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Bisphosphonates: Preclinical Review

JONATHAN R. GREEN
Novartis Pharma AG, Basel, Switzerland

Key Words. Bisphosphonates Zoledronic acid Bone resorption Apoptosis Antitumor effects Mevalonate pathway

L EARNING O BJECTIVES
After completing this course, the reader will be able to:

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1. Describe the mechanism of action of first-generation and nitrogen-containing bisphosphonates.
2. Explain how the mechanism of action of the bisphosphonates might directly affect tumor growth.
3. Discuss how the bisphosphonates might be incorporated into both the prevention and treatment of cancer.

CME Access and take the CME test online and receive 1 hour of AMA PRA category 1 credit at CME.TheOncologist.com

A BSTRACT
Bisphosphonates effectively inhibit osteoclast-medi-
ated bone resorption and are integral in the treatment of
benign and malignant bone diseases. The evolution of bis-
phosphonates over the past 30 years has led to the devel-
opment of nitrogen-containing bisphosphonates (N-BPs),
which have a mechanism of action different from that of
the nonnitrogen-containing bisphosphonates. Studies
conducted over the past decade have elucidated the
mechanism of action and pharmacologic properties of the
N-BPs. N-BPs exert their effects on osteoclasts and tumor
cells by inhibiting a key enzyme in the mevalonate path-
way, farnesyl diphosphate synthase, thus preventing pro-
tein prenylation and activation of intracellular signaling
proteins such as Ras. Recent evidence suggests that
N-BPs also induce production of a unique adenosine
triphosphate analogue (Apppi) that can directly induce
apoptosis. Our increased understanding of the pharma-
cologic effects of bisphosphonates is shedding light on the
mechanisms by which they exert antitumor effects. As a
result of their biochemical effects on protein prenylation,
N-BPs induce caspase-dependent apoptosis, inhibit
matrix metalloproteinase activity, and downregulate
v3 and v5 integrins. In addition, zoledronic acid
(Zometa; Novartis Pharmaceuticals Corp.; East
Hanover, NJ and Basel, Switzerland) exerts synergistic
antitumor activity when combined with other anticancer
agents. Zoledronic acid also inhibits tumor cell adhesion
to the extracellular matrix and invasion through
Matrigel and has antiangiogenic activity. A growing
body of evidence from animal models demonstrates that
zoledronic acid and other bisphosphonates can reduce
skeletal tumor burden and prevent metastasis to bone.
Further studies are needed to fully elucidate these bio-
chemical mechanisms and to determine if the antitumor
potential of bisphosphonates translates to the clinical set-
ting. The Oncologist 2004;9(suppl 4):3-13

INTRODUCTION achieve therapeutic concentrations. The pioneering work of

Bisphosphonates are ideally suited for the treatment of Fleisch and colleagues showed that bisphosphonates not only
metabolic bone disease because they bind avidly to the bone inhibit dissolution of hydroxyapatite crystals, but also affect
mineral at sites of active bone metabolism, where they osteoclast metabolism and function [1-3]. Bisphosphonates

Correspondence: Jonathan R. Green, Ph.D., Novartis Pharma AG, Klybeckstrasse 141, WKL-125.901, CH-4002 Basel,
Switzerland. Telephone: 41-61-696-4415; Fax: 41-61-696-3849; e-mail: jonathan.green@pharma.novartis.com Received
July 19, 2004; accepted for publication August 3, 2004. AlphaMed Press 1083-7159/2004/$12.00/0

