The anti-tumour activity of bisphosphonates

CANCER TREATMENT REVIEWS 2002; 28: 305–319 doi:10.1016/S0305-7372(02)00095-6, available online at http://www.idealibrary.com on

LABORATORY–CLINIC INTERFACE

The anti-tumour activity of bisphosphonates H. L. Neville-Webbe1, I. Holen2 and R. E. Coleman3 1

Department of Clinical Oncology, Cancer Research Centre, Sheffield S10 2SJ, UK; 2Division of Genomic Medicine, Cancer Research Centre, Sheffield S10 2SJ, UK; 3Medical Oncology, Cancer Research Centre, Sheffield S10 2SJ, UK

Bisphosphonates are stable analogues of pyrophosphate (PPi), an endogenous regulator of bone mineralisation. A number of placebo-controlled trials have demonstrated their positive impact on skeletal-related events (SRE) that occur as a consequence of metastatic or myelomatous bone disease. Based upon their chemical structure bisphosphonates can be classified into nitrogen-containing bisphosphonates, (N-bisphosphonates) (for example zoledronate and pamidronate) and non-nitrogen containing (for example, clodronate and etidronate), which more closely resemble PPi. Clinical trials investigating bisphosphonates in the preventative setting have shown bisphosphonates to not only delay occurrence of bone metastases in certain cancers, but in one trial, occurrence of non-osseous lesions was delayed, and survival was prolonged. Other trials however have shown the opposite. Likewise, in animal models of cancer and metastases, conflicting results have been obtained. In vitro work has concentrated on bisphosphonates direct action upon tumour cells and has found a variety of antitumour effects such as apoptosis induction, inhibition of cell growth, inhibition of invasive behaviour and inhibition of angiogenic factors. Furthermore it would appear that bisphosphonates have the potential to enhance anti-tumour activity of known cytotoxic drugs. Ongoing research aims to assess this further, in addition to determining more precisely the role of adjuvant bisphosphonates in cancers such as breast and prostate cancer. c s

2002 Elsevier Science Ltd. All rights reserved.

Key words: Bisphosphonates; anti-tumour activity; adjuvant bisphosphonates; bone metastases; apoptosis; anti-angiogenic; synergy.

INTRODUCTION Bisphosphonates are stable analogues of pyrophosphate (PPi) (Figure 1). Pyrophosphate has a P–O–P structure, two phosphate groups linked by an oxygen atom. Bisphosphonates have a P–C–P structure, a geminal (central) carbon atom replacing the oxygen. Side chains R1 and R2 are attached to the carbon atom (Figure 2) and influence the bisphosphonates’ ability to bind to bone, and also their anti-resorptive ability. Bisphosphonates containing a primary nitrogen atom in the R2 side-chain (for example pamidronate) are more potent than non-nitrogen bisphosphonates, whilst modifying the primary

Correspondence to: H. L. Neville-Webbe, Clinical Research Fellow, Department of Clinical Oncology, Cancer Research Centre, Weston Park Hospital, Whitham Road, Sheffield S10 2SJ, UK. Tel.: +44-114-226-5000; Fax: +44-114-271-3781; E-mail: [email protected]

amine to form a tertiary amine (for example ibandronate) increases potency. The most potent bisphosphonates to date appear to be those containing a tertiary amine within a ring structure, such as zoledronate. (Figure 3). Bisphosphonates bind avidly to hydroxyapaptite bone mineral surfaces and are selectively internalised by osteoclasts where they inhibit their activity (1). NBisphosphonates inhibit the mevalonate pathway (2), (Figure 4), whilst non-N-bisphosphonates are metabolically incorporated into non-hydrolysable analogues of ATP (3), both mechanisms ultimately leading to osteoclast apoptosis, with consequent reduction in tumour cell-induced bone resorption and destruction. Bisphosphonates have an established place in the management of malignancies with a predilection for skeletal involvement and evidence for bisphosphonates usage in the treatment of malignant bone disease is well established. More recent work has directed attention towards the effect of bisphosphonates on tumour cells

c 2002 ELSEVIER SCIENCE LTD. ALL RIGHTS RESERVED. 0305-7372/02/$ - see front matter s

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Figure 1 Structure of pyrophosphate. Pyrophosphate has two phosphate groups linked by an oxygen atom.

Figure 2 Structure of bisphosphonates. Bisphosphonates have a PCP structure. A hydroxyl group in R1 enhances binding to bone. A primary nitrogen atom in R2 confers anti-resorptive potency. Modifying the primary amine to form a tertiary amine further enhances potency.

H. L. NEVILLE-WEBBE ET AL.

lesions developing and decreased proportion experiencing bone secondaries. A trial of 133 women by Kanis et al. (4) first showed that oral clodronate, 1600 mg daily given to women with advanced breast cancer, but no radiological evidence of bony spread, reduced the number of skeletal metastases developing, and consequent skeletal complications, compared to placebo (32 vs. 63, respectively, p < 0:005). There was also a non-significant trend for the number of patients developing bony metastases to be lower in the treated group (15 vs. 19). Clodronate has also been shown to significantly reduce skeletal complications in multiple myeloma (5). In a subgroup analysis of those patients without skeletal fractures at presentation, 1600 mg daily of clodronate significantly improved both median survival, (59 months {95% CI 43–71 months} vs. 37 months {95% CI 31–52 months} for placebo), and 5-year survival, (46% vs. 35% for placebo) (see Table 1).

Adjuvant trials

Figure 3 Structure of zoledronate. Zoledronate contains a tertiary amine within an imidazole ring, making zoledronate one of the most potent available bisphosphonate to date.

directly, particularly within the bone microenvironment (Figure 5). There is evidence of direct anti-tumour activity in animal models of malignant disease, in in vitro models, and also some encouraging clinical observations. Such direct effects on tumour cells, in addition to bisphosphonates inhibitory effect on osteoclasts would provide a strong rationale for the use of adjuvant use of bisphosphonates in those malignancies with a predilection to metastasise to bone.

