The main cause of treatment failure for cancer patients is metastasis, a multistep
and complex process leading to the formation of secondary tumours from the original
cancer. Breast cancers preferentially metastasise into the skeleton, causing specific
clinical complications: hypercalcaemia, bone fractures and pain (Paget, 1889; Mundy,
1997). Bone metastases in breast cancer account significantly for considerable morbidity
and mortality. Indeed, 80% of patients with metastatic breast cancer will develop
bone metastases and survive for approximately 2 years (Coleman and Rubens, 1987).
Bisphosphonates (BPs) have proven useful in the treatment of bone metastases in breast
cancer patients and have improved their quality of life in a number of clinical trials
(Theriault et al, 1999; Lipton et al, 2000). In the first years of BP use, the efficacy
of these substances was thought to lie entirely in the antiproliferative and apoptotic
effects on osteoclasts (Hughes et al, 1995; Selander et al, 1996). However, it is
now suggested that these favourable effects may involve not only the inhibition of
osteoclast-mediated bone resorption but also a direct antitumour effect on cancer
cells (reviewed by Senaratne and Colston, 2002). Additionally, BPs have been reported
to prevent attachment and spreading of breast and prostate cancer cells onto bone
matrices (Boissier et al, 1997). A recent study using an in vitro model of cell invasion
has also suggested that BPs may inhibit the early event in the formation of bone metastases
(Boissier et al, 2000). Furthermore, several investigations have reported the inhibition
of cell growth and cell survival by BPs on breast cancer cells (Fromigue et al, 2000;
Senaratne et al, 2000; Hiraga et al, 2001; Jagdev et al, 2001).
While all these data support a direct action on tumour cells in vitro and is animal
models, the molecular mechanism(s) of BP action remain(s) unclear. Two classes of
BPs have been described. The first generation consists in non-nitrogen-containing
BPs that are metabolised in nonhydrolysable ATP analogues, whereas the more recent
generations, including zoledronic acid (ZOL), are nitrogen-containing BPs (N-BPs).
One possible mechanism by which N-BPs mediate their effects is based on their ability
to inhibit some of the enzymes involved in the mevalonate (MVA) pathway, such as farnesylpyrophosphate
synthase (van Beek et al, 1999; Bergstrom et al, 2000) and/or geranylgeranylpyrophosphate
synthase (Coxon et al, 2000), thus blocking the generation of isoprenoid compounds,
farnesyl pyrophosphate (FPP) and/ or geranylgeranyl pyrophosphate (GGPP), respectively.
These intermediates are required for post-translational prenylation (farnesylation
and geranylgeranylation) of key regulatory proteins, a step that is needed to their
attachment to the plasma membrane, where they are fully active and can exert their
biological function (Sinensky, 2000). Consequently, the identification of the proteins
that became unprenylated following N-BP treatment should be of great interest in understanding
the anticancer action of N-BPs.
Among all the potential candidate's proteins, the small GTPases of Ras and Rho families
could be attractive targets for two reasons. First, their prenylation is a functional
requirement: farnesylation of Ras and geranylgeranylation of Rho proteins occur, respectively,
through the transfer of a 15-carbon farnesyl isoprenoid from FPP by the action of
farnesyltransferase enzyme (FTase) and one or two 20-carbon geranylgeranyl isoprenoids
from GGPP by the action of geranylgeranyltransferase enzyme (GGTase). Second, the
small GTPases of Ras and Rho families are widely involved in human tumorigenesis and
metastasis, either through constitutive activation caused by mutations or by overexpression
(Adjei, 2001; Sahai and Marshall, 2002). Then, rendering insensitive to regulatory
signals, they lead to uncontrolled cell proliferation, inhibition of apoptosis and
enhanced angiogenesis, all main aspects of tumour development. Furthermore, Ras and
Rho proteins are also involved in carcinoma cell motility (reviewed by Oxford and
Theodorescu, 2003). For example, activated Ras was shown to stimulate migration of
breast cancer cells (Keely et al, 1999). Additionally, the regulation of the actin
cytoskeleton by Rho GTPases has been implicated in promoting a variety of cellular
processes such as changes in morphology, motility and adhesion that contribute to