The Oncologist 2004;9(suppl 4):3-13 www.TheOncologist.com

4 Bisphosphonates: Preclinical Review
are released during bone resorption and are internalized by
osteoclasts, leading to inhibition of bone resorption and
induction of osteoclast apoptosis [4-6].
There is now extensive preclinical evidence that bis-
phosphonates also have antitumor activity, as evidenced by
reduced proliferation and viability of tumor cell lines in
vitro and reduced skeletal tumor burden and slower pro-
gression of bone lesions in animal models. Several mecha-
nisms have been proposed to explain these observations.
Bisphosphonates may render the bone a less favorable
microenvironment for tumor cell growth by reducing
tumor-induced osteolysis and local release of growth fac-
tors, and bisphosphonates may also have direct antitumor
effects. Bisphosphonates inhibit proliferation and induce
apoptosis of a variety of human tumor cell lines in vitro [7-
17]. Bisphosphonates also inhibit tumor cell adhesion to the
extracellular bone matrix, inhibit invasion of tumor cells
through Matrigel, and have antiangiogenic and immuno-
modulatory activities. Consistent with these findings,
bisphosphonates have been shown to inhibit the formation
or progression of bone metastases and/or reduce skeletal
tumor burden in a variety of animal models. The mecha-
nisms responsible for the observed antitumor effects of
bisphosphonates are beginning to be elucidated.
MECHANISM OF ACTION
Bisphosphonates accumulate in the mineralized bone
matrix and are released during bone resorption. First-gener-
ation, nonnitrogen-containing bisphosphonates are metabo-
lized by osteoclasts to nonhydrolyzable cytotoxic ATP
analogues [18-21]. For example, clodronate is metabolized
to AppCC12p, which, at high concentrations, inhibits mito-
chondrial ATP/adenosine diphosphate (ADP) translocase,
thereby causing loss of the mitochondrial membrane poten-
tial and direct induction of apoptosis [22-24]. The high
affinity of bisphosphonates for bone mineral and subse-
quent uptake by activated osteoclasts during bone resorp-
tion ensures that cytotoxic concentrations of these
metabolites only accumulate within osteoclasts. However,
they may also accumulate within tumor cells growing in the
bone because tumor cells stimulate osteolysis.
In contrast, nitrogen-containing bisphosphonates (N-BPs),
which include pamidronate (Aredia; Novartis Pharma-
ceuticals Corp.; East Hanover, NJ), alendronate (Fosamax;
Merck and Company, Inc.; West Point, PA), ibandronate
(Bondronat; Hoffmann-La Roche Inc.; Nutley, NJ), rise-
dronate (Actonel; Proctor and Gamble Pharmaceuticals, Inc.;
Cincinnati, OH), and zoledronic acid (Zometa; Novartis
Pharmaceuticals Corp.), affect osteoclast activity and survival
through a different mechanism. After internalization, these
compounds inhibit a key enzyme, farnesyl diphosphonate
HMG CoA
HMG CoA
Statins
reductase
Mevalonate
Mevalonate
pyrophosphate
Dimethylallyl Isopentenyl
pyrophosphate+ AMP Apppi
pyrophosphate
Geranyl
pyrophosphate
N-BPs FPP-synthase
Farnesyl
pyrophosphate Farnesol
Dolichol
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Geranyl-Geranyl Squalene
pyrophosphate Protein
farnesylation
Ubiquinone
Cholesterol
Protein geranyl-
geranylation
Figure 1. Schematic representation of the mevalonate pathway and
the effects of nitrogen-containing bisphosphonates. Abbreviation:
HMG CoA = 3-hydroxy-3-methylglutaryl coenzymeA. Adapted with
permission from Gober et al. [25].
(FPP) synthase, in the biosynthetic mevalonate pathway (Fig.
1) [5, 25-28]. As a result, N-BPs interfere with a variety of
cellular functions essential for the bone-resorbing activity
and survival of osteoclasts [28, 29]. Several intermediates in
this pathway, including farnesyl pyrophosphate and geranyl-
geranyl pyrophosphate, are required for the posttranslational
modification (i.e., prenylation) of guanosine triphosphate-
binding proteins such as Ras, Rho, and Rac [30]. These sig-
naling molecules are involved in the regulation of cell
proliferation, cell survival, and cytoskeletal organization [26,
28, 31, 32]. In particular, inhibition of protein prenylation
and Ras signaling within osteoclasts leads to defects in intra-
cellular vesicle transport [33]. As a result, osteoclasts cannot
form a tight-sealing zone or ruffled borders, which are
required for bone resorption.
Recent studies have shown that N-BPs may have yet
another mechanism of action. As shown in Figure 1 [25, 34],
N-BPs also induce production of an intracellular ATP ana-
logue known as Apppi (triphosphoric acid 1-adenosin-5-yl
ester 3-[3-methylbut-3-enyl] ester), which may directly
induce apoptosis similar to AppCC12p (i.e., a metabolite of
clodronate) [34]. Inhibition of FPP synthase results in accu-
mulation of isopentenyl diphosphonate, which can be
metabolized to Apppi. The ability of various bisphospho-
nates (at 0.1-M concentrations) to inhibit recombinant
human FPP synthase activity (Fig. 2) [35] correlates well
with their ability to induce production of Apppi in J774
Green 5

in the FPP synthase assay correlate closely with their relative

100
potencies in terms of inhibiting vitamin D3-induced calcium
release from mouse calvaria cultures [36]. These data suggest
FPP synthase (% control)

