Enhanced tumor regression and tissue repair when zoledronic acid is
combined with ifosfamide in rat osteosarcoma
D. Heymanna, B. Orya, F. Blancharda, M-F. Heymannb, P. Coipeaua, C. Charriera,
S. Couillauda, J.P. Thieryc, F. Gouina,d,T, F. Redinia
aUniversite´ EA 3822; INSERM ERI 7, Physiopathologie de la Re´sorption Osseuse et The´rapie des Tumeurs Osseuses Primitives, Faculte´ de Me´decine,
1 rue Gaston Veil, 44035 Nantes cedex 1, France
bDe´partement de Pathologie, Hoˆ pital Nord Laennec, 44 800 St Herblain, France
cCentre He´pato-Biliaire, Hoˆ pital Paul Brousse, 94800 Villejuif, France
dService d’Orthope´die, CHU Hoˆ tel Dieu, 1 place Alexis Ricordeau, 44000 Nantes, France
Received 4 November 2004; revised 11 February 2005; accepted 25 February 2005
Available online 13 May 2005
Abstract
The efficacy of zoledronic acid (ZOL), with or without the anticancer drug ifosfamide (IFO), was tested on primary bone tumor growth
using a rat-transplantable model of osteosarcoma. The effects on bone remodeling and tumor growth were analyzed by radiography, microcomputed
tomography (micro-CT), and histological staining. The in vitro effects of ZOL were studied by proliferation, apoptosis, and cell
cycle analyses on the osteosarcoma cells OSRGA compared to rat primary osteoblasts. Treatment with ZOL was effective in preventing the
formation of osteolytic lesions that developed in bone sites and in reducing the local tumor growth, as compared to the untreated rats. The
combination of ZOL and IFO was more effective than each agent alone in preventing tumor recurrence, improving tissue repair, and
increasing bone formation as revealed by the analysis of trabecular architecture. In vitro studies demonstrated that ZOL was more potent
against the OSRGA cell line than osteoblasts (with a half-maximal inhibitory effect on proliferation seen at 0.2 and 20 AM, respectively), the
ZOL-induced inhibition of OSRGA proliferation being due to cell cycle arrest in S-phase. No effect on OSRGA apoptosis could be observed
in vitro, as assessed by Hoechst staining and caspase-1 and -3 activation. In situ cell death was determined by TUNEL staining on tumor
tissue sections. No significant difference in TUNEL-positive cells could be observed between ZOL-treated and -untreated rats. This is the
first report of the anti-bone resorption and antitumoral activities of zoledronic acid in a rat model of osteosarcoma, and its beneficial
association with an antitumoral chemotherapeutic drug in preventing tumor recurrence.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Bone tumor; Bisphosphonate; Chemotherapy; Osteolysis
Introduction
Osteosarcoma is the most frequent primary bone tumor,
it develops mainly in the young, the median age of
diagnosis being 18 years. The current strategy for treatment
of high-grade osteosarcoma is based on neo-adjuvant
chemotherapy, delayed en-bloc wide resection, and adjuvant
chemotherapy adapted to the histologic profile assessed on
tumor tissue removed during surgery [1]. Despite recent
improvements in surgery and the development of different
regimens of multidrug chemotherapy over the past 25 years,
survival remains around 55–70% after 5 years [2,3]. The
prognosis is worse with non-extremities localization,
advancing age, radio-induced osteosarcoma, and those
arising from Paget’s disease of bone, representing 40% of
the entire osteosarcoma population. In addition, patients
with metastatic osteosarcoma at the time of diagnosis have
poor survival statistics (30% at 5 years). This poor
8756-3282/$ – see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2005.02.020
* Corresponding author. Service d’Orthope´die, CHU Hoˆ tel-Dieu, 1 place
Alexis Ricordeau, 44000 Nantes, France. Fax: +33 2 40 41 28 60.
E-mail address: francois.gouin@chu-nantes.fr (F. Gouin).
Bone 37 (2005) 74 – 86
www.elsevier.com/locate/bone
prognosis of osteosarcoma warrants new therapeutic strategies
to improve the overall rate of survival, especially in
high-risk sub-groups.
As evidenced for bone metastases, a vicious cycle
between osteoclasts, bone stromal cells/osteoblasts, and
cancer cells has been hypothesized during the progression
of primary bone tumors [4]. Accordingly, suppression of
osteoclasts would be a primary approach to inhibit local
cancer growth. Several investigators have enhanced the
potential effect of osteoclast-regulating drugs on tumor
growth. Among these drugs, bisphosphonates (BPs) are an
important class of molecules for the treatment of bone
diseases with different molecular mechanisms of action.
Nitrogen-containing BPs, such as alendronate or zoledronic
acid, inhibit bone resorption by preventing the prenylation
of GTP binding proteins such as ras and raf in osteoclasts
by inhibition of farnesyl diphosphate synthase in the HMG
CoA mevalonate pathway [5], whereas non-nitrogen-containing
BPs such as clodronate are metabolized to nonhydrolyzable
analogs of ATP [6]. The final common result
is the induction of osteoclast apoptosis. BPs act by
inhibiting the recruitment, proliferation, and differentiation
of preosteoclasts, or by impeding the resorptive activity of
mature osteoclasts [7–10]. They also shorten the life span
of osteoclasts by inducing their apoptosis [11]. Previous
studies revealed that BPs have the ability to reduce the
osteolytic bone resorption associated with multiple myeloma
and breast cancer [12,13]. Zoledronic acid has also
shown efficacy in cancer metastases to bone due to prostate
cancer and other solid tumors, demonstrating that this BP
can reduce skeletal morbidity in both osteolytic and
osteoblastic diseases [14,15]. Moreover, recent preclinical
data show that BPs can act on tumor cells by inhibiting
tumor cell adhesion to mineralized bone as well as tumor
cell invasion and proliferation [16,17]. BPs also induce
tumor cell apoptosis and stimulate gy T cell cytotoxicity
against tumor cells [18]. For example, it has been reported
that the BP zoledronic acid has a clear direct antitumor
activity on breast cancer cells in vitro [19]. In the same
study, the commonly used antineoplastic agent, paclitaxel,
potentiated the antitumor effects of zoledronic acid, or vice
versa, ZOL enhances the cytostatic effect of paclitaxel.