ANTI-TUMOUR ACTION: EVIDENCE FROM THE CLINICAL SETTING (TABLE 1) Advanced disease trials Clinical trials of advanced cancer patients with no evidence of bone metastases, receiving bisphosphonates, have shown a reduction in number of new

Trials have assessed the role of adjuvant bisphosphonate in primary operable breast cancer. Between 1989 and 1995, Powles et al. (6) randomised 1069 women with early breast cancer to receive oral clodronate 1600 mg daily or placebo, for 2 years, within 6 months of surgery. The women received adjuvant chemotherapy and hormonal therapy as deemed appropriate. The two arms had a similar proportion of oestrogen and/or progesterone receptor positive tumours. During the on-medication period, (2 years), less women receiving clodronate developed metastases compared to the placebo arm (12 vs. 28, respectively, HR ¼ 0.44; 95% CI ¼ 0.22–0.86, p ¼ 0:016). However, with longer follow-up the difference in incidence of bone metastases between the two groups became non-significant (63 patients in clodronate group vs. 80 patients in placebo group, developed a bone metastasis, (HR ¼ 0.77; 95% CI ¼ 0.56–1.08; p ¼ 0:127). One implication of this could be that there is a need for longer or even indefinite, bisphosphonate therapy, to maintain an effect upon metastases. There was also no significant difference in incidence of non-bone metastases between the two arms (clodronate group ¼ 112, placebo group ¼ 128, p ¼ 0:257). Importantly however, there was a significant reduction in mortality for the clodronate-treated group: during the follow-up period 98 patients died in the clodronate arm, compared to 128 patients in the placebo arm (p ¼ 0:047). Diel et al. (7) investigated a smaller trial of 302 women with primary breast cancer, randomised to oral clodronate, 1600 mg daily for 3 years, or control, both groups receiving standard surgical and medical

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Figure 4 The mevalonate pathway and proposed mechanism of action for N-BP induction of apoptosis. (FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate).

treatment. This trial however selected patients deemed at high risk for future bony relapse, by randomising only those women with evidence of

Figure 5 Proposed mechanisms for the anti-tumour effects of bisphosphonates. 1, direct effects on tumour growth and survival; 2, direct inhibition of bone resorption; 3, indirect effects on primary tumour through inhibition of bone resorption.

bone marrow tumour cells. (The same group had previously found this was a risk factor for later distant metastases (8).) This trial found after 3 years follow-up that the number of women developing bony metastases in the clodronate group was approximately half that of the control group (3.1 vs. 6.3, respectively) Significantly, the incidence of visceral metastases was also lower in the clodronate group, and overall survival was prolonged. The observation that visceral metastases were reduced implied a direct effect of clodronate on tumour cells that was not dependent upon its effect via the bone microenvironment. Updated follow-up for this trial (9) revealed that the prophylactic effect of clodronate, though weaker, was maintained as regards bone metastases and overall survival, but not for prevention of visceral metastases. This is important, as it is possible a longer treatment period, (as with the trial by Powles et al.), would enable the preventative

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TA B L E 1 Summary of clinical trials of adjuvant oral clodronate, 1600 mg daily, for patients with breast cancer (placebo controlled) Authors

Number of patients

Duration of clodronate Bone metastases Non-bone metastases

Kanis et al. (4) Powles et al. (6)

133 1069

3 years 2 years

Reduced Reduced

Diel et al. (7)

302

2 years

Reduced

Mardiak et al. (11) Saarto et al. (10)

73 299

2 years 3 years

No change No change

Survival

No change Increased OS for clodronate group Reduced Increased OS for clodronate group No change No change Increased in clodronate Decreased OS and group DFS for clodronate group No change

OS, overall survival; DFS, disease-free survival.

effect on all metastases and survival, to be maintained for a longer period. This positive data conflicts with the trial by Saarto et al. (10). 299 women (pre- and post-menopausal) with node positive early breast cancer were randomised to clodronate 1600 mg daily or control, plus surgery and adjuvant cytotoxic/hormonal treatment. With a follow-up of 5 years bone metastases were found equally in both groups. Intriguingly, non-osseous distant and local metastases were found to be greater in the clodronate group (43% of clodronate-treated patients vs. 25% control group, p ¼ 0:007), and overall survival was worse. Though the two arms were well balanced, there were more progesterone receptor negative tumours in the clodronate group. After adjusting for this the clodronate group still had a worse outcome. Despite this, bone, as a site of first relapse was more frequent in the control group compared to the clodronate group, implying that clodronate delivered some positive effect on the skeleton. A further, small, trial involving 73 patients with locally advanced breast cancer but no evidence of bony metastases (11), using adjuvant oral clodronate 1600 mg/day, found bone metastases developed in more patients receiving bisphosphonate compared to placebo, and median time to their appearance was shorter in the clodronate arm. However these differences were not significant and there were no significant differences in 5-year survival rates between the two groups, leading the authors to conclude a lack of benefit for adjuvant clodronate in the adjuvant setting. Thus the evidence from the completed clinical trials remains conflicting. However most of these trials were designed to show bisphosphonate benefit in the prevention of bone metastases and the effect on visceral metastases and overall survival were additional discoveries. A large adjuvant study of oral clodronate run by the NSABP is recruiting rapidly, and adjuvant trials with potent N-bisphosphonates will commence shortly.

ANTI-TUMOUR ACTION: EVIDENCE FROM IN VIVO MODELS (TABLE 2) Animal models provide a more useful and flexible setting than the clinical for the testing of bisphosphonate potency and anti-tumour potential. Animal models may be specifically designed to reflect the pattern of human tumour behaviour, for example, the 4T1 orthotopic murine tumour model (12). This model mimics the human situation of the establishment of a primary tumour with subsequent development of bony metastases, visceral metastases, and death. A further advantage of animal models is that single variables can be manipulated to address particular issues, a situation that is obviously impossible in the human setting (see Table 2). Sasaki et al. (13) used a nude mouse model for breast cancer and bony metastases: MDA-MB-231 breast cancer cells were injected into the left cardiac ventricle, with the development of osteolytic bone metastases approximately 4 weeks later. The N-bisphosphonate risedronate (4 lg/animal/day, s.c.) was administered either after osteolytic lesions had developed (day 17) for 10 days, continuously for 28 days from the point of tumour cell inoculation, or 7 days before tumour cell injection. In all groups risedronate inhibited the development of bone metastases, and for those mice receiving continuous bisphosphonate, survival was increased compared to untreated mice. However, whilst risedronatetreated animals had a significant decrease in bone tumour-load, they also had greater amount of metastatic invasion into soft tissues surrounding bone, compared to untreated animals. In contrast, the experimental bisphosphonate YH529, when started the same day as MDA-MB-231 breast cancer cell inoculation into nude mice, (20 lg/ day/s.c. for 4 weeks) non-osseous metastases were reduced, in addition to bone lesions, and tumour burden in bone (14).

