Preclinical Profile of Zoledronic Acid in Prostate Cancer Models

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1 European Urology Supplements European Urology Supplements 3 (2004) Preclinical Profile of Zoledronic Acid in Prostate Cancer Models Jonathan R. Green* Novartis Pharma AG, WKL Postfach, CH-4002 Basel, Switzerland Abstract Objective: To describe the preclinical data from animal models of prostate cancer demonstrating the pharmacologic effects of zoledronic acid on bone metabolism and tumor growth. Methods: Published data were reviewed. Results: In animal models, zoledronic acid has been shown to decrease tumor-induced osteolysis, prevent bone metastasis, and reduce skeletal tumor burden. In androgen-deficient mice injected intracardially with PC-3 human prostate cancer cells, zoledronic acid reduced bone metastases. In a model of prostate tumors growing in the tibiae of mice, zoledronic acid significantly inhibited growth of both osteolytic and osteoblastic tumors and reduced circulating levels of prostate-specific antigen. Data from preclinical studies demonstrate that zoledronic acid has effects not only on osteoclasts, but also on osteoblasts and tumor cells. Zoledronic acid can disrupt the interactions between tumor cells and the bone microenvironment that promote tumor growth in the bone. Zoledronic acid has also been shown to inhibit proliferation and induce apoptosis of human prostate cancer cell lines in vitro and has enhanced antitumor activity when combined with taxanes. In addition, zoledronic acid appears to inhibit tumor cell invasion of the extracellular matrix and angiogenesis. Conclusions: Zoledronic acid has potent antitumor effects in prostate cancer models of bone metastases. Further preclinical studies are needed to fully elucidate the biochemical mechanisms responsible for this activity. # 2004 Elsevier B.V. All rights reserved. Keywords: Antitumor; Prostate cancer; Zoledronic acid 1. Introduction Bisphosphonates are potent inhibitors of osteoclastmediated bone resorption and effectively inhibit tumorinduced osteolysis in patients with bone metastases. As a result, these agents decrease the destructive bone resorption associated with malignant bone disease, thereby reducing skeletal morbidity. There is now extensive in vitro and in vivo preclinical evidence that bisphosphonates also have antitumor activity, as evidenced by reduced skeletal tumor burden and reduced formation of new bone metastases in animal models. Several mechanisms have been proposed to explain these observations. Bisphosphonates may render the bone a less favorable microenvironment for tumor cell * Tel ; Fax: address: jonathan.green@pharma.novartis.com. colonization by reducing tumor-induced osteolysis and thereby decreasing local release of bone-derived growth factors. In addition, bisphosphonates appear to have direct antitumor effects. Bisphosphonates have been shown to inhibit proliferation and induce apoptosis of a variety of human tumor cell lines in vitro [1 10], including prostate cancer cell lines [11,12]. Bisphosphonates also inhibit tumor cell adhesion and invasion of the extracellular bone matrix [13 16], and zoledronic acid has demonstrated antiangiogenic activity [17,18]. Recently zoledronic acid demonstrated a statistically significant reduction in skeletal complications in patients with bone metastases from advanced prostate cancer. Heretofore, other bisphosphonates including pamidronate, which has demonstrated efficacy in patients with breast cancer had failed to demonstrate clinical benefit in patients with advanced /$ see front matter # 2004 Elsevier B.V. All rights reserved. doi: /j.eursup

2 J.R. Green / European Urology Supplements 3 (2004) prostate cancer, presumably because of the different pathophysiology of bone lesions associated with these tumors. Prostate cancer typically forms osteoblastic lesions characterized by increased formation of new bone. But these lesions are also associated with significant increases in osteolysis as evidenced by high levels of bone resorption markers [19,20]. Moreover, new bone formation is uncoupled from osteolysis and does not contribute to bone strength [21]. Therefore, prostate cancer patients with bone metastases are at risk for fractures and other skeletal complications, including severe bone pain requiring palliative radiotherapy. A study of bone resorption markers in prostate cancer patients with osteoblastic lesions has recently demonstrated the correlation between high levels of bone resorption markers and an increased risk of skeletal