Moreover, the combination of BP with anticancer agents
such as uracil and tegafur caused an enhanced reduction of
bone metastases compared to UFT (tegafur/uracil) alone
[20]. Two in vitro studies on the effects of BPs alone on
osteosarcoma have reported inhibition of human osteosarcoma
cell growth by pamidronate and clodronate [21,22]. A
recent work from Evdokiou et al. reported that ZOL
reduced the cell number of different human osteogenic
sarcoma cell lines, but no synergistic effect was evidenced
when ZOL was combined with chemotherapeutic agents
[23]. Thus, the synergistic effect of combining a bone
regulating factor with conventional chemotherapy should be
further investigated on the development of primary tumors
at skeletal sites. To perform this study, a rat-transplantable
osteosarcoma model was used that mimics human osteosarcoma
development at the temporal and physiological
levels, presenting aspects of both tumor proliferation and
bone remodeling [24]. Hence, the purpose of this study was
to evaluate the efficacy of the combination of zoledronic
acid (ZOL: anti-bone resorption bisphosphonate) with
ifosfamide (IFO: conventional chemotherapy) on local
tumor growth, histological response, and animal survival
using a rat-transplantable model of osteosarcoma.
Materials and methods
The osteosarcoma model
The osteosarcoma was initially induced by a local
injection of colloidal radioactive 144Cerium in rats [25].
The evolution of the tumor is comparable at the temporal
(ratio 1:100 between rats and humans) and physiological
levels to the development of human osteosarcoma. The
tumor can be re-grafted as described below and maintained
in vivo for many months, or fragments can be frozen until
re-utilization. Lung metastases are observed in 75–90% of
rats bearing advanced malignant bone tumors.
Treatment with zoledronic acid and/or ifosfamide: effect on
local tumor growth and bone remodeling
Four-week-old male Sprague–Dawley rats (IFFACREDO,
L’Arbresle, France) were housed under pathogen-
free conditions at the Experimental Therapy Unit
(Medicine Faculty of Nantes, France), in accordance with
the institutional guidelines of the French Ethical Committee
and under the supervision of authorized investigators. For
the implantation, the rats were anaesthetized by inhalation
of a combination isoflurane/air (1.5%, 1 L/min) associated
with an intra-muscular injection of Imalgene (100 mg/kg,
Merial Lab., Lyon, France). Using a right tibial approach,
the periostum of the diaphysis was opened and resected
along a length of 5 mm, the underlying bone was intact. A
10-mm3 fragment of osteosarcoma was placed contiguous to
the exposed bone surface without periostum, and the
cutaneous and muscular wounds were sutured. Tumors
appeared at the graft site approximately 7–10 days later.
Rats bearing growing tumors with a volume >1200 mm3,
which were considered as progressive tumors, were
individually identified and assigned to the control or
treatment group (6–8 animals/group) and treated with
ZOL and/or IFO. ZOL, kindly provided as the disodium
hydrate by Pharma Novartis AG (Basel, Switzerland), was
administered at 100 Ag/kg s.c. twice a week, starting at day
11 after tumor implantation. IFO was supplied by Baxter
Lab. (France) and administered i.p. (15 mg/kg) to tumorbearing
rats at 24-h intervals on days 20, 21, and 22 after
tumor implantation (Fig. 1). Rats in the control group
received the same volume of the drug-formulating vehicle
D. Heymann et al. / Bone 37 (2005) 74–86 75
with the same schedule as the treated animals. The animals
were weighed twice a week. At that time, the tumor volume
was calculated from the measurement of two perpendicular
diameters using a caliper. Each tumor volume (V) was
calculated according to the following formula: 0.5 L
(S)2, where L and S are, respectively, the largest and
smallest perpendicular tumor diameters. Relative tumor
volumes (RTVs) were calculated from the formula: RTV =
(V42/V25), where V42 is the tumor volume on day 42 and V25
the tumor volume at day 25. Radiographic analyses were
performed once a week. The animals were sacrificed 6
weeks after tumor implantation, except for spontaneous
death, or when the tumor became too bulky and when the
life of the animal was threatened. At the time of autopsy,
right legs were kept for radiographic, histological studies
and micro-architectural parameter quantification. Lung
tumor dissemination was assessed by analyzing the number
and the size of tumor foci. In one experiment, animals were
treated with ZOL for more than 4 weeks as mentioned above
(from day 11 to day 42), then the rats were left for a further
6 weeks after the end of ZOL treatment to evaluate tumor
recurrence.
Treatment of the rats with ZOL: effect on survival
To determine the effect of ZOL on survival in the
osteosarcoma model, an experiment similar to that described
above was performed. Sixteen rats were implanted with
osteosarcoma tumor fragments: 8 were treated with vehicle
and 8 with ZOL from day 11 after tumor implantation.
Treatment continued until each animal showed signs of
morbidity, which included cachexia, respiratory distress, or
animals bearing too bulky tumors (one diameter >40 mm),
at which point they were sacrificed.