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TA B L E 2 Anti-tumour activity of bisphosphonates in animal models Authors

Animal model used

Cell line used

Bisphosphonate

Tumour burden in bone

Non-bone tumour burden

Sasaki et al. (13) Sasaki et al. (14)

Nude mouse Nude mouse

Breast cancer cell line Breast cancer cell line

Reduced Reduced

Increased Decreased

Krempien et al. (15) Mundy et al. (16) Kostenuik et al. (17)

Rat 4T1 mouse Rat

Walker carcinosarcoma Breast cancer cell line Walker carcinosarcoma

Risedronate Experimental BP (YH529) Clodronate Zoledronate Pamidronate

No change Reduced Increased

No change No change No change

The prophylactic effect of clodronate was investigated using a rat model whereby the Walker carcinosarcoma 256B cell line was implanted intraosseously. The bisphosphonate was given at a dose of either 30 mg/kg body weight/s.c. for 5 days, following which tumour cells were implanted into the rats 1, 3 or 7 weeks after treatment, or bisphosphonate was administered at a lower dose of 2.5–5.0 mg/kg body weight/s.c./daily for 21 days, with tumour cell implantation occurring on day 22. Both treatment regimes inhibited formation of bone lesions, but tumour burden and non-osseous tumour weight and size were not affected (15). Mundy et al. (16) assessed the effects of the more potent N-bisphosphonate zoledronate, using the 4T1 murine model. In this study, a single 0.3 lg dose was given intravenously 7 days after the primary tumour was established. The mice were sacrificed on day 28. Zoledronate was found to decrease not only osteolysis compared to the extensive bony destruction in untreated mice, but also to reduce bone tumourburden. However no survival advantage was gained in the zoledronate-treated mice. Much of the data from animal models is encouraging, but not conclusive. Kostenuik et al. (17) investigated the effect of the N-bisphosphonate pamidronate. Male Fischer rats received pamidronate (0.5 mg/kg body weight/day, s.c. for 7 days) followed by intramuscular injection of Walker 256 cancer cells, with sacrifice occurring 14 days later. The pamidronate-treated animals had a 2.6-fold increase in skeletal tumour burden, with non-bone metastatic disease being unaffected, compared to untreated animals. It is thus apparent that most of the studies of antitumour effects of bisphosphonates using animal models have either had no effect on extra osseous metastases or a deleterious effect. However it would appear that bisphosphonates not only affect osteoclasts in the bone, but also appear to directly affect tumour cells, albeit within the bone microenvironment. Bisphosphonates may induce tumour cell death directly, or cut-off their ‘survival factors’ such as transforming growth factor b (TGF-b) and insulinlike growth factor-1 (IGF-1), which are released

from the resorbing bone. TGF-b has been shown to stimulate tumour cell production of parathyroid hormone-related peptide, PTH-rP, which promotes osteoclastic bone resorption (18), thus creating a selfperpetuating circle of tumour-induced osteolysis.

ANTI-TUMOUR ACTION: EVIDENCE FROM IN VITRO MODELS (FIGURE 6) The growth and metastatic spread of a tumour is a complex multistep process (Figure 7). For a tumour to metastasise it must grow at its primary site, stimulate neoangiogenesis, migrate, adhere and invade basement membrane and extracellular matrix (ECM) proteins, evade destruction by the immune system, spread to distant sites, where tumour cells must again proliferate and survive the local environment. In vitro studies have the advantage of separately addressing the many steps of cancer cell progression and survival, and the possible biological of bisphosphonates on these processes (see Figure 6).

BISPHOSPHONATES INHIBIT ADHESION AND SPREADING OF TUMOUR CELLS Malignant tumours have the ability to adhere to other structures/organs and adhesion is necessary for effective spreading and invasion of the tumour cells. In 1996 van der Pluijm et al. (19) showed that bisphosphonates have the ability to potentially reduce tumour burden in bone, by inhibition of cell adhesion, at which time it was thought bisphosphonates only had an inhibitory effect on osteoclasts. Cortical and trabecular bone slices were pre-treated with a number of different bisphosphonates (varying concentrations 106 –104 M) for 18 h, followed by seeding of breast cancer cells on top. It was shown that N-bisphosphonates (pamidronate, olpandronate, alendronate and ibandronate) dose-dependently inhibited the adhesion and spreading of human breast cancer cells, MDA-MB-231, to bone

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Figure 6 Anti-tumour effects of bisphosphonates.

Figure 7 Steps in tumour development affected by bisphosphonates.

matrix, whereas the non-N-bisphosphonates (clodronate and etidronate) had little or no effect. Ibandronate was found to be the most potent bisphosphonate in both inhibition of adhesion and spreading of the tumour cells. This study implies prophylactic bisphosphonates may have a role in preventing tumour seeding in bone, by virtue of interference with tumour cell adhesion to bone. On the untreated bone slices tumour cells were capable of adhering and spreading within 5 h. In contrast, bisphosphonate pre-treatment of adjacent non-skel-

etal tissue had no effect on tumour cell adhesion to tissues surrounding bone. It is possible that bisphosphonates interfere with specific bone attachment proteins of breast cancer cells, such as b1 and b3 integrin subunits (20), whilst different attachment proteins may be responsible for non-bone attachment. In a slightly different study (21) where breast cancer and prostate cancer cells were pre-treated with bisphosphonate (ibandronate, NE-10244, {antiresorptiveactive pyridinium analogue of risedro-