invasion and metastasis of cancer cells in different models, either in vitro or in
vivo (Jaffe and Hall, 2002). In colon carcinoma cells, expression of a dominant-negative
RhoA resulted in the attenuation of cell invasion stimulated by the integrin α6β4
(O'Connor et al, 2000). In the same way, cells transformed by the activated RhoA gene
(del Peso et al, 1997) or cells expressing a constitutively active form of RhoA (Yoshioka
et al, 1998) have greatly promoted invasive ability, contributing to the acquisition
of a metastatic phenotype in vivo. Finally, we recently demonstrated that RhoA activation
contributes to breast cancer cell aggressivity (Denoyelle et al, 2003). Therefore,
it would be interesting to determine whether the anticancer action of N-BPs could
be related to the inhibition of Ras and/or RhoA prenylation, following the decrease
of FPP and/or GGPP synthesis. Recently, a part of the enigma was resolved by a study
demonstrating that ZOL-mediated apoptosis in breast cancer cells may be initiated
by inhibition of Ras prenylation (Senaratne et al, 2002). However, apoptosis of these
cancer cells occurs only at high concentrations (100 μ
M), higher than pharmacological concentrations. Although one study suggested that
the concentration of BP could reach as high a value as 800 μ
M at the osteoclast–bone interface (Sato et al, 1991), others reported that an estimated
concentration of BP is about 1 μ
M in the metastatic tumour nest in bone (Usui et al, 1997). Thus, it is uncertain
whether this proposed mechanism of action is also relevant for the clinical effect
of N-BPs. In contrast, only very low concentrations of ZOL (10−12–10−6 M) are able
to inhibit cancer cell invasion (Boissier et al, 2000). Recently, it was proposed
that this inhibitory effect was mediated by the inhibition of the mevalonate pathway
in a prostate cancer cell line, as it was reversed by geranylgeraniol (GGOH) and farnesol
(FOH) (Virtanen et al, 2002). In the present study, we thus attempted to analyse whether
the impairment of Ras and/or RhoA prenylation could be a potential mechanism by which
ZOL mediated its anti-invasive effect on the aggressive MDA-MB-231 breast cancer cell
line. In addition, matrix metalloproteinases (MMPs) and urokinase-type plasminogen
activator (u-PA)/u-PA receptor (u-PAR) contribute to cancer cell invasion (Blasi,
1999). It was already demonstrated that the inhibition of MDA-MB-231 cell invasion
is not explained by a decrease in MMP secretion as it was observed for concentrations
higher than required for cell invasion inhibition (Boissier et al, 2000). Then, the
effect of ZOL was examined on u-PA/u-PAR expression in MDA-MB-231 cells.
Next, based on the observation that BPs reduce the release of bone-derived growth
factors and cytokines associated with bone resorption, which have the potential to
attract cancer cells to bone, it is possible that BP therapy may prevent the development
of bone metastases. Two clinical trials of patients with breast cancer demonstrated
that BPs, given in adjuvant setting, reduce the incidence of skeletal metastasis with
a consequent improvement in survival (Diel et al, 1998; Powles et al, 2002). However,
other authors reported opposite results (Saarto et al, 2001). Recently, it was demonstrated
that cancer cells use the stromal cell-derived factor 1 (SDF-1)/chemokine receptor
of SDF-1 (CXCR-4) pathway to spread to bone (Taichman et al, 2002). CXCR-4 is greatly
expressed on malignant breast cancer cells in comparison to normal epithelial cells
(Müller et al, 2001). In addition, a high level of SDF-1 was noted in bone as well
as in all target organs for breast-cancer metastasis and, in Nude mice, a neutralising
anti-CXCR-4 antibody induces a significant inhibition of breast-cancer metastasis
in vivo, indicating a major role of the SDF-1/CXCR-4 pathway in the metastatic process.
Therefore, we evaluated the effect of ZOL on CXCR-4 expression and on the chemotactic
effect induced by SDF-1.
Finally, it is well established that a ‘vicious cycle’ between cancer cells and osteoclasts
favours cancer-induced osteolysis (Okada et al, 2000). It was demonstrated that invasive
mammary cell lines, which constitutively express inducible Cox-2, enhance osteoclast
formation through the production of high levels of prostaglandins E2 (PGE2) and subsequently
induce an increase of osteolysis (Ono et al, 2002). We thus examined whether ZOL could
affect the constitutive expression of Cox-2 in MDA-MB-231 cells.