complications. Patients with high levels of N-telopeptide (100 nmol/mmol creatinine) had a 5- fold increased risk of skeletal complications and a 4-fold increased risk of death compared with patients with low N-telopeptide (<50 nmol/mmol creatinine) [22]. Preclinical evidence also suggests that bone resorption is an important initiating event in the formation of bone metastases from prostate cancer. Zoledronic acid is a member of a class of compounds known as nitrogen-containing bisphosphonates (N-BPs). Unlike early generation bisphosphonates, N-BPs contain a nitrogen atom in their R 2 side chain and have a unique mechanism of action that involves inhibition of protein prenylation [23 26]. Zoledronic acid is also unique among the currently approved N-BPs in that it contains 2 nitrogen atoms. This chemical feature may explain the greater clinical activity of zoledronic acid compared with other N-BPs. 2. Effects of zoledronic acid on bone metabolism Zoledronic acid has been studied in a range of in vitro and in vivo preclinical models to determine its pharmacologic effects on bone metabolism [27,28]. Two models of osteoclast-mediated bone resorption have demonstrated the potent pharmacologic activity of zoledronic acid compared with a variety of other bisphosphonates tested in these assays (Table 1) [27]. The in vitro model of calcium release from mouse calvaria in response to vitamin D 3 demonstrated that zoledronic acid was 10-fold more potent than ibandronate, 100-fold more potent than pamidronate, and 200-fold more potent than clodronate. The second model was an in vivo assay of vitamin D 3 -induced hypercalcemia in thyroid-parathyroidectomized rats. Table 1 Inhibition of vitamin D 3 -induced calcium release from mouse calvaria in vitro and hypercalcemia in the thyroid-parathyroidectomized (TPTX) rat Compound Mouse calvaria TPTX rat Mean IC 50 a, mm (no. experiments) Mean SEM ED 50 b, mg/kg (no. experiments) Zoledronic acid (5) (7) Risedronate 0.01 (2) (4) Ibandronate 0.02 (3) (5) Alendronate 0.05 (2) (3) Olpadronate 0.06 (4) (4) Pamidronate 0.2 (6) (15) Clodronate 0.4 (3) (5) Etidronate 4.0 (3) >20,000 (3) IC 50 : inhibitory concentration at 50%; SEM: standard error of the mean; ED 50 : median effective dose. a Concentration of bisphosphonate in the culture medium required to inhibit 1,25-(OH) 2 vitamin D 3 -induced calcium release by 50%. b Dose of bisphosphonate, given on 4 consecutive days SC, required to inhibit 1,25-(OH) 2 vitamin D 3 -induced hypercalcemia by 50%. Adapted from J Bone Miner Res 1994:9: with permission of the American Society for Bone and Mineral Research [27]. The results shown in Table 1 represent the daily subcutaneous (SC) dose of each bisphosphonate required to reduce serum calcium levels by 50% compared with control rats. Again, zoledronic acid proved to be the most potent bisphosphonate in this assay, with a median effective dose of 0.07 mg/kg, which was 850-fold lower than that of pamidronate. Zoledronic acid was also approximately 20-fold more potent than ibandronate and risedronate. Zoledronic acid demonstrated dose-dependent effects on hypercalcemia in this assay and achieved complete normalization of serum calcium at a dose of 1.4 mg/kg. The results obtained from these in vitro and in vivo assays showed a close correlation coefficient of 0.97, indicating that the primary physiologic effect of bisphosphonates in vivo is inhibition of calcium release from the bone rather than altered renal or intestinal transport of calcium Bone resorption under physiologic conditions To assess the effects of zoledronic acid on bone metabolism in vivo, short-term experiments were carried out in intact growing male rats, and long-term experiments were carried out in estrogen-deficient female rats and monkeys [27,29 31]. Radiographic analysis of growing male rats showed a dose-dependent increase in the bone density of the proximal tibial metaphysis in animals treated with either zoledronic acid or pamidronate. Detailed histomorphometric analysis of the proximal tibia revealed a normal mineralization profile and dose-dependent increases in cancellous bone [32]. These data indicate that pamidronate and