Histological analysis
Right tibias and femora were fixed in 10% buffered
formaldehyde, decalcified by electrolysis, and after embedding
in paraffin, 5-Am-thick sections were mounted on glass
slides. Sections were stained with hematoxylin-eosin-safran
(HES) or with Masson trichrom. Analysis of necrotic and
fibrotic areas was performed on each tumor section using a
Leica Q500 image analysis system.
Micro-architectural quantification
Analysis of architectural parameters was performed using
the high-resolution X-ray micro-CT system for small animal
imaging SkyScan-1072 (SkyScan, Aartselaar, Belgium).
Relative volume (BV/TV) and specific surface (BS/BV) of
the tibiae [total bone (cortical + trabecular) or trabecular
bone] were quantified for each group and compared to the
tibia of control rats.
Cell proliferation
Rat osteosarcoma cells (OSRGA) and primary rat
osteoblasts were cultured in DMEM supplemented with,
respectively, 5% and 10% FBS, 1% glutamine, and
maintained at 37-C in a humidified atmosphere with 5%
CO2. The rat OSRGA cell line was derived from the
osteosarcoma used in the present study. Replicate subconfluent
cell cultures in 96-well plates were treated for 1–3
days with increasing concentrations of ZOL (107 to 104
M, diluted in PBS). Cell viability was determined by a cell
proliferation reagent assay kit using sodium 3V[1-(phenylaminocarbonyl)-
3,4-tetrazolium]-bis(4-methoxy-6-nitro)-
benzene sulfonic acid hydrate (XTT) (Roche Molecular
Biomedicals, Mannheim, Germany). Cell viability was also
assessed by trypan blue exclusion.
Induction of apoptosis
Programmed cell death was monitored microscopically
following Hoechst and trypan blue staining. OSRGA or
primary osteoblasts were seeded at 104 cells/well in a 24-
well plate and cultured for 24 h as described above before
being incubated with ZOL at indicated concentrations
during 24, 48, and 72 h. Trypsinized cells were then
resuspended in the presence of Hoechst no. 33258
staining (10 Ag/ml; Sigma) for 30 min at 37-C. Cells
Fig. 1. Summary of the experimental protocol. Osteosarcoma tumors were implanted contiguous to the tibia of male Sprague–Dawley rats on day 0. Zoledronic
acid (ZOL) was administered at 100 Ag/kg s.c. twice a week, starting on day 11 after tumor implantation. IFO was administered i.p. (15 mg/kg) to tumorbearing
rats at 24-h intervals on days 20, 21, and 22 after implantation. All of the mice were sacrificed at 6 weeks.
76 D. Heymann et al. / Bone 37 (2005) 74– 86
were then observed by UV microscopy (Leica, Wetzlar,
Germany).
Induction of apoptosis was also investigated by cleavage
of caspase-1 and -3 substrates in supernatants of cultures
with or without ZOL treatment. OSRGA cells or osteoblasts
were seeded at 15 103 cells/well (in a 24-well plate), then
incubated with ZOL (1 and 10 AM) for 24, 48, and 72 h.
Cells incubated with 1 AM staurosporine for 6 h were used
as positive controls. At the end of the incubation period, the
cells were lysed with 50 Al of RIPA buffer for 30 min. The
cells were then scraped off and protein content was
quantified in parallel samples using the BCA (bicinchominique
acid + Copper II sulfate) assay. Caspase 1 and 3
activity was assessed on 10 Al of the cell lysate with the kit
CaspACEi Assay System (Fluorometric, Promega, Madison,
USA) following the manufacturer’s instructions.
TUNEL assay for apoptosis
Apoptotic cells in the osteosarcoma were detected with
an in situ cell death detection kit (Roche Diagnostics,
Mannheim, Germany), based on the terminal-deoxynucleotidyl
transferase-mediated dUTP nick-end labeling method
(TUNEL). After formaldehyde-fixed and paraffin-embedded
tissue sections had been deparaffinized, specimens were
digested with proteinase K at 37-C for 15 min and washed
with PBS. Endogenous peroxidase activity was quenched
with 3% hydrogen peroxide in PBS for 5 min, then the
sections were immersed in the terminal deoxynucleotidyl
transferase reaction mixture containing enzyme and biotinlabeled
dUTP at 37-C for 1 h in the dark. Avidin peroxidase
was applied to the sections to detect labeled nucleotides.
Binding was localized with aminoethylcarbazole (AEC) and
the sections were lightly counterstained with hematoxylin.
Four to six sections per animal were prepared for staining of
apoptotic cells. The number of TUNEL-positive cells was
counted by microscopic examination with a 40 objective
lens, and indices were determined as the mean percentages
of positive cells among total cells.
Cell cycle analysis
Confluent cultures of OSRGA and osteoblasts were
incubated for the indicated times (24, 48, or 72 h) with or
without 10 Ag/ml ZOL, trypsinized, washed twice, and
lysed in PBS containing 0.12% Triton X-100, 0.12 mM
EDTA, and 100 Ag/ml ribonuclease A. Then 50 Ag/ml
propidium iodide was added for each sample for 20 min at
4-C in the dark. The intensity of propidium iodide labeling
was measured by flow cytometry (FACScan, BD Biosciences)
using the CellQuest software.
Statistics
The unpaired Mann–Whitney test was used to assess
differences in tumor progression between IFO-, ZOL-, or
IFO + ZOL-treated and vehicle-treated control groups.
Results with P < 0.05 were considered significant.
Statistical evaluation of the in vitro proliferation data was
performed by Student’s t test.
