proc Natl Acad Sci USA 1996; 93: 3068–3073.[Abstract/Free Full Text]

43. Van Beek E, Pieterman E, Cohen L et al. Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates. Biochem Biophys Res Commun 1999; 264: 108–111.[CrossRef][ISI][Medline]

44. Bergstrom JD, Bostedor RG, Masarachia PJ et al. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch Biochem Biophys 2000; 373: 231–241.[CrossRef][ISI][Medline]

45. Zhang FL, Casey PJ. Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem 1996; 65: 241–269.[CrossRef][ISI][Medline]

46. Marshall CJ. Protein prenylation: a mediator of protein–protein interactions. Science 1993; 259: 1865–1866.[ISI][Medline]

47. Zhang D, Udagawa N, Nakamura I et al. The small GPT-binding protein, Rho p21, is involved in bone resorption by regulating cytoskeletal organization in osteoclasts. J Cell Sci 1995; 108: 2285–2292.[Abstract/Free Full Text]

48. Luckman SP, Hughes DE, Coxon FP et al. Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translationalprenylation of GTP-binding proteins, including Ras. J Bone Miner Res 1998; 13: 581–589.[ISI][Medline]

49. Van Beek E, Lowik C, Van der Pluijm G, Papapoulos S. The role of geranylgeranylation in bone resorption and its suppression by bisphosphonates in fetal bone explants in vitro: a clue to the mechanism of action of nitrogen-containing bisphosphonates. J Bone Miner Res 1999; 14: 722–729.[ISI][Medline]

50. Adami S. Bisphosphonates in prostate carcinoma. Cancer 1997; 80: 1674–1679.[CrossRef][ISI][Medline]

51. Sasaki A, Boyce BF, Story B et al. Bisphosphonate risedronate reduces metastatic human breast cancer burden in nude mice. Cancer Res 1995; 55: 3551–3557.[Abstract]

52. Hall DG, Stoica G. Effect of the bisphosphonate risedronate on bone metastases in a rat mammary adenocarcinoma model system. J Bone Miner Res 1994; 9: 221–230.[ISI][Medline]

53. Saarto T, Blomqvist C, Virkkunen P, Elomaa I. Adjuvant clodronate treatment does not reduce the frequency of skeletal metastases in node-positive breast cancer patients: 5-year results of a randomized controlled trial. J Clin Oncol 2001; 19: 10–17.[Abstract/Free Full Text]

54. Hauschka PV, Mavrakos AE, Iafrati MD et al. Growth factors in bone matrix. Isolation of multiple types by affinity chromatography on heparin sepharose. J Biol Chem 1986; 261: 12665–12674.[Abstract/Free Full Text]

55. Pfeilshifter J, Mundy GR. Modulation of type ß transforming growth factor activity in bone cultures by osteotropic hormones. Proc Natl Acad Sci USA 1987; 84: 2024–2028.[Abstract]

56. Hiraga T, Nakajima T, Ozawa H. Bone resorption induced by a metastatic human melanoma cell line. Bone 1995; 16: 349–356.[CrossRef][ISI][Medline]

57. Guise TA. Parathyroid hormone-related protein and bone metastases. Cancer 1997; 80: 1572–1580.[CrossRef][ISI][Medline]

58. Guise TA, Yin JJ, Taylor SD et al. Evidence for a causal role of parathyroid hormone-related protein in pathogenesis of human breast cancer-mediated osteolysis. J Clin Invest 1996; 98: 1544–1549.[Abstract/Free Full Text]

59. Yoneda T. Cellular and molecular mechanisms of breast and prostate cancer metastases to bone. Eur J Cancer 1998; 34: 240–245.[CrossRef][ISI][Medline]

60. Boonekamp PM, Lowik CW, van der Wee-Pals LJ et al. Enanchment of the inhibitory action of APD on the transformation of osteoclast precursors into resorbing cells after dimethylation of the amino group. Bone Miner 1987; 2: 29–42.[ISI][Medline]