3 18 J.R. Green / European Urology Supplements 3 (2004) zoledronic acid cause a net increase in bone density in normal animals without adversely effecting bone mineralization. Two long-term experiments in estrogen-deficient rats and monkeys have further shown that zoledronic acid preserves bone mass, architecture, and strength under physiologic conditions that would normally lead to substantial bone loss [29 31].In ovariectomized rats, weekly SC injections of zoledronic acid (1.5 and 7.5 mg/kg/week) for 1 year completely preserved bone mineral density and maintained the mechanical strength of the femur and vertebrae [31]. Similarly, long-term treatment of ovariectomized adult rhesus monkeys with zoledronic acid (0.5 to 12.5 mg/kg/ week SC) for 69 weeks was well tolerated and dosedependently decreased bone turnover and bone loss without adversely affecting bone mineralization [29]. Consistent with these preclinical data, zoledronic acid (4 mg every 3 months) was recently shown to prevent bone loss in men with prostate cancer receiving androgen-deprivation therapy (ADT) [33]. After 1 year, patients treated with zoledronic acid had increased bone mineral density (BMD) compared with baseline and significantly greater BMD compared with the placebo group, which exhibited substantial bone loss as a result of ADT. These studies have important implications with respect to maintaining bone health and reducing fracture risk in patients receiving androgen-ablative therapies for prostate cancer Inhibition of osteoclasts and osteoclastogenesis The mechanism by which zoledronic acid and other N-BPs inhibit osteoclast activity has been elucidated in recent years. These compounds inhibit farnesyl diphosphonate synthase an important enzyme in the mevalonate pathway that is involved in the posttranslational modification (i.e., prenylation) of signaling molecules (eg, Ras and Rho) that are involved in a variety of essential functions, including membrane trafficking and cytoskeletal organization [23,25, 34 36]. Consequently, N-BPs interfere with a variety of cellular functions essential for the bone-resorbing activity and survival of osteoclasts [37,38]. For example, in vitro studies have shown that osteoclasts exposed to N-BPs are unable to form a tight sealing zone or ruffled borders as a result of defects in intracellular vesicle transport, and this effect was reversed when protein prenylation was restored [39]. In addition, N-BPs induce apoptosis of osteoclasts [5,40]. Recent evidence from in vitro studies and animal models has demonstrated that N-BPs also inhibit osteoclastogenesis and recruitment of osteoclast progenitors to the bone by a mechanism involving inhibition of protein prenylation [41,42]. This has important implications with regard to tumor growth in bone. As a result, fewer mature osteoclasts accumulate at sites of tumor growth, and tumor-induced osteolysis is minimized. A recent in vitro study suggested a potential mechanism to explain this effect. Low concentrations of both pamidronate and zoledronic acid were shown to increase production of osteoprotegerin (OPG) from primary human osteoblasts [43]. Zoledronic acid caused time- and dose-dependent increases in OPG production by human osteoblasts with a maximum effect after 72 hours. OPG inhibits osteoclastogenesis by antagonizing the activity of receptor activator of nuclear factor-kb (RANK) ligand [44,45]. RANK ligand is normally expressed on the surface of osteoblasts and, when it binds to its receptor (RANK) on the surface of osteoclast precursors, it induces them to differentiate into mature osteoclasts [46]. Therefore, up-regulation of OPG would inhibit differentiation and maturation of osteoclasts. Consistent with this proposed mechanism, it has been demonstrated that administration of OPG inhibits osteoclastogenesis and formation of bone metastases in mice [47,48]. One such experiment demonstrated that OPG inhibited metastasis of the prostate cancer cell line C4-2B to bone when it was administered at the same time as tumor cell inoculation [48]. Moreover, in a murine model of human myeloma, treatment with a specific inhibitor of RANK ligand (RANK-Fc) inhibited myeloma cell growth and survival in the bone [49]. Similarly, N-BPs inhibit formation of bone metastases in a variety of animal models. These studies suggest that osteoclastogenesis and initiation of bone resorption are important for tumor cells to establish bone metastases Effects on osteoblasts Although few studies have examined the effects of bisphosphonates on osteoblasts, it appears that N-BPs may affect the proliferation and maturation of human osteoblasts. This has important implications with respect to the effects of bisphosphonates in patients with prostate cancer. These studies have shown that N-BPs, including pamidronate, ibandronate, and zoledronic acid, had little effect on osteoblast proliferation or survival but had significant effects on osteoblast differentiation as evidenced by increased type I collagen synthesis [50,51]. Pamidronate and zoledronic acid were shown to increase total cellular protein, alkaline phosphatase activity, and type I collagen secretion in human osteoblast cultures and to increase the rate of cell mineralization [51]. Therefore, in addition to inhibiting bone resorption, zoledronic acid and other N-BPs appear to enhance the bone-forming activity of osteoblasts. This activity in conjunction