ANTI-TUMOUR ACTIVITY OF BISPHOSPHONATES

nate}, pamidronate and clodronate), a dose-dependent inhibition of adhesion to bone (mineralised and unmineralised) was observed. Again ibandronate was the most potent compound in achieving this, and clodronate the least. The anti-adhesion effect was not due to cytotoxicity, as the bisphosphonates were not cytotoxic at doses capable of inhibiting adhesion. The bisphosphonates had no effect on adhesion of normal cells (fibroblasts) to bone matrix, implying again that bisphosphonates may have a direct anti-tumour action albeit within the bone environment. Furthermore, while PC-3 prostate cancer cells have been shown to adhere well to ECM proteins, such adhesion is significantly inhibited by pretreatment of the PC-3 cells with a non-toxic dose (10 lM) of alendronate for 24 h. Adhesion to fibronectin, collagen types I and IV, and laminin was most effectively inhibited (22) though inhibition was found to be less than alendronate’s inhibition of invasion.

BISPHOSPHONATES INHIBIT TUMOUR CELL INVASION, AND POSSIBLY MIGRATION Using migration and invasion assays Boissier et al. (23) found that pre-treatment with bisphosphonates for 24 h, inhibited the invasive ability of MDA-MB231 breast cancer cells and PmPC3 prostate cancer cells, in a dose-dependent manner. Up to 60–90% inhibition of invasion was achieved, with zoledronate being the most potent and clodronate the least so. To rule out such effective inhibition being the result of bisphosphonate-induced apoptosis of cancer cells, thus rendering cells incapable of invasion, the group showed that for zoledronate, at a concentration that inhibited invasion, apoptosis was not induced. However they did not find any effect of bisphosphonates on cell migration. In contrast Virtanen et al. (22) found that PC-3 cells pre-treated with alendronate or clodronate for 24 h, significantly reduced migration of the cells, as compared to control treatment. At concentrations <100 lM alendronate and clodronate had no effect upon the growth rate of PC-3 cells and thus a concentration of 10 lM was used for the invasion and migration assays. The addition of mevalonate pathway intermediaries, geranylgeraniol, trans–transfarnesol and mevalonate to alendronate pre-treated cells significantly, though not completely, reversed the effects of alendronate on migration. As expected addition of such intermediaries to clodronate-treated cells did not reverse the effects of clodronate. In fact the addition of trans–trans-farnesol potentiated the effect of clodronate-induced inhibition of migration.

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The same group also found that the mevalonate pathway is important for PC-3 cells ability to invade, as pre-treatment of PC-3 cells with 10 lM mevastatin (b-hydroxy-b-methylglutaryl-CoA reductase inhibitor) significantly inhibited invasion of the cells through artificial membrane, Matrigel. This inhibitory effect was reversed by mevalonate pathway intermediaries, implying protein geranylgeranylation and/or farnesylation is necessary for PC-3 cell invasion. Pre-treatment of PC-3 cells with 10 lM of alendronate significantly inhibited invasion compared to control, and addition of geranylgeraniol or trans–trans-farnesol reversed the alendronate inhibitory effect. Pre-treatment with 10 lM of clodronate also significantly inhibited cell invasion, but again addition of geranylgeraniol or trans–trans-farnesol did not oppose clodronate’s effect, but in fact potentiated it. These results would imply that not only do N-bisphosphonates affect inhibition of migration and invasion of prostate cancer cells by virtue of their effect upon the mevalonate pathway, but that the latter pathway would also seem to be important for such cancer cells to migrate and invade. Various bisphosphonates (including pamidronate, alendronate and clodronate) have also been found to dose-dependently reduce the invasion of human fibrosarcoma cell lines (HT1080) and human melanoma cell lines (C8161) (24). When these cell lines were cultured alone they could effectively invade the Matrigel membrane, but this was reduced in the presence of bisphosphonates, including clodronate. Both of these tumours have high potential for early and rampant metastatic spread and corresponding poor prognosis in the clinical setting. Beyond treatment of early stage disease, no therapy has shown great efficacy in the advanced stages for these malignancies.

BISPHOSPHONATES INDUCE APOPTOSIS OF TUMOUR CELLS AND REDUCE TUMOUR CELL GROWTH Shipman et al. (25) found that the N-bisphosphonate incadronate (YM175) induced apoptosis of human myeloma cells, through the same mechanism by which N-bisphosphonates induce osteoclast apoptosis. Bisphosphonates induce osteoclast apoptosis by inhibition of the mevalonate pathway, (the same pathway responsible for cholesterol synthesis), leading to loss of important signalling proteins(see Figure 1).These signalling proteins are required for many cell types, so it is not entirely surprising that bisphosphonate may induce tumour cell apoptosis again by interfering with the mevalonate pathway. The main target of bisphosphonate action has re-

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cently been found to be farnesyl diphosphatate synthase (FPP) (26), leading to loss of prenylated proteins. The latter are required for post-translation lipid modification (that is, prenylation) of signalling GTPases, such as Ras, Rho and Rac. [Reviewed by Rogers et al. (27).] Furthermore, inhibition of the mevalonate pathway in osteoclasts would appear to ultimately lead to apoptosis through activation of caspase-3/caspase-3 like proteolytic enzymes (28). Caspase activation is considered essential for apoptosis, with certain caspases initiating the caspase cascade leading to activation of those caspases, such as caspase-3, involved in the execution phase of apoptosis (29). Zoledronate-induced apoptosis of MCF-7 human breast cancer cells has also been found to be through inhibition of the mevalonate pathway (30). Addition of the mevalonate pathway intermediary geranylgeraniol inhibited apoptosis to levels very near to that of control. Initiation of the caspase cascade in response to apoptotic stimuli is induced by release of mitochondrial cytochrome c into the cytosol (31). It is thus possible that the main mechanism underlying N-bisphosphonate induced apoptosis is the inhibition of the mevalonate pathway, leading to loss of geranylgeranylated proteins Rho, Ras and Rac, and ultimately the activation of caspase activity and cell death through apoptosis. Very recent work (32) would indicate that for zoledronate-treated breast cancer cells (MCF-7 and MDA-MB-231) there is evidence for decreased generation of FFP. This leads to decreased prenylation of Ras, as indicated by impaired Ras membrane localisation, (which is necessary for the functional integrity of Ras). In this study, zoledronate-induced apoptosis was associated with a time- and dose-related increase of mitochondrial cytochrome c into the cytosol, with a corresponding increase in caspase-3 cleavage products. This would imply that for zoledronate, (and presumably other N-bisphosphonates) inhibition of the mevalonate pathway leads to a deregulation of signalling proteins such as Ras, resulting in induced cytochrome c release into the cytosol, with subsequent initiation and activation of the caspase cascade, and an irreversible commitment of the cell to undergo apoptosis. Conversely it is also possible that inhibition of the mevalonate pathway may lead to apoptosis through additional mechanisms. Benford et al. (33) found that apoptosis induced by alendronate in J774 cells (a macrophage-like cell line) could be significantly, but not completely, inhibited by co-incubation with the caspase-3 inhibitor, Z-DEVD-fmk. Co-treatment with Z-DEVD-fmk did completely inhibit the activity of caspase-3 like proteases. This would imply that whilst caspase-3/caspase-3 like proteases are involved in N-bisphosphonate induced apoptosis of