61. Lowik CW, van der Pluijm G, van der Wee-Pals LJA et al. Migration and phenotypic transformation of osteoclast precursors into mature osteoclasts: the effect of a bisphosphonate. J Bone Miner Res 1988; 3: 185–192.[ISI][Medline]

62. Colucci S, Minielli V, Zambonin G et al. Alendronate reduces adhesion of human osteoclast-like cells to bone and bone protein-coated surfaces. Calcif Tissue Int 1998; 63: 230–235.[CrossRef][ISI][Medline]

63. Boissier S, Magnetto S, Frappart L et al. Bisphosphonates inhibit prostate and breast carcinoma cell adhesion to unmineralized and mineralized bone extracellular matrices. Cancer Res 1997; 57: 3890–3894.[Abstract]

64. Van der Pluijm G, Vloedgraven H, van Beek E et al. Bisphosphonates inhibit the adhesion of breast cancer cells to bone matrices in vitro. J Clin Invest 1996; 98: 698–705.[Abstract/Free Full Text]

65. Boissier S, Ferreras M, Peyruchaud O et al. Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases. Cancer Res 2000; 60: 2949–2954.[Abstract/Free Full Text]

66. Senaratne SG, Pirianov G, Mansi JL et al. Bisphosphonates induce apoptosis in human breast cancer cell lines. Br J Cancer 2000; 82: 1459–1468.[ISI][Medline]

67. Peyruchaud O, Winding B, Pecheur I et al. Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions. J Bone Miner Res 2001; 16: 2027–2034.[ISI][Medline]

68. Magnetto S, Boissier S, Delmas PD, Clezardin P. Additive antitumor activities of taxoids in combination with the bisphosphonate ibandronate against invasion and adhesion of human breast carcinoma cells to bone. Int J Cancer 1999; 83: 263–269.[CrossRef][ISI][Medline]

69. Selander KS, Monkkonen J, Karhukorpi EK et al. Characteristics of clodronate-induced apoptosis in osteoclasts and macrophages. Mol Pharmacol 1996; 50: 1127–1138.[Abstract]

70. Jadgev SP, Coleman RE, Shipman CM et al. The bisphosphonate, zoledronic acid, induces apoptosis of breast cancer cells: evidence for synergy with paclitaxel. Br J Cancer 2001; 84: 1126–1134.[CrossRef][ISI][Medline]

71. Senaratne SG, Mansi JL, Colston KW. The bisphosphonate zoledronic acid impairs membrane localisation and induces cytochrome c release in breast cancer cells. Br J Cancer 2002; 86: 1479–1486.[CrossRef][ISI][Medline]

72. Fromigue O, Lagneaux L, Body JJ. Bisphosphonates induce breast cancer cell death in vitro. J Bone Miner Res 2000; 15: 2211–2221.[ISI][Medline]

73. Shipman CM, Rogers MJ, Apperley JF et al. Bisphosphonates induce apoptosis of human myeloma cell lines: a novel antitumor activity. Br J Haematol 1997; 98: 665–672.[ISI][Medline]

74. Shipman CM, Croucher PI, Russell RG et al. The bisphosphonate incadronate (YM175) causes apoptosis of human myeloma cells in vitro by inhibiting the mevalonate pathway. Cancer Res 1998; 58: 5294–5297.[Abstract]

75. Aparicio A, Gardner A, Tu Y et al. In vitro cytoreductive effects on multiple myeloma cells induced by bisphosphonates. Leukemia 1998; 12: 220–229.[CrossRef][ISI][Medline]

76. Mackie PS, Fisher JL, Zhou H, Choong PF. Bisphosphonates regulate cell growth and gene expression in the UMR 106-01 clonal rat osteosarcoma cell line. Br J Cancer 2001; 84: 951–958.[CrossRef][ISI][Medline]

77. Riebeling C, Forsea AM, Raisova M et al. The bisphosphonate pamidronate induces apoptosis in human melanoma cells in vitro. Br J Cancer 2002; 87: 366–371.[CrossRef][ISI][Medline]

78. Lee MV, Fong EM, Singer FR, Guenette RS. Bisphosphonate treatment inhibits the growth of prostate cancer cells. Cancer Res 2001; 15: 2602–2608.