4 J.R. Green / European Urology Supplements 3 (2004) with the well-documented effects of bisphosphonates on osteoclast-mediated bone resorption could help to maintain the normal homeostasis of the bone. 3. Antitumor effects against prostate cancer 3.1. In vitro studies In addition to its effects on osteoclasts and osteoblasts, zoledronic acid has potent antitumor and antiangiogenic effects and, therefore, may have the potential to reduce tumor burden in bone via a more direct antitumor mechanism. Bisphosphonates have demonstrated dose- and time-dependent antitumor activity (cytostatic and apoptotic effects) against a variety of human tumor cell lines in vitro [1 10,52], including prostate cancer cell lines [11,12,53]. In vitro studies with different prostate cancer cell lines (i.e., PC-3, LNCaP, and Du145) have shown that zoledronic acid inhibits proliferation, reduces cell viability, induces apoptosis, and causes cell-cycle arrest [11,12,53]. The effects of zoledronic acid and pamidronate (10 to 100 mm) on the viability of these cell lines is shown in Fig. 1 [53]. Although these cell lines have different phenotypes, zoledronic acid dose-dependently decreased viability to nearly zero in all cases. In the studies reported by Corey et al. [11],zoledronicacid (14 mm) inhibited proliferation of PC-3 and LNCaP cells by up to 70% after 4 days, and a 5-fold higher concentration (68 mm) induced apoptosis and caused cell-cycle arrest. The androgen-independent PC-3 cell line, which lacks functional p53, underwent rapid apoptosis and cell-cycle arrest in S phase, whereas the androgen-dependent LNCaP cells with wild-type p53 exhibited a more delayed apoptotic response and G 1 phase arrest. Therefore, the status of p53 in prostate tumor cells appears to influence their response to N-BPs. These in vitro studies have also been extended using a more physiologic culture system that mimics the normal microenvironment of prostate tumors growing in bone. In these experiments, prostate epithelial cells were co-cultured with bone marrow stromal cells and colony formation was assessed [54]. Zoledronic acid potently inhibited colony formation in this assay with a maximal effect at 50 mm. At this concentration, clodronate and pamidronate had little effect. In vitro studies have also shown that the combination of zoledronic acid with standard anticancer drugs, including taxanes, results in synergistic apoptotic effects on a variety of tumor cell lines [55,56]. For example, in 72-hour cultures of MCF-7 breast cancer cells, the combination of zoledronic acid (10 mm) plus paclitaxel (2 nm) resulted in a 4-fold enhancement of apoptosis compared with either agent alone (Fig. 2) [55,57]. However, this synergy between zoledronic acid and taxanes is not restricted to breast cancer cell lines. The combination of low concentrations of zoledronic acid (12.5 or 25 mm) plus subtherapeutic concentrations of docetaxel (1 ng/ml) also demonstrated additive and dose-dependent effects on the viability of PC-3 prostate cancer cells [58]. If these in vitro findings translate to the clinical setting, it may be possible to reduce the dose of taxanes and improve tolerability without reducing efficacy. Recent studies with DU-145 prostate cancer and MCF-7 breast cancer cell lines have also demonstrated that the combination of zoledronic acid with the cyclooxygenase-2 inhibitor SC236 had additive inhibitory effects on the growth of these tumor cells [59,60] Animal models These in vitro findings are supported by data from animal models showing that bisphosphonates can reduce skeletal tumor burden [3,11,49,61 65]. These studies have focused primarily on models of multiple myeloma, breast, and prostate cancer and, using radio- Fig. 1. Effects of zoledronic acid and pamidronate on viability of human prostate cancer cell lines. *Indicates statistically significant reductions from baseline (p < 0.001). Adapted with permission from Oades et al. [53]. Fig. 2. Synergistic effect of zoledronic acid plus paclitaxel on apoptosis of MCF-7 breast cancer cells in vitro. Data from Jagdev et al. [55] and reproduced with permission from Clézardin [57].