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J774 cells, their activation is not essential. In contrast, in J774 cells co-incubated with N-bisphosphonate (pamidronate, ibandronate and alendronate) and geranylgeraniol, for 24 h, apoptosis was prevented, an effect that was present still at 48 h. Senaratne et al. (34) was the first group to show that N-bisphosphonates and clodronate could directly reduce human breast cancer cell viability, and induce apoptosis, in vitro. In this study the human breast cancer cell lines MCF-7, MDA-MB-231, and Hs 578T were incubated for 4 days with various bisphosphonates, and viability and apoptosis was assessed in comparison to control. Zoledronate was the most effective, causing a 50% reduction in cell viability, with a concentration of 15 lM for MDAMB-231 cells, 20 lM for MCF-7 cells, and 3 lM for Hs 578T cells, compared to control. Clodronate could also reduce MDA-MB-231 cell viability by 50%, though at 700 lM, > 2000 lM and 1000 lM, for the three cell lines, respectively. 24 h exposure to pamidronate 100 lM was sufficient to induce loss of cell viability at 4 days. The effects of bisphosphonate on apoptosis are relatively fast, as illustrated by a study showing that zoledronate can reduce cell number (to approximately 77% of control), with a corresponding increase in the number of apoptotic cells (8:05  0:6% compared to 1:23  0:5% in untreated cells) in MCF-7 cells, following just 2 h of incubation (30). There are some indications that bisphosphonates are tumour cell specific, as viability of non-malignant cells, 3T3 mouse embryo fibroblasts, were not significantly affected by up to 100 lM of zoledronate, (34). In contrast pamidronate at 100 lM induced an 83% inhibition of growth of breast tumour cells (MDA-231 line, MCF-7 line and Hs 578T line) relative to control cultures. Pamidronate (at 100 lM) and zoledronate (at 50 lM) were also shown to directly induce apoptosis of the breast cancer cells (MDAMB-231 line), (34) either assessed visually or by measurement of DNA fragmentation, a reproducible outcome measurement of apoptosis. Zoledronate at 10 lM induced 3.6-fold increase in fragmented DNA, compared to control, whilst pamidronate at this concentration had no effect. With a 100 lM of each, zoledronate was able to induce 84% DNA fragmentation (6.4-fold compared to control), and pamidronate 32% (2.4-fold compared to control). Pamidronate also significantly affects the antiapoptotic oncoprotein, bcl-2, as measured by Western analysis (34). The ratio of this protein with its pro-apoptotic counterpart, bax, may influence a cell’s susceptibility to undergo or not undergo, apoptosis (35), in part due to the inhibitory effect of the bcl-2 family upon cytochrome c release and consequent inhibition upon the caspase cascade activation. MDA-MB-231 cells treated with 100 lM of pamidr-

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onate caused a time-dependent decrease in bcl-2 protein, with a corresponding decrease in bcl-2:bax ratio, with no effect upon bax protein itself. By day 4 the decline in bcl-2:bax ratio was 50% that of control. Furthermore the same group recently showed (32) that zoledronate-induced apoptosis could be suppressed by over-expression of bcl-2 in MDA-MB-231 cells, further implying that interaction exists between this anti-apoptotic oncoprotein and N-bisphosphonates. As mentioned above, caspase activity, in particular caspase-3, plays a major role in executing apoptosis. Caspase activation causes proteolytic cleavage of the 116 kDa poly(ADP)-ribose polymerase (PARP) protein to smaller fragments 85 and 30 kDa. MDA-MB-231 cells treated with 100 lM of pamidronate or zoledronate caused a time-dependent decrease in full-length, 116 kDa PARP, and a corresponding increase in the smaller 30 kDa fragment. By 72 h of bisphosphonate treatment there was virtual disappearance of the 116 kDa fragment, with a concomitant appearance of the 30 kDa cleaved product, as assessed by Western analysis, thus implying the N-bisphosphonates induce both apoptosis and caspase activation (34). Caspase-3/caspase-3 like involvement has also been implicated in ibandronate-induced apoptosis of breast cancer cells in vitro (36). Treatment of MDAMB-231 breast cancer cells with 100 lM of ibandronate for 72 h clearly induced apoptosis, as shown by electrophoretic analysis of internucleosomal DNA fragmentation. Apoptosis was due to an increase in capsase-3 activity, as determined by measurement of the proteolytically cleaved substrate (Ac-DEVDpNA). Furthermore, the caspase inhibitor Z-VADfmk inhibited, though not completely, the DNA fragmentation induced by ibandronate. Bisphosphonates have also shown anti-tumour activity against prostate cancer cells. Lee et al. (37) found that pamidronate at 100 lM caused significant decreases in prostate cancer cell (PC3, LNCaP, and DU145 cell lines) number after 48 h of treatment, compared to control. This was largely due to necrosis as opposed to induction of apoptosis. At lower concentrations pamidronate induced cytostasis. In contrast to data obtained from breast cancer cells, 100 lM of zoledronate did not cause a significant decrease in cell number (though this concentration of zoledronate would in breast cancer cells (30)), and only minimally induced cell death. Even in the presence of a cell-survival environment (serum) both bisphosphonates were able to induce cell death and/or cytostasis. Furthermore, exposure of cells to 25–50 lM of zoledronate for 12–24 h, followed by up to 72 h incubation of cells without bisphosphonate, was sufficient to inhibit prostate cancer cell growth, in the presence of serum. In this