79. Wood J, Bonjean K, Ruetz S et al. Novel antiangiogenic effects of the bisphosphonate compound zoledronic acid. J Pharmacol Exp Ther 2002; 302: 1055–1061.[Abstract/Free Full Text]

80. Wood J, Schnell C, Green JR. Zoledronic acid, a potent inhibitor of bone resorption, inhibits proliferation and induces apoptosis in human endothelial cells in vitro and is anti-angiogenic in a murine growth factor implant model. Proc. Am Soc Clin Oncol 2000; 19: 664 (Abstr 2620).

81. Fournier P, Boissier S, Filleur S et al. Bisphosphonates inhibit angiogenesis in vitro and testosterone-stimulated vascular regrowth in the ventral prostate in castrated rats. Cancer Res 2002; 62: 6538–6544.[Abstract/Free Full Text]

82. Santini D, Vincenzi B, Avvisati G et al. Pamidronate induces modifications of circulating angiogenic factors in cancer patients. Clin Cancer Res 2002; 8: 1080–1084.[Abstract/Free Full Text]

83. Haas W, Pereira P, Tonegawa S. {gamma}/{delta} cells. Annu Rev Immunol 1993; 11: 637.[CrossRef][ISI][Medline]

84. De Libero G. Sentinel function of broadly reactive human {gamma}{delta} T cells. Immunol Today 1997; 18: 22–26.[CrossRef][ISI][Medline]

85. Fisch P, Malkovsky M, Kovats S et al. Recognition by human V{gamma}9/V{delta}2 T cells of a GroEL homolog on Daudi Burkitt’s lymphoma cells. Science 1990; 250: 1269.[ISI][Medline]

86. Bukowski JF, Morita CT, Tanaka Y et al. V{gamma}9/V{delta}2 TCR-dependent recognition of nonpeptide antigens and Daudi cells analyzed by TCR gene transfer. J Immunol 1995; 154: 998–1006.[Abstract/Free Full Text]

87. Kunzmann, Bauer E, Feurle J et al. Stimulation of {gamma}{delta} T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 2000; 96: 384–392.[Abstract/Free Full Text]

88. Kunzmann V, Bauer E, Wilhelm M. {gamma}{delta} T cell stimulation by pamidronate. N Engl J Med 1999; 340: 737–738.[Free Full Text]

89. Spets H, Georgii Hemming P, Siljason J et al. Fas/APO-1 (CD95)-medi-ated apoptosis is activated by interferon-{gamma} and interferon-{alpha} in interleukin-6 (IL-6)-dependent and IL-6-independent multiple myeloma cell lines. Blood 1998; 92: 2914.[Abstract/Free Full Text]

90. Miyagawa F, Tanaka Y, Yamashita S, Minato N. Essential requirement of antigen presentation by monocyte lineage cells for the activation of primary human {gamma}{delta} T cells by aminobisphosphonate antigen. J Immunol 2001; 166: 5508–5514.[Abstract/Free Full Text]

91. Ferrarini M, Consogno G, Rovere P et al. Inhibition of caspases maintains the antineoplastic function of {gamma}{delta} T cells repeatedly challenged with lymphoma cells. Cancer Res 2001; 61: 3092–3095.[Abstract/Free Full Text]

92. Powles T, Paterson S, Kanis JA et al. Randomized, placebo-controlled trial of clodronate in patients with primary operable breast cancer. J Clin Oncol 2002; 20: 3219–3224.[Abstract/Free Full Text]

93. Saad F, Gleason DM, Murray R et al. A randomized, placebo-controlled trial of zoledronic acid in patients with hormone-refractory metastatic prostate carcinoma. J Natl Cancer Inst 2002; 94: 1458–1468.[Abstract/Free Full Text]