75 that inhibition of FPP synthase is a central mechanism by

which N-BPs inhibit osteoclast-mediated bone resorption.
Moreover, osteoclasts isolated from animals treated with N-
50
BPs show a profound suppression of FPP synthase activity
[37]. Therefore, FPP synthase appears to be one of the key
25
* molecular targets of N-BPs. Inhibition of protein prenylation
in cancer cells is also thought to be responsible for many of
* the observed antitumor effects of N-BPs.
0 *
Clodronate Ibandronate Risedronate Zoledronic acid
EVIDENCE OF ANTITUMOR EFFECTS

Figure 2. The effects of equimolar concentrations (0.1 m) of In Vitro Studies

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bisphosphonates on recombinant human FPP synthase activity.
Bisphosphonates have demonstrated direct antitumor
*p < 0.001. Adapted with permission from Dunford et al. [35].
activity against a variety of tumor cell lines at concentrations
ranging from 5-2,000 M [38]. Bisphosphonates cause dose-
cells. In this assay, clodronate serves as a negative control and time-dependent inhibition of proliferation and induce
with little or no effect on FPP synthase activity, and it apoptosis of myeloma, breast cancer, prostate cancer, pancre-
induces no Apppi production. Of the N-BPs tested, zole- atic cancer, lung cancer, and osteosarcoma cell lines in vitro
dronic acid demonstrated the highest potency in terms of [7-17, 39]. For example, in studies with the human MDA-
both inhibition of FPP synthase activity (Fig. 2) [35] and MB-231 breast cancer cell line, clodronate, pamidronate, and
production of Apppi [34]. The implication of this work is zoledronic acid demonstrated dose-dependent effects on cell
that N-BPs, by inhibiting FPP synthase, can potentially viability, with 50% inhibitory concentration (IC50) values of
induce apoptosis of osteoclasts and tumor cells by at least 700, 40, and 15 M, respectively [12]. This effect was not
two distinct pathways. caused by calcium chelation and appeared to be specific to
It has been demonstrated that the potency of various tumor cells. Similar results have been observed with other
N-BPs with respect to inhibition of FPP synthase activity breast cancer cell lines. For instance, dose-dependent
also correlates well with their observed potency in terms of increases in the proportion of apoptotic cells have been
inhibiting bone resorption in vitro (Table 1) [35, 36]. Among observed when Hs 578T cells were incubated with zoledronic
the N-BPs tested, zoledronic acid was the most potent acid [12]. Studies with MCF-7 cells have further shown that
inhibitor of FPP synthase activity, followed by risedronate the effects of zoledronic acid on tumor cell viability and apop-
and ibandronate. The relative potencies of these three N-BPs tosis could be reversed by incubation with geranylgeraniol,
indicating that inhibition of protein prenylation can induce
tumor cell apoptosis [39]. In vitro studies with several differ-
Table 1. Relative potencies of bisphosphonates with respect to ent prostate cancer cell lines, including PC-3 and LNCaP
inhibition of FPP synthase and bone resorption activity
23.1, have shown that zoledronic acid inhibits proliferation,
FPP synthase Bone resorption induces apoptosis, and causes cell-cycle arrest in a dose-
Bisphosphonate IC50 (M)a IC50 (M)b
dependent manner [40, 41]. These are just a few examples of
Zoledronic acid 0.02 0.002
the antitumor activity of bisphosphonates against a wide vari-
Risedronate 0.10 0.01 ety of human and murine tumor cell lines, and in every
Ibandronate 0.31 0.02 instance where several bisphosphonates have been tested,
Alendronate 0.50 0.05 zoledronic acid has demonstrated the most potent activity.
Pamidronate 0.85 0.2 In vitro studies have also shown that combining N-BPs
a
Mean values calculated from dose-response plots of inhibition of with a variety of standard anticancer agents results in addi-
FPP synthase in J774 cell homogenates based on three experiments. tive or synergistic antitumor effects against a range of
Data from Dunford et al. [35]. tumor cell lines [39, 42-49]. Jagdev et al. [39] were the first
b