Results
Histological characterization of osteosarcoma
The tumor mass was characterized by large mesenchymal
cells possessing nuclear chromatin condensations and
clear cytoplasm (Figs. 2A and B). Large necrotic foci were
observed inside the tumor mass and were surrounded by
cells with nuclear chromatin condensation and dense
cytoplasm which were considered as dead cells (Fig.
2A). Areas of proliferating tumor cells (presence of numerous
mitoses) were detected at the periphery of the tumor
mass or between necrotic foci (Fig. 2B). Where the tumor
cells were weakly differentiated (osteoblastic type), they
induced new bone span formation (Fig. 2B, insert) and
recruited numerous osteoclasts in contact with the bone
initially formed (Fig. 2C). Thus, a strong cortical reaction
characterized by a great number of activated osteoclasts
and an intense bone remodeling activity was observed in
contact with the tumor tissue. Cancellous bone invaded by
tumor cells was also completely remodeled (data not
shown). Moreover, invasion of articular cartilage by tumor
cells induced cartilage damage and new bone formation
(Fig. 2C, insert). Sacrificed animals possessed lung metastasis
(Fig. 2D). The pulmonary tissue was invaded by
tumor foci characterized by central necrosis, high-grade
proliferating tumor cells, and numerous venous emboli
(Fig. 2D, insert).
Protocol design
ZOL treatment began 11 days after tumor implantation,
when the tumor volume exceeded 1200 mm3 to avoid
spontaneous tumor regression. This treatment regimen was
selected to establish whether inhibition of bone resorption
by bisphosphonate could reduce local osteosarcoma
growth. Preliminary experiments were conducted to
determine the optimal dose of IFO to induce partial tumor
regression: 1 15 mg/kg, 1 30 mg/kg, 3 15 mg/kg,
3 30 mg/kg, and 3 60 mg/kg. Doses of 3 30 and
3 60 mg/kg induced strong inhibition of tumor growth,
with a decrease in the tumor volume of more than 90%.
Below these doses (1 15, 1 30, and 3 15 mg/kg),
tumor volume was reduced by IFO, with complete
regression 12 days after IFO injection (3 15 mg/kg)
in 50–75% cases, but tumor recurrence was observed 22
days after IFO injections (data not shown). Thus, a dose
of 3 15 mg/kg was chosen in studies to investigate
whether ZOL could reverse the tumor recurrence associated
with IFO treatment.
D. Heymann et al. / Bone 37 (2005) 74–86 77
ZOL and IFO strongly increase bone formation in
osteosarcoma-bearing rats
Radiographs taken at the time of sacrifice, i.e., 6 weeks
after tumor implantation, are shown in Figs. 3A–D. In
control rat tibiae, high bone remodeling was observed at the
tumor implantation site, resulting in maximal cortical
destruction and intensive interactions between altered bone
tissue and tumor cells (Fig. 3A). In the ZOL-treated group,
osteolytic lesions were rarely observed (Fig. 3B) and the
metaphyses of long bones exhibited high bone density
reflecting inhibition of bone resorption and retention of the
primary spongiosa (arrows, Fig. 3B). Radiographs of the
tibiae from IFO-responsive and IFO + ZOL-treated animals
revealed no osteolytic lesions (Figs. 3C and D).
By combining micro-CT and 3D image registration, we
could follow the bone remodeling associated with osteosarcoma
development. The tibia from tumor-bearing rat was
characterized by large remodeling activities as compared to
control tibia: deep re-structuring of the cortical bone with a
reduction of its thickness and paralleled enhancement of
trabecular bone (Figs. 4A and B). Micro-architectural
parameters [Bone Volume (BV)/Total Volume (TV) and
Bone Surface (BS)/TV] were calculated and compared
between each group of treated animals (Fig. 4 and Table 1).
When animals were treated with IFO, ZOL, and IFO+ ZOL, a
significant increase in bone mass due to increase in bone
formation was observed both at the cortical and trabecular
levels (Figs. 4C–E), which was confirmed by the quantification
of trabecular relative bone volume: respectively, +24,
48% and 76% as compared to tumor control tibia (Table 1).
The same effects were observed when the relative total bone
volume was considered: +12, 19% and 32%, respectively. It
can be deduced from histological analysis that the increased
bone mass upon treatment is woven bone.
IFO increased by itself the relative bone volume, but to a
lesser extent than ZOL alone (+12 and 19%, respectively,
for the total bone, and +24 and 48% for the trabecular bone),
suggesting that IFO alone could block the vicious cycle and
indirectly decreased bone resorption.
ZOL prevents the tumor recurrence observed with IFO
alone
Treatment of tumor-bearing animals with IFO led to a
partial response: an inhibition of tumor progression was
observed in all animals, but 28% showed tumor recurrence
42 days after tumor implantation (Fig. 5A). The association
of IFO with ZOL led to a complete regression of the tumor
in 100% of cases, preventing the possible tumor recurrence
observed with ifosfamide alone (Fig. 5A). The relative
tumor volumes calculated between days 25 and 42 for the
Fig. 2. Histological characterization of the osteosarcoma model after 6 weeks implantation (HES stain). Osteosarcoma fragments were implanted in contact to
the tibia of male Sprague–Dawley rat. (A) Large necrotic foci (T) observed inside the tumor mass; (B) Area of proliferating tumor cells presenting numerous
mitoses (arrows); new bone formation induced by tumor cells (insert). (C) Intense remodeling activity in contact with tumor tissue (oc: osteoclast); invasion of
articular cartilage by tumor cells (insert). (D) Presence of lung metastasis (T) with numerous venous emboli (insert). Original magnification: 5 (C insert and
D), 10 (D insert), 20 (A and B insert), 40 (B and C).