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regard zoledronate was more potent than pamidronate. Most of the evidence suggesting bisphosphonates have direct anti-tumour activity is accrued from in vitro or murine in vivo models. One group however (38) looked at the effect of bisphosphonates upon fresh human breast tumour samples. Zoledronate, pamidronate, ibandronate and clodronate at varying concentrations (105 –103 M) were incubated with fresh tumour samples over 24–144 h. For concentrations between 103 and 104 M all the bisphosphonates demonstrated a time-related cytotoxic effect on fresh tumour samples, with zoledronate being the most potent and clodronate the least so. Whilst data exists for the mechanisms of action of bisphosphonates on osteoclastic bone resorption in myeloma and in the metastatic setting, the possible effects of bisphosphonates on the much rarer primary malignant bone tumours remains to be clarified. Osteosarcoma growth causes destruction of bone through proteolytic mechanisms and/or osteoclastic bony destruction (39). In vitro treatment with both pamidronate (108 –104 M) or clodronate (106 –102 M), for up to 4 days, were able to inhibit proliferation of UMR 106-01 (rat osteosarcoma cell line), in a time- and dose- dependent manner (39). Pamidronate at a concentration of > 104 M and clodronate at a concentration of > 102 M, was cytotoxic to the cells. Pamidronate between 1  106 and 1  105 M, and clodronate between 1  104 and 1  102 M, could inhibit cell growth, whereas higher doses were cytotoxic. Following 24 h treatment with pamidronate (at 105 M), there were a greater proportion of apoptotic cells compared to control. Despite the effect on cell growth and induction of apoptosis, this concentration of pamidronate had no effect on the levels of osteoblast-derived inhibitor of osteoclastic activity, osteoprotegerin (OPG). OPG is a regulator of osteoclast activity, and effectively inhibits osteoclast action when OPG acts as a decoy receptor binding to receptor activator of NF-jB ligand (RANKL) on osteoblast cells, thus preventing its interaction with RANK. The former is a final mediator, (in the presence of M-CSF) for osteoclast development. However pamidronate at the above concentration down-regulated expression of RANKL mRNA by the osteosarcoma cells. If osteosarcoma cells recruited osteoclasts in vivo (as suggested by these cells producing RANKL in vitro) then bisphosphonates may act by interrupting the release of osteoclast-differentiating factors from osteosarcoma cells, in addition to acting upon the latter directly. Furthermore at the same concentration pamidronate also significantly reduced osteopontin (OPN) mRNA expression. OPN is expressed by osteoblasts, being involved in adherence of the

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latter to the extracellular bone matrix (39). However OPN over-expression has been found in various primary malignant tumours and this may be a reflection of malignant potential (39). Hence it is of interest that pamidronate has been shown to downregulate OPN expression in vitro. Sonnemann et al. (40) assessed the effects of pamidronate on 7 different human osteosarcoma cell lines. Pamidronate inhibited malignant cell growth in a dose- and time-dependent manner. After 72 h 50 lM pamidronate reduced proliferation by up to 73%. Pamidronate was shown to only very weakly affect growth of non-malignant fibroblasts, implying bisphosphonates might have activity specific to the malignant clone.

BISPHOSPHONATES INHIBIT MATRIX METALLOPROTEINASE (MMP) ACTIVITY MMPs are a family of zinc-dependent endopeptidases, and are involved in normal and pathological tissue remodelling. They can be loosely divided into four groups based upon substrate specificity (reviewed in (41)): Those MMPs implicated particularly in cancer growth and metastasis are: (i) Interstitial collagenases {MMP-1 and -13}; (ii) Gelatinases {MMP-2 and -9}; (iii) Stromelysins {MMP-3}; and various membrane-type MMPs. Not only are they implicated in the ability of tumour cells to invade and metastasise, more recent work has shown MMPs are also involved in the growth of the primary tumour itself, in initiation and maintenance of growth of metastatic foci, and in angiogenesis (24). N-bisphosphonates (zoledronate, ibandronate) and non-N-bisphosphonates (clodronate) can inhibit MMP-2, -9 and -12 proteolytic activity (23) in a dosedependent manner, and all were equi-potent in this regard. Such bisphosphonates-induced inhibition of MMP activity was completely reversed by the addition of zinc (50 lM), implying that the ability of bisphosphonates to chelate divalent ions is the mechanism whereby bisphosphonates inhibit MMP activity. Teronen et al. (24) found that clodronate dosedependently inhibited neutrophil MMP-8-mediated degradation of b-casein and type I collagen substrates. Clodronate also inhibited activity of MMP-9 and -2. Alendronate and pamidronate dose-dependently inhibited activity of MMP-3, (closely linked to various malignancies), and others (MMP-1, -2, -8, -9, -12, and -20). Likewise zoledronate dose-dependently inhibited MMP-3, -13, and -20 activity. At concentrations capable of inhibiting various MMP activity the bisphosphonates were not cytotoxic, implying a directed inhibition rather than induction

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of cell death (42) Whilst Derenne et al. (43) found zoledronate significantly reduced production of MMP-1 by bone marrow stromal cells stimulated by IL-1b, zoledronate (and pamidronate less so), up regulated in vitro MMP-2 secretion by bone marrow stromal cells. This could have clinical implications, as MMP-2 has been implicated in the bone resorption and metastatic process. Combining a bisphosphonate with a MMP inhibitor may prevent such unwanted stimulation of MMP activity. Nude mice inoculated with the human breast cancer cell line MDA-MB-231 and treated with ibandronate and the tissue inhibitor of MMP-2 (TIMP-2) did not develop osteolytic bone lesions. (44) Mice receiving bisphosphonate alone or TIMP-2 alone developed a reduced number of osteolytic bone lesions compared to control mice. Furthermore survival was increased in mice receiving TIMP-2 alone or both bisphosphonate and TIMP-2.