Inhibition of 1,25-dihydroxyvitamin D3-induced calcium release to show that the combination of zoledronic acid (10 M)
from mouse calvaria in vitro. Data represent means of several plus paclitaxel (Taxol; Bristol-Myers Squibb; Princeton,
experiments. Data from Green et al. [36].
NJ; 2 nM) enhanced apoptosis of MCF-7 breast cancer cells
6 Bisphosphonates: Preclinical Review
fourfold compared with either agent alone. Similar results
were reported when ibandronate or zoledronic acid were
combined with epirubicin (Ellence; Pharmacia and
Upjohn; Portage, MI) plus docetaxel (Taxotere; Aventis
Pharmaceuticals Inc.; Bridgewater, NJ) [50]. In cultures of
primary breast cancer cells, the concentrations of epirubicin
plus docetaxel were gradually reduced to suboptimal levels
by serial dilution until there was little or no inhibition of
tumor cell growth with these two drugs alone. However, the
addition of 15 M ibandronate or zoledronic acid resulted in
35% and 70% inhibitions of tumor cell growth, respectively.
Combinations of zoledronic acid with taxanes also have
demonstrated synergistic antitumor activity against prostate
cancer cell lines. The combination of zoledronic acid
(12.5 or 25 M) with suboptimal concentrations of doc-
etaxel (0.1 ng/ml) demonstrated additive and dose-depen-
dent cytotoxic effects on PC-3 prostate cancer cells at 72
hours [45]. In addition, recent studies with DU-145 prostate
cancer and MCF-18 breast cancer cell lines have demon-
strated that the combination of zoledronic acid with the
cyclooxygenase-2 inhibitor SC236 had additive inhibitory
effects on tumor cell growth [48, 49]. These and many other
studies have shown that N-BPs can enhance the cytotoxic
and cytostatic effects of standard anticancer agents used to
treat a variety of solid tumors. Recently, zoledronic acid
also has been shown to possess antileukemic activity
against a Philadelphia chromosome-positive cell line, and
the combination of zoledronic acid with imatinib mesylate
(Gleevec; Novartis Pharmaceuticals Corp.) demonstrated
synergistic antileukemic activity [51]. These findings are
intriguing given that Bcr-Abl stimulates the Ras signaling
pathway.
Two recent studies with breast cancer cell lines have
begun to shed light on the possible mechanisms underlying
the observed synergy between N-BPs and anticancer agents.
In the first of these studies, MCF-7 breast cancer cells were
incubated with zoledronic acid (25 M) and doxorubicin
(Adriamycin; Pharmacia and Upjohn; Peapack, NJ; 0.05
M), and the effects of different sequences and incubation
periods were tested [43]. The only combination that resulted
in synergistic enhancement of apoptosis was doxorubicin for
24 hours followed by zoledronic acid for 1 hour, whereas
zoledronic acid administered either before doxorubicin or
together with doxorubicin did not increase apoptosis (Fig. 3)
[43]. This suggests that exposure to doxorubicin at a concen-
tration that was not cytotoxic sensitized tumor cells to the
cytotoxic effects of zoledronic acid. A similar but opposite
effect of sequencing was also reported by the same group
when zoledronic acid (25 M) was combined with tumor
necrosis factor (TNF)-related apoptosis inducing ligand
(TRAIL) at a concentration of 10 ng/ml. Among five combi-
nations tested, the only combination that produced synergis-
tic apoptotic effects was zoledronic acid for 48 hours fol-
lowed by TRAIL for 24 hours. This combination yielded
14.7% apoptotic cells compared with 0.7% for zoledronic
acid alone and 2.7% for TRAIL alone (p < 0.001). The
intriguing results of these in vitro studies suggest a novel
approach to enhance the synergy between N-BPs and
chemotherapeutic agents in the clinical setting.
Animal Models
These in vitro findings are supported by data from many
animal models showing that the newer N-BPs can significantly
reduce the number and size of osteolytic lesions in tumor-bear-
ing mice, reduce skeletal tumor burden, induce tumor cell
apoptosis in bone lesions, reduce serum levels of tumor mark-
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ers, and prevent formation of bone metastases (Table 2) [9, 40,