5 20 J.R. Green / European Urology Supplements 3 (2004) Fig. 4. Bone histomorphometric analysis to tibiae injected with either (A) PC-3 or (B) LuCaP 23.1 human prostate cancer cells from animals treated with vehicle or zoledronic acid either at the time of tumor cell injection (prevention) or after bone tumors were established (treatment). Data are presented as percent tumor volume versus total tissue volume. *p < yp < Adapted with permission from Corey et al. [11]. graphic, histologic, and histomorphometric techniques, have clearly shown that bisphosphonates can inhibit the formation or progression of bone metastases and/or reduce skeletal tumor burden. These effects are observed when the bisphosphonate is administered either at the time of tumor cell inoculation (prevention setting) or after bone metastases are established (treatment setting). In vivo studies in a prostate cancer model have recently been reported [11]. In these studies PC-3 and LuCaP 23.1 cells were injected directly into the tibiae of mice. In this model, PC-3 cells form osteolytic lesions whereas LuCaP cells form osteoblastic lesions more typical of human prostate cancer. The treatment group received zoledronic acid (5 mg SC twice weekly) either at the time of tumor cell injection or after tibial tumors were established (7 days for PC-3 and 33 days for LuCaP tumors). Treatment with zoledronic acid significantly inhibited growth of both osteolytic and osteoblastic metastases by radiographic analysis compared with untreated control mice (Fig. 3) [11]. In animals injected with PC-3 cells, skeletal tumor volume was significantly reduced in both the prevention (p = 0.029) and treatment (p = 0.022) settings (Fig. 4A) [11]. Likewise, in animals injected with LuCaP cells, zoledronic acid also reduced skeletal tumor volume in both the prevention (p = 0.001) and treatment (p = 0.009) settings (Fig. 4B) [11]. Therefore, zoledronic acid reduces skeletal tumor burden associated with both lytic and blastic lesions. In animals injected with LuCaP cells, zoledronic acid also significantly decreased serum levels of prostatespecific antigen in both the prevention (p = 0.02) and treatment (p = 0.01) settings (Fig. 5) [11], showing that zoledronic acid has clinically relevant effects on a surrogate marker for tumor burden. However, zoledronic acid did not inhibit growth of SC tumors in this model. These in vivo studies provide compelling evidence of the potential of zoledronic acid to reduce tumor burden in bone and inhibit formation and progression of bone metastases in prostate cancer models. This could have important implications with respect to the clinical benefit of zoledronic acid in patients with prostate cancer. Zoledronic acid significantly reduced skeletal complications in patients with bone metastases from advanced prostate cancer. Zoledronic acid is also Fig. 3. Radiographic imaging of mouse tibiae bearing osteolytic PC-3 or osteoblastic LuCaP 23.1 tumors from control animals and animals treated with zoledronic acid (5 mg SC twice weekly). Zoledronic acid was administered either at the time of tumor cell injection (prevention) or after tumors were established (treatment). Adapted with permission from Corey et al. [11]. Fig. 5. Blood concentration of prostate-specific antigen (PSA) over time in animals bearing LuCaP 23.1 tibial tumors and treated with zoledronic acid either at the time of tumor cell injection (prevention) or after bone tumors were established (treatment). Data expressed as mean standard error of the mean at each time point. Adapted with permission from Corey et al. [11].