78 D. Heymann et al. / Bone 37 (2005) 74– 86
whole of the series [control group (n = 35), ZOL-treated
group (n = 15)] was significantly decreased in ZOL-treated
animals as compared to the control group: respectively 2.46 +
3.19 vs. 3.44 + 5.2 (29%, P < 0.05, Fig. 5B). IFO treatment
strongly reduced the overall tumor progression with a relative
tumor volume of 0.6 + 0.7 [83% compared to the control,
P < 0.001 (n = 14)], whereas IFO + ZOL totally inhibited
tumor proliferation with a relative tumor volume of 0 +
2.61 [100% compared to the control, P < 0.001; Fig. 5B
(n = 15)].
ZOL markedly diminishes metastasis dissemination and
enhances survival
At the time of autopsy, the number and size of lung
metastases were assayed. No metastases were detected in
animals treated with ZOL and/or IFO that present
significant inhibition of tumor progression (not shown).
Furthermore, tumor recurrence could not be observed up to
6 weeks after the end of ZOL treatment (not shown). To
evaluate the effect of tumor burden on survival, osteosarcoma-
bearing rats were treated with ZOL in separate
experiments and the time of onset of morbidity was
assessed. The results demonstrate a significant delay in
morbidity onset in ZOL-treated rats as compared to the
control group, as shown in Fig. 5C for one representative
series.
The combination of ZOL with IFO improves tissue repair as
compared to each agent alone
Histological analyses demonstrated that the residual
bone mass of animals treated with the combination of
ZOL and IFO was mainly composed of an extensive
fibrosis with small foci of calcified necrosis compared to
the other groups which were characterized by a greater
necrotic area. As revealed by collagen Masson trichrom
staining, untreated tumor tissues presented with around
50% necrosis without a detectable fibrotic component
(Fig. 6A). IFO treatment resulted in both necrosis and
fibrosis at the site of implantation (respectively 20% and
25%, Fig. 6B) and a strong macrophage reaction
phagocyting necrotic tissues (data not shown), whereas
ZOL-treated tissues exhibited extensive necrotic tissue
associated with a weak fibrotic component (50% necrosis
vs. 10% fibrosis, Fig. 6C). The combination IFO + ZOL
showed extensive fibrosis associated with small calcified
necrotic foci and residual macrophage polycaryons (40%
fibrosis with 20% necrosis, Fig. 6D).
ZOL inhibits OSRGA cell proliferation in vitro
Osteosarcoma cells are more sensitive to ZOL than
osteoblasts
To determine whether the antitumor activity of ZOL
observed in vivo could be mediated by a direct
antitumor effect on cell proliferation, the effect of ZOL
was assessed in vitro on the proliferation of osteosarcoma
cells OSRGA compared to that of primary
osteoblasts (Fig. 7A). The XTT viability test showed
that ZOL was more potent against the OSRGA cell line
than primary osteoblasts, with a half-maximal inhibitory
effect on proliferation seen at 0.2 and 20 AM,
respectively (Fig. 7A). To determine whether these
effects were due to inhibition of cell proliferation and/
or induction of cell death, the time-dependent effects of
ZOL were assessed by counting viable cells, as assessed
by trypan blue exclusion, over a 72-h period in OSRGA
cells cultured with or without 10 AM ZOL. The results
demonstrated a 85% induction of cell death in the
presence of 10 AM ZOL after 24 h of culture as
compared to osteoblasts where no cell death was observed
(Fig. 7B).
Fig. 3. Radiograph of Sprague –Dawley rat tibias 6 weeks after
implantation with osteosarcoma. (A) Untreated group. (B) 100 Ag
zoledronate treatment group (ZOL, at 100 Ag/kg s.c. twice a week, starting
at day 11 after tumor implantation). (C) 15 mg ifosfamide treatment (IFO,
i.p. to tumor-bearing rats at 24-h intervals on days 20, 21, and 22 after
implantation), (D) Combined zoledronate + ifosfamide treatment group
(arrow: zoledronate-induced high bone density).
D. Heymann et al. / Bone 37 (2005) 74–86 79
ZOL-induced cell death is not mediated by caspase
activation
To determine whether the ZOL-induced death in OSRGA
cells was caused by apoptosis, Hoechst staining and
caspase-1 and -3 activation were investigated. Hoechst
staining showed no modification of nuclear morphology in
the presence of ZOL as compared to control cells (data not
shown). Concerning the caspase-1 and -3 activity in
OSRGA cells and osteoblasts, the results showed that
ZOL does not induce any activation of caspases in either
OSRGA cells or osteoblasts (not shown). TUNEL staining
of tumor tissue sections showed no significant differences in
TUNEL-positive cells from untreated and ZOL-treated rats
(not shown).
ZOL induces S-phase arrest in OSRGA cells
Flow cytometry of DNA content was performed to
identify cell cycle perturbations following treatment with
ZOL over a 72-h period in OSRGA cells compared to
primary rat osteoblasts. In osteoblasts, in which ZOL did not
cause a loss of cell viability, the cell cycle profiles were not
modified in the presence of 10 AM ZOL for 48 or 72 h of
Fig. 4. Comparison of micro-CT scans of rat tibiae 6 weeks after implantation with osteosarcoma, treated with vehicle, IFO, ZOL, or both drugs. (A) Control
tibia, (B) Control tumor (untreated group), (C) 15 mg ifosfamide treatment group (IFO, i.p. to tumor-bearing rats at 24-h intervals on days 20, 21, and 22 after
implantation), (D) 100 Ag zoledronate treatment group (ZOL, at 100 Ag/kg s.c. twice a week, starting at day 11 after tumor implantation), (E) Combined
zoledronate + ifosfamide treatment group. Hemi-sagittal sections are shown.