52-60]. Some models have even shown effects on visceral
tumors and an improvement in survival of tumor-bearing
mice, but these findings have been less consistently observed.
Animal studies have focused on models of multiple myeloma,
breast cancer, and prostate cancer. Using radiographic, histo-
logic, and histomorphometric techniques, these studies have
shown that N-BPs can inhibit the formation or progression of
bone metastases and/or reduce skeletal tumor burden.
Bisphosphonates have been administered either at the time of
tumor cell inoculation (i.e., prevention setting) or after bone
metastases are established (i.e., treatment setting).
Several models of multiple myeloma have been estab-
lished and have consistently shown reductions in tumor-
induced osteolysis and skeletal tumor burden by N-BPs [52,
53, 59]. For example, in the 5T2 model of murine myeloma,
treatment with zoledronic acid (120 g/kg s.c. twice weekly)
after osteolytic lesions were established significantly
reduced development of new osteolytic lesions, reduced the
bone surface occupied by osteoclasts and inhibited tumor-
induced osteolysis, and reduced skeletal tumor burden as
evidenced by serum paraprotein levels [53]. Moreover,
treatment with zoledronic acid at the first sign of circulating
15
Apoptosis (%)
10
13.6%
5
1.5% 1.7%
0
Control DOX alone ZOL alone DOX + DOX then ZOL then
50 nM, 24 hours 25 M, 1 hour ZOL ZOL DOX
Figure 3. Synergistic apoptotic effect of zoledronic acid (ZOL) in
combination with doxorubicin (DOX) on MCF-7 breast cancer cells
in vitro. Adapted with permission from Neville-Webbe et al. [43].
Green
Table 2. Animal models demonstrating antitumor effects of zoledronic acid
Study Tumor cells
Yaccoby et al. [52] Primary human myeloma cells
Croucher et al. [53] 5T2 murine myeloma
Green et al. [54] MDA-MB-231 human breast cancer
Peyruchaud et al. [55] MDA-MB-231 human breast cancer
Hiraga et al. [56] 4T1 murine mammary carcinoma
Hiraga et al. [9] MDA-MB-231 human breast cancer
Nobuyuki et al. [57] 4T1 murine mammary carcinoma
Corey et al. [40] PC-3 and LuCaP 23.1 prostate cancer
Adapted with permission from Green [58].
paraprotein significantly prolonged disease-free survival in
those mice.
Numerous studies in breast cancer models have also
been reported. Zoledronic acid was shown to inhibit pro-
gression of established bone metastases and development of
new bone metastases in two models of breast cancer [54,
55]. In nude mice injected with human MDA-MB-231
breast cancer cells and allowed to develop osteolytic lesions,
mice treated with zoledronic acid (0.2, 1.0, or 5.0 g/day
s.c.) had significantly less radiographic bone lesion area, by
>80%, than controls [54]. Ibandronate (1.0 g/day) and
alendronate (10 g/day) resulted in nonsignificant reduc-
tions in bone lesion area (65% and 55% reductions, respec-
tively). These findings were confirmed in another
independent study using highly sensitive in vivo imaging of
MDA-MB-231 cells genetically engineered to express green
fluorescent protein [55]. Similar studies have been con-
ducted with ibandronate in nude mice bearing MDA-MB-
231 breast cancer cells, which typically form both adrenal
and bone metastases. Treatment with ibandronate (4 g/day
s.c.) inhibited the radiographic progression of established
osteolytic lesions and decreased skeletal tumor burden com-
pared with controls [9]. However, administration of iban-
dronate at the time of tumor cell inoculation resulted in a
twofold increase in adrenal tumor load [61]. In a murine
breast cancer model, treatment with zoledronic acid
(5 g/day) for 7 days after injection of 4T1 murine mam-
mary tumor cells (i.e., prevention setting) markedly
decreased the formation of new bone metastases at day 28
[54]. The observed decrease in radiographic bone lesion
area was accompanied by an increase in the number of apop-
totic osteoclasts and apoptotic tumor cells in bone lesions.
7
Bisphosphonate Dose and schedule
Zoledronic acid 100 g/kg/week s.c.
Pamidronate 1.3 mg/kg every 2 weeks s.c.
Zoledronic acid 120 g/kg twice weekly s.c.
Zoledronic acid 0.2-5.0 g/day s.c.
Ibandronate 1.0 g/day s.c.
Alendronate 10 g/day s.c.