NEW AND FUTURE DIRECTIONS Recent and ongoing research into the potential antitumour activities of bisphosphonates is investigating possible anti-angiogenic effects of bisphosphonates, as neo-angiogenesis is a prerequisite for cancer cell growth and spread. The combining of bisphosphonates with other agents known to have cytotoxic activity is also a growing area of interest, with data now emerging that some bisphosphonates, zoledronate in particular, combined with anticancer drugs, results in synergistic anti-tumour effects.

BISPHOSPHONATES HAVE ANTI-ANGIOGENIC PROPERTIES Neoangiogenesis is necessary for tumour growth, survival and spread to distant sites (45). Vascular endothelial growth factor (VEGF) is a potent angiogenic cytokine, specifically inducing proliferation of endothelial cells and increasing microvessel permeability (46). Breast cancer cells have been found to produce VEGF, with higher circulating levels measured in patients with advanced breast cancer compared to healthy controls and benign breast disease (47). VEGF has also been found to be a survival factor for breast cancer cells (48) by upregulating bcl2 expression, and thus inhibiting tumour cell apoptosis. In node-negative breast cancer, VEGF is a strong independent predictor of relapse-free survival (49) and overall survival (50). High tumour levels of VEGF predicts for poor efficacy of treatment with tamoxifen and chemotherapy in advanced breast cancer (51).

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Basic fibroblastic growth factor (b-FGF) also induces angiogenesis, and is expressed in breast cancer tissue (52). Similar to VEGF, increased b-FGF expression has also been found to be an indicator for worse prognosis in (node-negative) breast cancer patients (52). Osteoblastic cells in the bone marrow produce both VEGF and b-FGF and vascularisation is needed for osteoclastic bone resorption. In fact, bone matrix proteins involved in bone resorption (OPN and bone sialoprotein) have been found to stimulate angiogenesis (53). Work previously carried out in our laboratory found that intravenous zoledronate (4 mg) or pamidronate (90 mg) induced a significant decrease in bone marrow plasma values of b-FGF (84:98  14:7% of baseline), and in VEGF (71:77  23:2% of baseline) (54). These values were measured at 3 days following zoledronate infusion. Treatment also induced a significant decrease in serum b-FGF three days after the infusion, (42.82  14.4% of baseline) and a (non-significant) decline in serum VEGF (95:04  19:2% of baseline). In contrast Santini et al. (55) found that pamidronate was able to induce decreases in serum VEGF of cancer patients with a variety of solid tumours (including NSCLC, breast, prostate and bladder cancers) that had metastasised to bone. Significant decreases in serum VEGF level were found 24 h post90 mg infusion of pamidronate, with further significant decreases at 2 days. The decrease in serum VEGF was also present at seven days after the infusion. Recent work has also revealed that zoledronate may have anti-angiogenic properties (56), causing a dose-dependent inhibition of proliferation of human endothelial cells stimulated by b-FGF, (IC50 value 4:2  0:4 lM), in vitro. At slightly higher concentrations, (IC50 value 6:9  0:4 lM), zoledronate had similar effects upon VEGF induced proliferation, whereas pamidronate at identical concentrations had no effect. At these concentrations zoledronate did not induce apoptosis. Furthermore, zoledronate and pamidronate (at 50 lM) both completely inhibited capillary sprouting in an ex vivo model of angiogenesis (aortic ring assay). For the first time it was also shown that zoledronate had anti-angiogenic properties in non-mineralised tissue. A murine in vivo model was used whereby angiogenesis was induced by subcutaneous implants (porous chambers containing bFGF or VEGF). Mice were treated with 1–100 lg/kg/day/s.c. of zoledronate, or 100– 1000 lg/kg/day/s.c. of pamidronate, for 6 days, starting 24 h prior to chamber implantation. Following sacrifice, the vascularised tissue induced to grow around the implant was measured for weight and blood content. Zoledronate dose-dependently inhibited the b-FGF induced angiogenic response, with ED50 values of 3.1 and 2.9 lg/kg, for tissue blood content and tissue weight parameters, re-

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spectively. Zoledronate’s effect upon the VEGF induced response was much less, with ED50 values of >100 and 24 lg/kg, for blood content and weight parameters, respectively. Pamidronate, (doses 10fold greater than zoledronate), induced a dose-dependent inhibition of bFGF-induced angiogenesis, giving ED50 values of 190 and 560 lg/kg for blood content and weight parameters, respectively.

BISPHOSPHONATES HAVE SYNERGISTIC AND ADDITIVE POTENTIAL WITH OTHER DRUGS Of particular interest is the potential for bisphosphonates to enhance the anti-tumour activity of known cytotoxic agents that are commonly used in the clinical setting. This reflects more accurately the clinical scenario, particularly in breast cancer. Such experiments have been designed to test this in vitro for breast and prostate cancer, and multiple myeloma. Paclitaxel (TaxolTM ) and docetaxel (TaxotereTM ) are taxoids that induce mitotic spindle abnormalities, and are commonly used in the management of breast cancer, both in the early and advanced setting. As early as 1996 Sterns and Wang (57), using a murine model, found that the combination of paclitaxel with alendronate significantly enhanced the anti-tumour efficacy of paclitaxel. Human prostate PC-3 ML subclones were injected into SCID mice. Pre-treatment of these mice with alendronate (0.04–0.1 mg/kg twice weekly or 0.1 mg/kg weekly) reduced the formation of bone metastases but nonosseous metastases were unaffected. However, alendronate pre-treatment (0.1 mg/kg once or twice weekly) in addition to paclitaxel (10–50 mg/kg/day once or twice weekly), not only prevented the formation of bone metastases, but also prevented the formation of non-osseous metastases, with a corresponding increase in survival rate for dual-treated mice. Similarly ibandronate has been shown to enhance the anti-tumour behaviour of both paclitaxel and docetaxel (58). Whilst ibandronate did not enhance the ability of the taxoids to decrease breast cancer cell (MDA-MB-231) survival or induce apoptosis, the combination of taxoid with ibandronate produced an additive effect on inhibition of cell-adhesion to bone slices. In invasion and migration assays both taxoids dose-dependently inhibited tumour cell invasion and migration, (docetaxel being 10 times more potent compared to paclitaxel). Ibandronate alone partially inhibited tumour cell invasion, but had no effect upon migration. In combination experiments ibandronate produced an additive effect on inhibition of cell invasion induced by the taxoids.