Table 1
Quantification by micro-CT scan of the bone remodeling activity in tibiae of rats treated or not with zoledronic acid, ifosfamide, or both
Tumor control (CT) Ifosfamide (IFO) Zoledronic acid (ZOL) Ifosfamide + Zoledronic acid (I + Z)
Total bone
BV/TV 0.57 0.64 (+12%) 0.68 (+19%) 0.75 (+32%)
BS/BV (1/mm) 7.72 4.98 (36%) 4.89 (37%) 4.51 (42%)
Trabecular bone
BV/TV 0.25 0.31 (+24%) 0.37 (+48%) 0.44 (+76%)
BS/BV (1/mm) 23.95 16.87 (30%) 11.60 (52%) 13.15 (45%)
Rats were treated with zoledronic acid (ZOL: 100 Ag/kg s.c. twice a week, starting at day 11 after tumor implantation), ifosfamide (IFO: 15 mg/kg administered
i.p. to tumor-bearing rats at 24-h intervals on days 20, 21, and 22 after implantation), or both. The relative bone volume (BV/TV) and the specific surfaces (BS/
BV) of tibiae from each group were calculated from 3D image registration data and compared to the vehicle-treated tumor control (CT). Two compartments
were studied: trabecular bone and total bone (trabecular + cortical bone).
80 D. Heymann et al. / Bone 37 (2005) 74– 86
incubation (Fig. 8). However, in OSRGA cells, ZOL caused
a 3-fold increase in the number of cells arrested in S-phase:
ZOL treatment resulted in S-phase arrest in the OSRGA
cells, exerting its effects at 48 h, with the number of cells in
S-phase increasing from 10% in control untreated cells to
25% or 32% in the corresponding 1 or 10 AM ZOL-treated
cells. This observation was concomitant with a reduction of
cells in G0/G1 and G2/M phases: 37% vs. 48% and 17% vs.
33%, respectively (Fig. 8). The cells in the apoptotic sub-
G0/G1 peak also increased from 9% to 14%.
It was previously suggested that a possible mechanism
for the BP effects on cell growth was the ability of these
compounds to act as calcium-chelating agents. We investigated
this possibility by the addition of equimolar
concentrations of EDTA with respect to the concentration
of ZOL that reduced cell viability by 50%. We found no
significant effect of EDTA on cell viability in both OSRGA
and primary osteoblasts, suggesting that calcium chelation
was not responsible for the observed effects (data not
shown).
Discussion
The vicious cycle that has been described in osteolytic
metastases consists of release of osteolytic mediators by
tumor cells, bone degradation, release of growth factors
from degraded bone, enhanced tumor cell growth, and
further release of osteolytic mediators [4]. Inhibitors of bone
resorption thus appear one of the more promising tools to
manage skeletal metastases. One can speculate that this
vicious cycle may also apply in the case of the primary bone
tumors, and that inhibitors of bone resorption such as
bisphosphonates may interfere with primary tumor development
at a skeletal site. Zoledronic acid was used in the
present study since its efficacy has been widely reported as a
bone anti-resorptive agent and also as a potent inhibitor of
skeletal complications associated with bone metastases. Its
combination with ifosfamide has been also studied as a
means to reinforce the anticancer potency of this drug,
which could then be used at lower doses. ZOL doses used in
the present study are justified as the clinical dose (4 mg IV
every 3–4 weeks) is equivalent to approximately 100 Ag/kg
of the research grade disodium salt used in this study.
However, even if dosing frequency of twice a week is
greater, it could be justified by the very aggressive nature of
the osteosarcoma and the short survival.
Initially, it was thought that the specific inhibition of
osteoclastic bone resorption is the only mechanism of action
of BPs, by which they are effective in the treatment of
cancer patients bearing bone metastases. However, evidence
is emerging from both preclinical and clinical studies to
suggest that BPs also have direct antitumor properties that
may contribute to their therapeutic efficacy in malignant
bone diseases [26]. Interestingly, the N-BP zoledronic acid
inhibited the development not only of osteolytic but also
osteoblastic bone lesions in a murine model of prostate
cancer [27,28]. Beyond induction of tumor cell apoptosis,
bisphosphonates can inhibit tumor growth via other
mechanisms: inhibition of cell growth, inhibition of tumor
cell adhesion and spreading, and inhibition of tumor cell
invasion [29]. However, the precise mechanism by which
BPs inhibit the growth of bone tumors or bone metastases is
not known: direct or indirect antitumor effect via osteoclast
inhibition and alteration of the bone microenvironment (the
seed-and-soil hypothesis)? Currently, the data are conflicting
and whether or not BPs possess anticancer effects is still
controversial. Using a rat-transplantable model of osteosarcoma,
we demonstrated here the efficacy of zoledronic acid
on primary bone tumor growth. Our results are in agreement
Fig. 5. Effect over time of IFO alone or combined with ZOL on tumor
volume after osteosarcoma implantation. Rats were treated by zoledronic
acid (ZOL, 100 Ag/kg s.c. twice a week, starting at day 11 after tumor
implantation) to ifosfamide (IFO administered i.p. at 15 mg/kg to tumorbearing
rats at 24-h intervals on days 20, 21, and 22 after implantation). (A)
Time course of the mean tumor volume from a representative series (IFO:
ifosfamide; IFO + ZOL: ifosfamide + zoledronate). TTP < 0.05. (B) Relative
tumor volume (RTV) between day 25 and day 42 for the whole series.
TTTP < 0.001. (C) Survival rate of a representative series of 8 animals
treated (ZOL) or not (CT) with 100 Ag/kg ZOL, as described in Fig. 1,
where day 0 represents the time of implantation.