Zoledronic acid 3 g/day 12 days s.c.
Zoledronic acid + 250 g/kg
UFT 20 mg/kg/day
Ibandronate 4 g/day s.c.
Zoledronic acid 0.5 or 5 g every 4 days i.v.
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Zoledronic acid 5 g/mouse twice weekly s.c.
Notably, the 4T1 mammary tumor model has provided
the first in vivo evidence of synergy between zoledronic acid
and chemotherapy. In animals with established orthotopic
tumors, the combination of zoledronic acid (250 g/kg) with
20 mg/kg/day of oral UFT (a combination of uracil and tega-
fur [4:1 molar ratio]) reduced skeletal tumor burden more
effectively than either agent alone [56]. Similarly, the com-
bination of ibandronate with doxorubicin (150 g/day) has
been investigated in the MDA-MB-231 model and was
shown to have additive antitumor effects in bone [61].
Studies in a prostate cancer model have also recently been
reported. In those studies PC-3 and LuCaP 23.1 cells were
injected directly into the tibia of mice [40]; PC-3 cells form
osteolytic lesions, and LuCaP 23.1 cells form osteoblastic
lesions. The treatment group received zoledronic acid (5 g
s.c. twice weekly) either at the time of tumor cell injection or
after tibial tumors were established (7 days for PC-3 tumors
and 33 days for LuCaP 23.1 tumors). Treatment with zole-
dronic acid significantly inhibited growth of both osteolytic
and osteoblastic metastases by radiographic analysis (Fig. 4)
[40] and also reduced skeletal tumor burden as evidenced by
a significant decrease in serum levels of prostate-specific
antigen in animals bearing LuCaP 23.1 tumors [40]. The
observed reduction in serum prostate-specific antigen levels
provides compelling direct evidence of the antitumor activity
of zoledronic acid in this animal model.
The potential of zoledronic acid to prevent bone metas-
tasis was also demonstrated in an animal model of prostate
cancer [62]. In that model, mice were injected with PC-3
cells, and the incidence of bone metastases was studied in
normal mice and in mice that were rendered androgen defi-
cient by surgical castration. Androgen-deficient mice devel-
8 Bisphosphonates: Preclinical Review
Figure 4. The effects of
zoledronic acid (5 g s.c.
twice weekly) on tumor
volume (assessed radi-
ographically) in mice with
tibial tumors from PC-3
Tumor volume (% tissue volume)
and LuCaP 23.1 prostate
cancer cell lines. Zole-
dronic acid was adminis-
tered either at the time of
tumor cell injection (i.e.,
prevention) or after tibial
tumors were established
(i.e., treatment), which
was 7 days postinjection
for PC-3 and 33 days
postinjection for LuCaP.
*p < 0.03; **p < 0.001.
Adapted with permission
from Corey et al. [40].
oped significantly more bone metastases than did intact con-
trol mice. This strongly suggests that excess bone resorption
caused by androgen ablation can stimulate metastasis of PC-
3 cells to the bone. This is consistent with current hypotheses
of tumor cell metastasis to bone. Moreover, daily treatment
with zoledronic acid significantly reduced the incidence of
bone metastases in both normal and castrated mice.
Although the majority of animal models suggest that the
primary antitumor effect of bisphosphonates is manifested in
the bone, where they reach the highest concentrations, pre-
liminary data from the 4T1 mammary tumor model have
demonstrated that zoledronic acid can also inhibit visceral
metastases [57]. 4T1 cells expressing luciferase were used to
quantitate tumor burden in mice. After tumor cell inocula-
tion, mice were treated with zoledronic acid (5.0 g every
4 days), which resulted in lower tumor burdens than in con-
trols not only in bone but also in the liver and lungs [57, 63].
Treatment with zoledronic acid also prolonged survival of
tumor-bearing mice. In the same model, ibandronate
(4 g/day s.c.) had no effect on lung or liver metastases or on
survival [61, 63]. As mentioned above, treatment of mice
bearing 5T2 myeloma cells with zoledronic acid (120 g/kg
s.c. twice weekly) also resulted in a significantly longer dis-
ease-free survival time (median, 47 days versus 35 days for
untreated controls; p < 0.01) [53].