6 J.R. Green / European Urology Supplements 3 (2004) currently being investigated in patients with early stage prostate cancer to determine if it will reduce the incidence of bone metastases. The potential of zoledronic acid to prevent bone metastasis has been elegantly demonstrated in an animal model of prostate cancer [66]. In this model, mice were injected intracardially with PC-3 cells and the incidence of bone metastases was studied in normal mice versus mice that were rendered androgen-deficient, thus mimicking the situation in patients receiving antiandrogen therapy. Mice that were surgically castrated developed significantly more bone metastases than did intact control mice. This strongly suggests that excessive bone resorption caused by androgen ablation can stimulate metastasis of PC-3 cells to the bone. Although it remains to be seen if this is true for other prostate cancer cell lines that form typical osteoblastic lesions, this is consistent with current hypotheses of how tumor cells colonize the bone. Moreover, daily treatment of both normal mice and castrated mice with zoledronic acid significantly reduced the incidence of bone metastases. This effect may be a result of reduced bone resorption or a direct antitumor effect. 4. Mechanisms of antitumor effects A variety of mechanisms have been proposed to explain the observed antitumor effects of zoledronic acid (Table 2) [11,13,14,17,40,43,66 68]. The majority of existing evidence from animal models points to inhibition of bone resorption as the primary antitumor mechanism; however, there is abundant in vitro evidence that bisphosphonates inhibit tumor cell proliferation and directly induce apoptosis of tumor cells. It has long been suggested that bisphosphonates may inhibit tumor growth in the bone microenvironment Table 2 Proposed antitumor mechanisms of zoledronic acid Mechanism Reference Indirect Inhibition of bone resorption and # release of Padalecki et al. [66] bone-derived growth factors Inhibition of osteoclastogenesis via " OPG Viereck et al. [43] Antiangiogenic effects Wood et al. [17] Direct # Proliferation of tumor cells Corey et al. [11] " Apoptosis of tumor cells Benford et al. [40] # Invasiveness Boissier et al. [13,14] # MMP activity/expression Corey et al. [11] Cytoskeletal disorganization Denoyelle [67] Modulation of a v b 3 expression Pécheur et al. [68] OPG: Osteoprotegerin; MMP: Matrix metalloproteinase. and the ability of tumor cells to colonize the bone by reducing the numbers and activity of osteoclasts, thus reducing the release of growth factors and making the bone less fertile soil [69]. Tumor cells secrete a variety of factors, including parathyroid hormone-related peptide, that stimulate bone resorption, which in turn leads to release of bone-derived growth factors such as transforming growth factor b, insulin-like growth factors, and fibroblast growth factor. These growth factors stimulate tumor cells to proliferate and to stimulate further bone resorption. As described above, evidence from animal models suggests that simply inhibiting osteoclastogenesis and osteoclast-mediated bone resorption can profoundly inhibit the formation of bone metastases [47,48,66]. However, this may not completely explain the observation that bisphosphonates can inhibit colonization of the bone and tumor growth in bone. Bisphosphonates clearly have direct antitumor activity as well. One of the primary mechanisms by which zoledronic acid and other bisphosphonates appear to exert direct antitumor effects is via induction of apoptosis [3,5 7,9,10]. Presumably, the concentration of bisphosphonates in the bone at sites of active bone metabolism would be sufficient to induce apoptosis of tumor cells growing in bone. Bisphosphonates appear to induce apoptosis of both osteoclasts and tumor cells alike by activation of caspases [5,40,52], and recent studies suggest that activation of caspasedependent apoptosis is caused by inhibition of Ras farnesylation [70] Inhibition of tumor cell adhesion and invasion of the extracellular bone matrix Bisphosphonates have also been shown to directly inhibit adhesion of tumor cells to the bone extracellular matrix and to inhibit the process of tumor cell invasion and metastasis [13 16]. An in vitro Matrigel TM -based invasion assay demonstrated that N-BPs inhibited the ability of human breast and prostate cancer cells to invade the extracellular matrix [13]. In this assay, subnanomolar concentrations of zoledronic acid (10 10 M) that did not inhibit tumor cell motility or induce significant apoptosis effectively inhibited tumor cell invasion into the Matrigel. Similar findings have also been reported for alendronate, and this effect was reversed by addition of geranyl geraniol and transtrans-farnesol [71]. One contributory mechanism may be inhibition of matrix metalloproteinase (MMP) activity, which is important for tumor cell invasion and migration. Bisphosphonates are broad-spectrum inhibitors of MMPs [13,72,73] and have been shown to inhibit the activity of MMPs produced by tumor cell