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The authors postulate that it is possible the bisphosphonate is contributing its effect through inhibition of MMP proteolytic activity, whereas taxoids prevent invasion through their effect upon migration inhibition and/or apoptosis induction. In contrast Jagdev et al. (30) was able to show that treatment of MCF-7 cells with the combination of zoledronate (10 lM) with paclitaxel (2 nM) causes a synergistic increase in the proportion of apoptotic cells. From dose–response studies and consequent isobologram analysis they clearly showed that this drug combination produced synergistic effects upon reducing cell number and inducing the number of apoptotic cells. They also found that the combination of zoledronate (100 lM) with the antioestrogen tamoxifen (0.01 lM or 0.1 lM) was synergistic in inducing apoptosis in MCF-7 cells (59). In murine models (heart injection model of bone metastases using the MDA-MB-231 cell line) treatment with ibandronate and the anthracycline doxorubicin suppressed bone and non-osseous (adrenal) metastases (60) more effectively than either agent alone. In an orthotopic murine model (4T1 mammary tumour cells inoculated into mammary fad pad subcutaneously) incadronate or zoledronate cotreatment with UFT, (a pro-drug of fluorouracil), inhibited the formation of bone, lung and liver metastases, in an additive fashion. UFT alone significantly decreased all these metastases as well. Synergy experiments have also been carried out using human myeloma cell lines, XG-1, U266 and IM-9 (61). The potent glucocorticoid dexamethasone is commonly used in the treatment of myeloma. Following 6 days incubation of myeloma cell lines with zoledronate alone, dexamethasone alone, or in both in combination, the following results were obtained. For zoledronate-treated cells 45.4% growth inhibition occurred, and 52.1% with dexamethasone. This compares to 78.6% growth inhibition with both drugs, though not an additive effect, is at least a more potent effect. This is in comparison to the effect upon apoptosis, where synergy between the two drugs was seen. Zoledronate (50 lM) alone and dexamethasone (10 lM) alone induced apoptosis in 7.3% and 8.4%, respectively, as compared to control cultures (3.9% apoptosis) However, the drugs in combination induced apoptosis in 24.3% of cells implying synergistic activity. Interestingly zoledronate has also shown additive potential with cyclo-oxygenase (COX) inhibitors. Witters et al. (62) found that zoledronate alone (3 lM) and COX-2 inhibitor SC236 (5 lM) alone caused 23% and 40% growth inhibition of prostate cancer cells (DU-145), respectively. When used in combination there was an additive inhibitory effect of 60%. COX (prostaglandin H synthase) is an important enzyme involved in the formation of prostacyclins, throm-

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boxanes and prostaglandins from arachidonic acid, and exists in two isoforms, the constitutive COX-1 and COX-2, expressed only in response to certain stimuli. The enzyme COX-2 is overexpressed in a variety of malignancies, including breast (63) and prostate cancer (64), and is associated with increased malignant potential. COX-2 derived prostaglandins have been shown to induce neoangiogenesis, with COX-2 detectable in tumour vasculature, as well as in the tumour itself. In addition COX-2 expression significantly correlates with VEGF expression in certain malignancies (65). Furthermore COX-2 over-expression may have an inhibitory effect upon apoptosis, as celecoxib (a specific COX-2 inhibitor) was found to induce apoptosis in prostate cancer cells (66). In breast cancer oestrogens are produced from androgens, this conversion involving the enzyme aromatase, and hence the use of aromatase inhibitors in managing breast cancer. However breast cancers also contain intratumoural aromatase activity and COX-2 expression correlates with expression of aromatase in breast cancer tissue (67). Prostaglandins, such as PGE2 , regulate the activity of aromatase (68), and high levels of PGE2 are found in breast tumours. Such high concentrations were found to correlate with hormone receptor negativity and greater malignant potential (69). COX-2 over-expression has also been reported for the human invasive breast cancer cell line MDA-MB-231, with very low levels in the oestrogendependent MCF-7 breast cancer cell line (70). Hence, it may be of particular interest in breast cancer research, to combine a potent bisphosphonate such as zoledronate, with its potential effect upon apoptosis and angiogenic factors, with specific COX-2 inhibitors, with their potential effect also upon apoptosis, angiogenesis, and oestrogen expression.

CONCLUSIONS Whilst many questions remain unanswered, there does appear to be increasing evidence supporting indirect and direct anti-tumour activity, particularly of the N-bisphosphonates, but also the non-N-bisphosphonates. Clinical trials are planned to test the role of bisphosphonates in the adjuvant setting of those malignancies with a predilection for bone and highlight patient populations that will benefit most. Trials will need to be specifically designed and powered to enable survival analysis. In vivo and in vitro work will continue to define the specific antitumour mechanisms of bisphosphonates. Potential co-activity with other anti-tumour drugs is an important area that requires ongoing research, as this may more accurately reflect the clinical scenario, where patients often receive bisphosphonates in

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addition to anti-cancer agents. Of interest would be potential synergistic activity of different classes of bisphosphonate. Whilst the N-bisphosphonates are the most potent as regards inhibition of bone resorption, combination with non-N-bisphosphonates may contribute further anti-tumour activity, in part due to different mechanisms of action. Furthermore it is possible that non-N-bisphosphonates have influence on aspects of tumour activity that N-bisphosphonates do not. In particular N-bisphosphonates have been shown to induce increases in pro-inflammatory cytokines, such as IL-6 and TNF-a, whilst clodronate and etidronate do not (71). Such cytokines are also related to tumour growth and survival. Further work in these areas will define the roles of bisphosphonates, not only as inhibitors of bone resorption, but also as agents with specific or direct anti-tumour activities.

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