These animal models provide convincing evidence of the
potential of bisphosphonates, particularly the more potent
N-BPs, to reduce tumor burden in bone and inhibit formation
and progression of bone metastases in a variety of tumor mod-
els. It is less clear whether bisphosphonates have antitumor
activity beyond the bone, but it appears that zoledronic acid
may be sufficiently potent to inhibit extraskeletal tumor cell
growth and to prolong survival in some animal models.
PC-3 (lytic) LuCaP 23.1 (blastic)
80
60
40
*
*
20 **
**
0
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Vehicle Prevent Treat Vehicle Prevent Treat
Zoledronic acid: 5 g/mouse (twice a week)
MECHANISMS OF ANTITUMOR EFFECTS
The precise mechanisms responsible for the antitumor
effects of bisphosphonates are beginning to be elucidated.
Bisphosphonates appear to make the bone a less favorable
site for tumor cell growth via the inhibition of osteoclast-
mediated bone resorption and osteoclastogenesis, thereby
reducing the release of growth factors that stimulate tumor
growth in bone. In addition, bisphosphonates directly
inhibit tumor cell growth and survival and the ability of
tumor cells to colonize the bone. Either or both of these
mechanisms may be at work.
Apoptosis
One of the primary mechanisms responsible for the
direct antitumor activity of bisphosphonates is induction
of tumor cell apoptosis. Both N-BPs and non-N-BPs
appear to induce apoptosis of osteoclasts and tumor cells
by activation of caspases [9, 12-14, 16, 17, 64, 65]. One
mechanism by which bisphosphonates induce apoptosis
is through production of ATP analogues (either as direct
metabolites or as a result of inhibition of the mevalonate
pathway), which can disrupt mitochondrial ATP/ADP
translocase. A recent study investigating the mechanism
by which zoledronic acid induced apoptosis in human
breast cancer cell lines (MDA-MB-231 and MCF-7)
indicated that this response was associated with
cytochrome c release from the mitochondria and subse-
quent caspase-3 activation [66]. It appears that N-BPs
may induce cytochrome c release by modulating expres-
sion of Bcl-2, a key antiapoptotic regulatory protein
[66]. These events may be precipitated by inhibition of
Ras activation, which requires protein prenylation
(specifically farnesylation) [66].
Green
Inhibition of Tumor Cell Adhesion and Invasion of the
Extracellular Bone Matrix
Bisphosphonates have also been shown to inhibit adhe-
sion of tumor cells to extracellular matrix (ECM) proteins and
to inhibit the process of tumor cell invasion and metastasis
[67-70]. Using an in vitro Matrigel-based invasion assay,
Boissier et al. have shown that bisphosphonates inhibit the
ability of human breast and prostate cancer cells to invade the
ECM [67]. In this assay, zoledronic acid and ibandronate
caused dose-dependent inhibition of cell invasion through the
Matrigel at extremely low concentrations (1010 M) that did
not inhibit tumor cell motility or induce significant apoptosis.
Clodronate was approximately six orders of magnitude less
potent in the same assay. Furthermore, the combination of
ibandronate with taxanes enhanced the inhibitory effect on
MDA-MB-231 cell invasion [69].
One contributory mechanism may be the inhibition of
matrix metalloproteinase (MMP) activity, which is necessary
for tumor cell invasion of the ECM [67, 71, 72]. Bisphos-
phonates have been shown to inhibit the activity of MMPs
produced by tumor cell lines, and this seems to correlate with
reduced invasiveness in the Matrigel assay [71, 72]. For
example, zoledronic acid was shown to inhibit the production
of MMP-2 and MMP-9 by PC-3 cells [40]. These data sug-
gest a potential mechanism by which N-BPs may inhibit tumor
cell invasion of the bone but cannot explain the apparent
dependence of this effect on protein prenylation.
Recent studies suggest that inhibition of tumor cell
adhesion to ECM proteins and invasion through Matrigel
is dependent on inhibition of protein prenylation. In one
study, zoledronic acid was shown to dose-dependently
inhibit adhesion of MCF-7 and MDA-MB-231 cells to a
variety of matrix proteins (Fig. 5) [73], and this inhibitory