MATERIALS AND METHODS
Cell culture
A human breast carcinoma cell line MDA-MB-231 was used in this study. MDA-MB-231 cells
were grown in RPMI 1640 medium (Eurobio, Les Ulis, France), 10% foetal calf serum
(FCS, Costar, Brumath, France), 2 mM L-glutamine (Gibco BRL, New York, NY, USA), 100 IU ml−1
penicillin (Sarbach, Suresnes, France) and 100 μg ml−1 streptomycin (Diamant, Puteaux,
France). Cells were cultured at 37°C in a humidified 5% CO2 atmosphere. Then, adherent
cells were incubated with indicated concentrations of ZOL (Zometa®, Novartis) during
different periods. The role of MVA pathway enzymes was studied by treating cells with
100 μ
M MVA, 10 μ
M FOH (analogue of FPP), 10 μ
M GGOH (analogue of GGPP), or 10 μ
M squalene (SQUA) (Sigma, Saint Quentin Fallavier, France). The farnesyltransferase
inhibitor FTI-277, the geranylgeranyltransferase inhibitor GGTI-298 and the C3 exoenzyme
from Clostridium botulinum C3 transferase (C3 Exo), a specific inhibitor of RhoA,
were purchased from Calbiochem (San Diego, CA, USA).
Cell proliferation
For the proliferation assay, we used the minimal concentration of FCS (2%) to allow
sufficient viability of MDA-MB-231 cells. Briefly after trypsinisation, the cells
were seeded at a concentration of 5 × 104 cells per well in a 24-well plate (Costar,
Cambridge, MA, USA) and incubated with ZOL. Cell number was measured on day 3 for
MDA-MB-231 with a particle counter (Coulter Z1, Coultronics, France) after detachment
with a nonenzymatic cell dissociation solution (Sigma).
Flow cytometry analysis
Flow cytometry analysis was performed as previously described (Denoyelle et al, 2001).
Tumour cells were detached by a nonenzymatic cell dissociation solution and washed
twice in cold PBS. The phosphatidylserine expression on the surface of breast-cancer
cells was determined using an Annexin V commercial kit (Immunotech, Marseille, France).
Briefly, cells were incubated with 10 μl of a fluorescein–isothiocyanate (FITC)-conjugated
Annexin V. Staining with propidium iodide (0.3 μg ml−1) was performed to confirm the
absence of cell membrane permeability. u-PAR expression on MDA-MB-231 cells was determined
by direct immunofluorescence, while u-PA and CXCR-4 were detected by indirect immunofluorescence.
Approximately 5 × 105 cells were incubated for 15 min at 4°C with 5 μl of specific
antibodies (1 mg ml−1). After two washes, the cell suspension was immediately analysed
in a flow cytometer (EPICS XL-MCL, Coulter, USA), when antibody was directly conjugated
to phycoerythrin (u-PAR, Immunotech), while another 15 min incubation with an FITC-labelled
F(ab′)2 fraction of goat anti-mouse IgG1 (10 μg ml−1, Immunotech) was carried out
for the detection of u-PA (American Diagnostic, Greenwich, CT, USA) and CXCR-4 (R&D
Systems, Abingdon, UK) antibodies. Data are expressed as the percentage of fluorescent
cells or as the specific mean channel fluorescence intensity (MFI). Specific MFI was
calculated for each sample by subtracting the background MFI produced by an irrelevant
antibody from the MFI generated by the specific antibody.
Invasion assay on a Matrigel-coated membrane in a Transwell
An 8-μm-diameter Pore Transwell (Dutscher, Brumath, France) were coated with 500 μl
of Matrigel (Becton Dickinson Europe, Meylan, France) diluted at 100 μg ml−1. Breast
tumour cells were detached by the nonenzymatic cell dissociation solution, washed
twice with PBS, and 2 × 105 cells in RPMI 1640 with 0.2 mg ml−1 bovine serum albumin
(BSA, Sigma) were seeded in the upper chamber of the Matrigel-coated insert. The lower
chamber was filled with 1 ml of RPMI 1640 together with 2 mg ml−1 BSA and basic fibroblast
growth factor (bFGF, 20 ng ml−1) (R&D Systems) to induce invasion. The chemotactic
effect induced by SDF-1 was studied by realising a gradient, which was achieved by
adding SDF-1 (200 ng ml−1) in the lower compartment. After 18 h of incubation, the
nonmigrating cells in the upper chamber were gently scraped, and the adherent cells
present on the lower surface of the insert were stained by May–Grünewald–Giemsa and
counted by light microscopy, 10 fields (magnification × 200) were counted for each
insert. To verify that observed responses are dependent on CXCR-4 receptor binding,
the cells were incubated for 30 min at 4°C with 100 μg of the CXCR-4 antibody before
being seeded in the upper chamber.