D. Heymann et al. / Bone 37 (2005) 74–86 81
with previous studies that reported the efficacy of zoledronic
acid in the treatment of osteolytic lesions associated with
other types of cancer: multiple myeloma [30] and bone
metastases associated with prostate cancer [27,28] or breast
carcinoma [31]. A single publication reported the use of the
bisphosphonate alendronate for palliative management of
osteosarcoma in two dogs [32]. The authors observed that
both animals remained comfortable and survived for more
that 10 months after diagnosis, despite the fact that neither
primary tumor was resected. However, our present study is
the first to reveal an in vivo inhibitory effect of N-BP on
primary bone tumor and lung metastases development in a
rodent model. The inhibitory effect of BPs on cancer cells in
non-skeletal sites is still unclear in the literature. Diel et al.
have reported that breast cancer patients treated with
clodronate together with anticancer therapies show not only
decreased bone metastases but also reduced visceral
metastases and increased survival compared to patients
treated with conventional anticancer therapies alone [33].
However, a few years later, Saarto et al. published
contrasting data showing that clodronate increased visceral
metastases and had little effect on bone metastases [34].
Powles et al. have reported that clodronate significantly
inhibits bone metastases without affecting visceral metastases
[35]. Using the 4T1 mouse breast cancer model,
ibandronate was shown to reduce bone metastases but failed
to inhibit lung and liver metastases [36]. More recently,
using the same animal model, Yoneda et al. observed the
suppression of bone metastases by zoledronic acid and also
a significant reduction in tumor burden in lung and liver
[37].
The antitumor effects of BPs observed in vivo can be
partly explained by an inhibitory effect exerted by these
compounds on the proliferation and survival of a variety of
tumor cells themselves including those of breast [38–40],
prostate [27], multiple myeloma [41–43], and osteosarcoma
[21–23]. In our experimental model, we have also
demonstrated that ZOL induced a dose- and time-dependent
decrease in cell proliferation together with an induction of
cell death in OSRGA cells, whereas primary osteoblasts
were more resistant. Flow cytometry of OSRGA cells
demonstrated that the underlying mechanism of the ZOL
effect on cell proliferation involves inhibition of cell-cycle
progression mainly due to S-phase arrest. Unexpectedly, no
sign of apoptosis was observed in OSRGA cells as assessed
by Hoechst staining and caspase activation. No clear
evidence of in situ apoptosis could be observed by TUNEL
staining of tumor tissue sections from untreated and ZOLtreated
rats. However, cell cycle analysis did show a small
proportion of hypodiploid OSRGA cells in the apoptotic
sub-G0/G1 peak when the cells were treated with ZOL,
consistent with the onset of apoptosis. Complementary timelapse
experiments showed a huge increase in OSRGA cell
death from 20 h of treatment with 10 AM ZOL, whereas
Fig. 6. Collagen Masson trichrom staining of osteosarcoma implanted in Sprague–Dawley rats treated with IFO or ZOL alone or combined. Rats were treated
by zoledronic acid (ZOL, 100 Ag/kg s.c. twice a week, starting at day 11 after tumor implantation), ifosfamide (IFO administered i.p. at 15 mg/kg to tumorbearing
rats at 24-h intervals on days 20, 21, and 22 after implantation), or both. (A) Untreated tumor tissue presented approximately 50% necrosis. (B)
Ifosfamide treatment resulted in both necrosis (T) and fibrosis (arrowhead). (C) Zoledronate-treated tissues exhibited extended necrotic tissue (arrowhead). (D)
The combination IFO + ZOL showed extensive fibrosis (arrowhead) with small calcified necrotic foci (T) and residual macrophage polycaryons. Original
magnification: 20.
82 D. Heymann et al. / Bone 37 (2005) 74– 86
osteoblasts were not affected (not shown). In these experiments,
sensitive cells showed morphological changes
characterized by the formation of dense rounded apoptotic
bodies. We therefore hypothesize that the induction of cell
death in OSRGA cells by ZOL resembled ‘‘anoikis’’, a
special mode of apoptosis that occurs when adherent cells
detach or lose the particular attachment contacts with the
extracellular matrix that confer survival signals to the cells,
a process independent of caspase activation. It is now
becoming increasingly clear that apoptosis can occur in the
absence of caspase activation, as has been documented in a
number of recent studies [44]. Our results parallel those of
Evdokiou et al. who reported a ZOL-induced reduction of
cell number in a panel of human osteogenic sarcoma cell
lines, due either to cell cycle arrest in S-phase or to the
induction of apoptosis [23]. It seems that OSRGA cells
behave like MG-63 cells in the presence of ZOL, which
inhibits cell cycle progression and increases the proportion
of cells arrested in S-phase. This effect has also been
recently reported in another in vitro cancer model by Forsea
et al. who described an accumulation of melanoma cells in
the S-phase of the cycle after ZOL treatment [45]. Lee et al.
demonstrated a reduction of the of growth prostate cancer
cell lines by ZOL, with a major increase in cells present in
the G0/G1 and S phase [46]. These results show that ZOL
has direct effects on the proliferation and survival of
osteosarcoma cells in vitro, suggesting a new therapeutic
application for this compound in the case of primary bone
tumors, where new therapeutic strategies are warranted.
However, the precise molecular mechanisms implicated in
the ZOL-induced cell death need further investigation.