7 22 J.R. Green / European Urology Supplements 3 (2004) lines. For example, zoledronic acid was shown to inhibit the production of MMP-2 and MMP-9 by PC-3 cells [11]. However, this occurs at higher concentrations than those required to reduce invasion in the Matrigel assay [72,73]. Therefore, inhibition of MMPs is probably not the primary mechanism by which bisphosphonates inhibit tumor cell invasion. Studies using MDA-MB-231 breast cancer cells suggest that zoledronic acid also causes disorganization of the actin cytoskeleton by a mechanism involving inhibition of RhoA activity, which requires protein prenylation [67]. Consistent with results found in other studies, this effect was reversed by geranyl geraniol and could be mimicked by a Rhoselective inhibitor. Finally, N-BPs also appear to modulate expression of a number of cell adhesion molecules, such as a v b 3, resulting in decreased metastatic potential of tumor cells (reviewed by Drs. Per-Anders Abrahamsson and E. David Crawford in this supplement) [68].A small molecule inhibitor of a v b 3 was recently shown to effectively prevent metastasis of MDA-MB-231 breast cancer cells to bone [74]. Interestingly, a v b 3 integrin is also required for osteoclasts to adhere tightly to the bone and form resorption lacuna during active bone resorption. Therefore, effects on a v b 3 could have pleiotropic effects on bone resorption and tumor metastasis Antiangiogenic effects In vitro and in vivo studies have demonstrated that zoledronic acid also has antiangiogenic effects. Zoledronic acid dose-dependently inhibited the proliferation of human umbilical vein endothelial cells (HUVEC) in vitro [17], and inhibited capillary-like tubule formation by HUVEC in the Matrigel assay [18]. In addition, it has recently been reported that zoledronic acid decreases the survival of HUVEC by sensitizing them to tumor necrosis factor-induced programmed cell death [75]. In an animal model, zoledronic acid was shown to decrease revascularization (as measured by vessel area) of the ventral prostate gland in castrated rats treated with testosterone [18]. The observed inhibitory effect of zoledronic acid on endothelial cell proliferation, adhesion, and migration appears to be mediated, at least in part, by modulation of the expression of a v b 3 and a v b 5 integrins and the 67-kD laminin receptor [75,76]. Another potential mechanism that could contribute to the antiangiogenic effects of zoledronic acid is modulation of proangiogenic growth factors, including vascular endothelial growth factor and basic fibroblast growth factor. In cancer patients with bone metastases, treatment with zoledronic acid resulted in significant and sustained decreases in serum levels of vascular endothelial growth factor for up to 21 days [77]. 5. Conclusions and future directions Preclinical evidence suggests that zoledronic acid has antitumor activity against prostate cancer cells and prostate tumors growing in bone. A variety of potential mechanisms to explain these effects have been proposed. However, further research will be required to fully elucidate the molecular mechanisms involved and to determine the most effective dose and schedule of zoledronic acid to maximize its antitumor potential against prostate cancer, either alone or in combination with standard antineoplastic drugs such as taxanes. Recently a randomized placebo-controlled trial in men with hormone-independent prostate cancer demonstrated that treatment with 4 mg zoledronic acid (every 3 to 4 weeks for up to 2 years) resulted in statistically significant reductions in skeletal complications and bone pain. Correlative studies suggest that reducing skeletal complications, particularly fractures, may provide an indirect survival benefit in men with prostate cancer [78]. Studies are ongoing to further investigate the clinical benefit of zoledronic acid in patients with prostate cancer throughout the course of their disease. In men with early stage disease, zoledronic acid prevents bone loss associated with ADT, which may reduce the risk of bone metastasis. In addition, the available preclinical evidence suggests that zoledronic acid may have beneficial effects on tumor progression in bone. Therefore, zoledronic acid is being investigated for the prevention of bone metastasis in patients with early stage prostate cancer. References [1] Aparicio A, Gardner A, Tu Y, Savage A, Berenson J, Lichtenstein A. In vitro cytoreductive effects on multiple myeloma cells induced by bisphosphonates. Leukemia 1998;12: [2] Derenne S, Amiot M, Barille S, Collette M, Robillard N, Berthaud P, et al. 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