Separation of particulate and cytosolic fractions
After different incubation times with ZOL, separation of particulate and cytosolic
fractions was performed as previously described (Denoyelle et al, 2003). Briefly,
MDA-MB-231 cells were washed with cold PBS, lysed in ice-cold buffer that contained
phosphatase and protease inhibitors and centrifuged at 100 000 g for 30 min at 4°C.
The supernatant was collected as the cytosolic fraction. Pellets were homogenised
in the above-mentioned lysis buffer containing 2% Triton X-114 (Sigma) and centrifuged
at 800 g for 10 min at 4°C. The supernatant was collected and was referred to as the
membrane fraction. The protein concentration was determined according to the method
of Bradford using the Bio-Rad protein assay (Hercules, CA, USA).
Confocal microscopy analysis of actin cytoskeleton on MDA-MB-231 cells
MDA-MB-231 was cultured in four-well Glass Lab-Tek chamber slides (Nunc, Roskilde,
Denmark). The confocal microscopy analysis of actin filaments was performed after
an 18 h incubation with 1 μ
M ZOL as previously described (Denoyelle et al, 2001). Actin filaments were visualised
by phalloidin–tetramethylrhodamine–isothiocyanate (TRITC) conjugate (Sigma). Computer-assisted
image analysis of fluorescence was performed using a confocal microscopy scanning
laser microscope (Leica TCS, wavelength excitation 488 nm, emission 580 nm).
Western blotting
Equal amounts of protein extracts (50 μg) were subjected to PAGE (15% for RhoA, Ras
and 10% for Cox-2) under denaturing conditions (SDS–PAGE). Proteins were electrotransferred
onto polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech, Saclay,
France), as described (Denoyelle et al, 2001). Membranes were immunoblotted overnight
with RhoA or pan-Ras polyclonal antibody (1 : 500, from Santa Cruz, SA, USA) or Cox-2
monoclonal antibody (1 : 250, from Transduction laboratories), followed by incubation
with the appropriate horseradish peroxidase-conjugated secondary antibody (1 : 10 000,
Dako, Trappes, France), then developed with enhanced chemoluminescence reagent (ECL)
solution (Amersham Pharmacia Biotech) and exposed to Kodak X-OMAT films. For reprobing,
membranes were stripped with a solution containing 50 mM Tris-HCl (pH 6.8), 2% SDS
and 100 mM 2-mercaptoethanol for 30 min at 50°C. Blots were rehybridised with β-actin
polyclonal antibody (1 : 5000, Sigma) to control protein loading. Each immunoblot
is representative of three distinct experiments.
Total RNA extraction and RT–PCR
After the indicated incubation time of MDA-MB-231 cells with 1 μ
M ZOL, cells were detached and washed twice in PBS. Total RNA extraction was performed
using the SV Total RNA Isolation system (Promega, Madison, WI, USA) according to the
manufacturer's instructions. Primers were chosen using biomolecular sequence databases
(Genbank) and oligonucleotides were synthesised by ProOligos (Paris, France); the
sequences were as follows: cox-2 (forward primer, 5′-cggacaggattctatggaga-3′; reverse
primer, 5′-caatcatcaggcacaggagg-3′), GAPDH (forward primer, 5′-cggagtcaacggatttggtcgtat-3′;
reverse primer, 5′-cgctcctggaagatggtgatgg-3′). RT–PCR was carried out using the ‘Access
RT–PCR system’ (Promega) according to the manufacturer's instructions. After PCR,
15 μl of products and standard DNA ladder were run on a 1.5% agarose gel stained with
ethidium bromide. The predicted sizes for cox-2 and GAPDH PCR products were, respectively,
300 and 222 bp.
ELISA assay
The measurement of PGE2 was performed using an ELISA assay, according to the manufacturer's
instructions (R&D Systems).
Statistical analysis
Statistical significance was determined by the Student's t-test using the InStat Software
(Sigma).