The purpose of this study was also to check the possible
synergistic effect between ZOL and the anticancer drug
ifosfamide, which is currently used in the treatment of
human osteosarcoma [47,48]. Considering the quantification
of relative bone volume by micro-scanner analysis and
the measurement of tumor volumes, only additive effects
were demonstrated when ifosfamide and ZOL were combined,
but not synergy. This is probably due to the strong
inhibitory effect of ifosfamide alone that masks an eventual
synergistic effect when associated with ZOL. A few studies
have reported inconsistent results on the benefit of a
combination of ZOL and anticancer agents in vitro: two
studies in breast cancer cells [19] and more recently in
leukemic cell lines [49] reported synergistic effects on tumor
cell number and apoptosis when ZOL and paclitaxel were
combined. Similarly, results from Witters et al. showed
enhanced growth inhibition in human breast cancer cell
lines in the presence of both ZOL and docetaxel [50].
However, the treatment of a panel of human osteogenic
sarcoma cell lines with the combination of the chemotherapeutic
agents doxorubicin or etoposide with ZOL did
not significantly augment apoptosis in any of the cell lines
tested [23]. Unfortunately, we cannot test the combination
of ZOL with ifosfamide in vitro in the OSRGA cell line, as
this chemotherapeutic agent needs prior hepatic enzymatic
conversion to exert its cytostatic activity [51].
Concerning the in vivo combination of ZOL + ifosfamide
in our experimental model of rat osteosarcoma, we
demonstrated that this therapeutic combination induces a
huge increase in bone mass as revealed by the quantification
of micro-architecture parameters and an improvement of
tissue repair. Few data have reported the association
between a BP and an anticancer drug in vivo, and to our
knowledge, only three of them described this combination
in osteosarcoma and only with the aim of targeting
anticancer drugs to bone via the BP moiety. Two of them
analyzed the effects of new cisplatin-linked phosphonates
(compounds that contain both an osteotropic and an
anticancer moiety) in rat models of osteolytic bone metastasis
and transplantable osteosarcoma, revealing that
antineoplastic and osteotropic properties can be maintained
after addition of a cisplatin moiety to a triphosphonate or
bismethylenephosphonate [52,53]. The third paper published
by Hosain et al. described a methotrexate-bisphosphonate
conjugate containing a peptide bond that possessed
over five times greater antineoplastic activity against
osteosarcoma in experimental models compared to methotrexate
alone, but the conjugate also appeared to be three to
Fig. 7. In vitro effects of zoledronic acid on OSRGA and osteoblast cell
proliferation and caspase activation. (A) Proliferation of OSRGA (dotted
line) and osteoblastic (full line) cells was determined after exposure to
zoledronic acid (0– 104 M) for 3 days with 10% SVF using the XTT quick
proliferation kit as described in Materials and methods. Changes in
absorbance at 490 nm were measured and results are plotted as percentage
of untreated cell proliferation. (B) The viable cell number was determined
after trypan blue exclusion on OSRGA cells treated (dotted line) or not (full
line) with 10 AM ZOL for the indicated times. TTTP < 0.001. Statistical
evaluation of the data was performed by Student’s t test.
D. Heymann et al. / Bone 37 (2005) 74–86 83
four times more toxic than methotrexate, possibly due to a
myelosuppressive effect from increased delivery of methotrexate
to bone [54]. A recent case report revealed the
successful management of widespread osteosarcoma with a
combination of radio- and chemotherapy in a patient who
received bisphosphonates regularly [55], indicating the
benefit of tri-therapy in osteosarcoma. Beside osteosarcoma,
a unique study has been recently published on the in vivo
effects of the chemotherapeutic agent UFT (a combination
of tegafur and uracil) with or without zoledronic acid, in the
murine 4T1/luc model of breast cancer [20]. In this study,
the combination of UFT with ZOL enhanced the reduction
of bone metastases compared with UFT alone, suggesting
that combination with BP increases the anti-metastatic effect
of UFT. In our osteosarcoma model, the combination
ifosfamide-bisphosphonate led to more qualitative than
quantitative responses as this therapeutic regimen improves
tissue repair, as compared to ifosfamide or ZOL alone, with
a higher proportion of fibrotic tissue than necrosis at the
graft site. Previous data have reported stimulatory effects of
ZOL on osteoblast differentiation by enhancing type I
collagen secretion [56] that could explain part of the fibrosis
induced by the ZOL + IFO.
Taken together, these data demonstrate that treatment
with ZOL after osteosarcoma implantation not only prevents
the development of osteolytic lesions, but also inhibits
primary bone tumor growth by a potential direct antitumor
effect (as revealed by inhibitory effects on OSRGA cell
Fig. 8. Flow cytometry of ZOL-treated OSRGA cells compared to osteoblasts. OSRGA cells and osteoblasts were incubated for 24–72 h in the absence
(control) or presence of 1 or 10 AM ZOL. At each point, cells were harvested, fixed, and stained with propidium iodide. The positions on the histograms of the
hypodiploid sub-G0/G1, G0/G1, S, and G2/M peaks, and the percentage of cells in each of the cycle phases, in a representative experiment are indicated.
84 D. Heymann et al. / Bone 37 (2005) 74– 86
proliferation). The relevance of its combination with the
standard antitumor drug IFO resides in the histological
improvement of tissue repair and opens new areas in the
field of therapeutic combinations for the treatment of
primary bone tumors.
Acknowledgments
Zoledronic acid was kindly provided by Pharma Novartis
AG, Basel, Switzerland. We thank Dr Jonathan Green for
helpful discussions, Paul Pilet from the microscopy platform,
and Christelle Bailly, Agne`s Hivonnait, and Cyril Le
Corre from the Experimental Therapy Unit (IFR26, Nantes,
France). This work was supported by INSERM (Contrat de
Recherche Strate´gique no. 4CR06F), by the Ministe`re de la
Recherche (ACI no. TS/0220044), and the ? Comite´ des
Pays de Loire de la Ligue Contre le Cancer X.
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