effect was overcome by addition of either farnesol or ger-
anylgeraniol or by the addition of a broad spectrum caspase
inhibitor [73]. Similar findings have been reported for alen-
dronate. The inhibitory effect of alendronate on tumor cell
invasion through Matrigel was reversed by the addition
of geranylgeraniol and
trans-trans-farnesol
100
Figure 5. Percent adherent
cells versus controls when
Adherent cells (% control)
MDA-MB-231 human
breast cancer cells were
incubated with zoledronic 50
acid for 24 hours (0.1 or
100 M) before culture
on plates coated with vari-
ous extracellular matrix
proteins. Adapted with 0
permission from Pickering
et al. [73].
9
[74]. Therefore, inhibition of the mevalonate pathway and
induction of caspase activity are important for the
inhibitory effects of N-BPs on tumor cell adhesion to the
ECM and on invasiveness. Further, it has been shown that
an activating Ras mutation enhances adhesion of a normal
breast epithelial cell line to ECM proteins, suggesting that
increased Ras activation in response to growth factor recep-
tor signaling may increase the metastatic potential of breast
cancer cells [73]. Thus, by inhibiting protein prenylation
and Ras signaling, zoledronic acid should reduce the
metastatic potential of tumor cells.
Antiangiogenic Effects
In vitro and in vivo studies have further demonstrated that
zoledronic acid has antiangiogenic effects. In vitro assays
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with human umbilical vein endothelial cells (HUVECs) have
shown that zoledronic acid dose-dependently inhibited the
proliferation of HUVECs induced by fetal calf serum and
basic fibroblast growth factor (bFGF), and these findings have
been confirmed in vivo [75]. Systemic administration of zole-
dronic acid to mice resulted in potent inhibition of angiogen-
esis induced by s.c. implants impregnated with bFGF, with
a dose of 3 g/kg producing a 50% efficacy (ED50) [75]. It
has also been reported that zoledronic acid can reduce bone-
tumor-associated angiogenesis in the murine 5T2 myeloma
model [53]. In another series of experiments, zoledronic
acid, as well as ibandronate, risedronate, and clodronate,
inhibited formation of capillary-like tubules by HUVECs in
vitro [76]. In vivo, zoledronic acid and ibandronate, but not
clodronate, decreased revascularization (as measured by
vessel area) of the ventral prostate gland in castrated rats
treated with testosterone [76].
The inhibitory effect of zoledronic acid on endothelial
cell adhesion and migration appears to be mediated, at least
in part, by modulation of integrins (e.g., v3 and v5) that
are involved in angiogenesis [77, 78]. Interestingly, v3
integrin is also required for osteoclasts to adhere tightly to
the bone and form resorption lacunae during active bone
Control
0.1 M Zoledronic acid
100 M Zoledronic acid
24-hour treatment
Laminin Fibronectin Vitronectin Collagen I
10
resorption, and v3 expression confers on tumor cells a
greater propensity to metastasize to bone [79]. In fact, a
small molecule inhibitor of v3 was recently shown to
effectively prevent metastasis of MDA-MB-435 breast can-
cer cells to bone [80]. Therefore, effects on v3 could have
pleiotropic effects on bone resorption and tumor metastasis.
In addition, it has recently been reported that zoledronic
acid decreases the survival of HUVECs by sensitizing them
to TNF-induced programmed cell death [78]. Zoledronic
acid also appears to modulate serum levels of proangio-
genic growth factors such as vascular endothelial growth
factor and bFGF in cancer patients [81]. These studies sug-
gest a variety of potential mechanisms to account for the
observed antiangiogenic effects of bisphosphonates.
CONCLUSIONS AND FUTURE DIRECTIONS
There is now extensive in vitro and in vivo preclinical
evidence that bisphosphonates, particularly the more
potent N-BPs, have antitumor activity and can reduce
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ACKNOWLEDGMENT
Jonathan Green is a full time employee of Novartis
Pharma AG and holds stock in the company.
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