RESULTS
Apoptosis and inhibition of MDA-MB-231 cell proliferation requires high concentrations
of ZOL
The effects of various concentrations of ZOL on the growth rates of MDA-MB-231 cells
were studied by measuring the cell number in the absence or presence of ZOL at different
concentrations after a 3-day incubation. Furthermore, the effects of ZOL on apoptosis
were also assessed by flow cytometry using Annexin V conjugated to fluorescein, an
early indicator of apoptosis. Positive control was done using Taxol (a well-known
mitotic spindle toxin). ZOL had no effects on MDA-MB-231 cell proliferation at low
concentrations (<10 μ
M) (Figure 1
Figure 1
Effect of zoledronic acid (ZOL) on MDA-MB-231 cell proliferation. Cells (5 × 105)
were seeded per well and cells were counted in a particle counter after 3 days of
culture with a minimal concentration of FCS (2%) to ensure viability of the cells
in the presence or absence of indicated concentrations of ZOL. Results are mean±s.e.m.
of four independent experiments (**
P<0.01).
). In contrast, cell numbers were significantly reduced at a concentration of 100 μ
M ZOL. Likewise, ZOL did not induce apoptosis on this cell line for low concentrations,
whereas Annexin V binding was detected at the tumour cell surface when they were treated
with 100 μ
M ZOL (Table 1
Table 1
Zoledronic acid (ZOL) induces apoptosis in MDA-MB-231 cells only at high concentrations
ZOL (μ
M)
Control
0.01
0.1
1
10
100
Taxol
Annexin V
7.9±1.7
12.6±2.3
8.2±2.8
9.9±3.2
13.7±2.6
35.2
*±5.3
64.2
**±4.8
Propidium iodide
4.2±1.8
6.7±2.3
5.9±3.1
5.6±2.9
6.5±1.2
5.1±1.9
6.9±2.5
Confluent MDA-MB-231 cells were incubated for 18 h with ZOL at indicated concentrations.
Taxol was used as positive control. Results are the mean±s.e.m. of three independent
experiments and are expressed as the percentage of positive cells (Bold indicates
significance;
*
P<0.05,
**
P<0.01).
).
Inhibition of MDA-MB-231 cell invasion through Matrigel by low concentrations of ZOL
Under our conditions, as shown in Figure 2
Figure 2
Effect of increasing concentrations of zoledronic acid (ZOL) on MDA-MB-231 cell invasiveness
through Matrigel. MDA-MB-231 cells were treated for 18 h by indicated concentrations
of ZOL. After detachment and two washes in PBS, 2 × 105 cells were added in the upper
Transwell chamber coated with Matrigel, as described in the Materials and Methods
section. After 18 h incubation at 37°C, the cells in the upper part of the invasion
chamber were gently detached and cells that had traversed the filter were counted
by light microscopy after May–Grünewald–Giemsa coloration. In all, 10 fields (magnification
× 200) were counted for each insert. Data are expressed as the percentage (as compared
to control) of the mean±s.e.m. of five independent experiments. (*
P<0.05, **
P<0.01).
, MDA-MB-231 cells were highly invasive in Matrigel-coated chambers. Treatment of
MDA-MB-231 cells for 18 h with ZOL at low concentrations (from 10−7 M) inhibited MDA-MB-231
cell invasion in the in vitro invasion assay in a dose-dependent manner (Figure 2).
The number of invading cells was decreased by 62.1±3.8% (P<0.01) following treatment
with 1 μ
M ZOL. Consequently, a 1 μ
M concentration of ZOL was chosen for treatment of MDA-MB-231 cells for the following
experiments.
Inhibition of MDA-MB-231 cell invasion by ZOL is reversed by GGOH
It was reported that N-BPs inhibit enzymes of the MVA pathway, and thus the synthesis
of isoprenoid intermediates. To determine whether the inhibition of isoprenoids by
ZOL is responsible for its inhibitory effect on cell invasion, we tested whether the
inhibition of cell invasion by ZOL was reversed by the MVA pathway metabolites (MVA,
FOH, GGOH and SQUA). It was verified that the simple addition of these intermediates
has no effects on the invasion of untreated MDA-MB-231 cells (data not shown). The
invasion-suppressive effect of ZOL on MDA-MB-231 cells was circumvented by the addition
of 10 μ
M GGOH, which restores geranylgeranylation (the percentages of invading cells in comparison
to untreated cells were, respectively, for 1 μ
M ZOL-treated cells, 37.9±3.8 and 92.4±2.8% in the absence and presence of GGOH) (Figure
3
Figure 3
Effect of the mevalonate (MVA) pathway metabolites on zoledronic acid (ZOL)-induced
MDA-MB-231 cell invasion inhibition – Comparison with the effect of FTase inhibitor
(FTI-277), GGTase inhibitor (GGTI-298) and C3 exoenzyme. MDA-MB-231 cells were treated
for 18 h with 5 μg ml−1 C3 exoenzyme or 10 μ
M FTI-277 or 10 μ
M GGTI-298 or 1 μ
M ZOL in the presence or absence of different MVA pathway metabolites–100 μ
M mevalonate (MVA), 10 μ
M farnesol (FOH), 10 μ
M geranylgeraniol (GGOH) and 10 μ
M squalene (SQUA) and experiments were performed as indicated in Figure 2. Data are
expressed as the percentage (as compared to control) of the mean±s.e.m. of five independent
experiments. Significant difference from nontreatment control (**); from ZOL-treated
cells (##) (**,##
P<0.01).
). In contrast, MVA, SQUA and FOH (which restores farnesylation) did not reverse the
anti-invasive effect of ZOL. In addition, to confirm that the anti-invasive effect
of ZOL is mediated by inhibiting protein geranylgeranylation, we next examined whether
the GGTase I inhibitor, GGTI-298, mimicked the effect of ZOL. As shown in Figure 3,
treatment of MDA-MB-231 cells with GGTI-298 caused a significant decrease of cell
invasion, while FTI-277, an inhibitor of FTase, at the same concentrations, was devoid
of effect. Therefore, these results suggest that inhibition of geranylgeranylated
proteins (such as Rho) rather than farnesylated proteins (such as Ras) seems to be
important to explain the anti-invasive action of ZOL. Since the small GTPase RhoA
requires geranylgeranylation to promote cancer invasion, we suggested that RhoA could
represent a potential candidate to mediate ZOL effects. In support of this hypothesis,
it was found that the addition of Clostridium botulinum C3 transferase (C3 exoenzyme),
a widely accepted RhoA inhibitor (Sekine et al, 1989), mimicked ZOL action on MDA-MB-231
cells.
ZOL mediates MDA-MB-231 cell invasion inhibition by preventing RhoA translocation
from the cytosol to cell membrane
Since the small GTPase RhoA must be targeted to the plasma membrane for its activation
(Yoshioka et al, 1998), we examined the effect of ZOL on the translocation of RhoA
protein from the cytosol to the membrane fraction in MDA-MB-231 cells. In control
MDA-MB-231 cells, RhoA is predominantly associated with the cell membrane, suggesting
that in these cancer cells the overexpression of RhoA facilitates its translocation
from the cytosol to the cell membrane, as previously described (Yoshioka et al, 1998;
Fritz et al, 1999). In contrast, after treatment with 1 μ
M ZOL for 18 h, RhoA was almost found in the cytosol fraction and the amount of this
protein associated with the cell membrane was greatly reduced (Figure 4A
Figure 4
Zoledronic acid (ZOL) prevents the translocation of RhoA from cytoplasm to the cell
membrane in MDA-MB-231 cells (A), but has no effect on Ras localisation (B). (A) MDA-MB-231
cells were treated with ZOL (1 μ
M) in the presence or absence of GGOH (10 μ
M) or FOH (10 μ
M) for 18 h. C3 exoenzyme was used as positive control. (B) MDA-MB-231 cells were
treated or not for 18 h with 1 μ
M ZOL. Cells were then lysed and the membrane (M) and cytosolic (C) fractions were
separated by ultracentrifugation. RhoA (A) and Ras (B) were detected by immunoblotting
using RhoA and Ras polyclonal antibodies. These results are representative of three
independent experiments.
). In addition, we observed that the inhibiting effect of ZOL on the RhoA membrane
localisation was prevented by the addition of GGOH, but not FOH. C3 exoenzyme was
used as positive control. Therefore, these results suggested that RhoA seems to be
the main target of the inhibitory effect induced by ZOL on cell invasion. Additionally,
because it was recently suggested that the treatment of MDA-MB-231 with high concentrations
of ZOL inhibits generation of FPP leading to decreased prenylation of Ras (Senaratne
et al, 2002), we also analysed the effect of ZOL on the cellular localisation of Ras.
In control cells, Ras was predominantly associated with cell membrane fraction because
MDA-MB-231 cells are characterised by a Ras mutation, leading to its constitutive
activation. According to our results (see above), ZOL did not inhibit the translocation
of Ras from cytoplasm to the cell membrane at low concentration (1 μ
M) after 18 h of treatment (Figure 4B).
ZOL induces a disorganisation of actin cytoskeleton and a loss of stress fibres formation
in MDA-MB-231 cells
Since RhoA is engaged in cytoskeleton reorganisation to promote cancer invasion, we
studied the effect of ZOL on the morphology of MDA-MB-231 breast cancer cells. Untreated
MDA-MB-231 cells were flat and well spread. In contrast, ZOL induced dramatic morphological
changes characterised by a cell rounding and a disorganisation of actin cytoskeleton
accompanied by a loss of stress fibres formation, which were clearly evidenced by
confocal microscopy. Moreover, these morphological changes were rescued by the addition
of GGOH, but not FOH (Figure 5
Figure 5
Morphological changes and reorganisation of actin cytoskeleton on MDA-MB-231 cells
treated by zoledronic acid (ZOL). – Comparison with C3 exoenzyme. The actin cytoskeleton
of MDA-MB-231 cells was labelled by phalloidin-TRITC and analysed by confocal microscopy.
On untreated cells (A), note the actin stress fibres and cell spreading. Treatment
for 18 h with 1 μ
M ZOL (B) induced morphological changes of cells associated with disorganisation of
actin stress fibres. The modifications induced by ZOL were reversed by geranylgeraniol
(GGOH) (C) but not by farnesol (FOH) (D) and mimicked by C3 exoenzyme (E) (Scale bar=10 μ
M).
). Importantly, C3 exoenzyme mimicked the morphological changes induced by ZOL (Figure
5).
ZOL inhibits the expression of u-PAR but not of u-PA in MDA-MB-231 cells
As u-PA associated with its receptor (u-PAR) is required to stimulate invasion of
cancer cells (Blasi, 1999), we studied the effect of ZOL on the u-PA and u-PAR expressions
by flow cytometry. After an 18 h incubation with 1 μ
M ZOL, we observed a dose-dependent decrease of u-PAR antigen on the MDA-MB-231 cell
surface (60% decrease) (Table 2
Table 2
Effect of zoledronic acid (ZOL) on u-PA and u-PAR expression on MDA-MB-231 cells
ZOL (μ
M)
0.01
0.1
1
10
100
u-PA
93.6±4.3
97.2±5.6
84.8±4.6
73.7±7.3
67.3
*±5.8
u-PAR
96.6±5.1
64.2±9.4
43.6
**±4.6
45.7
*±7.1
39.3
**±4.9
MDA-MB-231 cells were incubated for 18 h with increasing concentrations of ZOL and
u-PA and its receptor u-PAR expressed at the membrane were analysed by flow cytometry.
Data are expressed as the percentage (as compared to the controls) of the mean fluorescence
intensity (MFI) of four separate experiments. (Bold indicates significance;
*
P<0.05,
**
P<0.01).
). In contrast, the inhibition of u-PA expression was only observed for 100 μ
M ZOL, but not at lower concentrations (Table 2).
Inhibition by ZOL of the chemotactic effect induced SDF-1 and CXCR-4 expression on
MDA-MB-231 cells
Since cancer cells use the SDF-1/CXCR-4 pathway to spread to bone (Taichman et al,
2002), we investigated whether ZOL may affect the SDF-1/CXCR-4 chemotaxis mechanism.
Firstly, we verified that the addition of SDF-1 to the lower chamber induced invasion
of MDA-MB-231 cells through a reconstituted basement membrane (Matrigel). The percentage
of cells that invade the Matrigel was at least four times higher when SDF-1 was added
in the lower chamber, indicating a potent chemotactic effect of SDF-1 on MDA-MB-231
cells (Figure 6
Figure 6
Zoledronic acid (ZOL) inhibits the chemotactic effect induced by SDF-1 on MDA-MB-231
cells – Partial reversion by geranylgeraniol (GGOH). Confluent MDA-MB-231 cells were
treated for 18 h with 5 μg ml−1 exoenzyme or 10 μ
M FTI-277 or 10 μ
M GGTI-298 or 1 μ
M ZOL in the presence or absence of GGOH and farnesol (FOH) and seeded into the upper
chamber of Transwell coated with Matrigel, and a gradient of SDF-1 (100 ng ml−1) was
established by placing the chemokine in the lower chamber. Then the invasion was measured
as described in Figure 2. The inhibitory effect of ZOL was compared with that of C3
exoenzyme and of neutralising antibody against CXCR-4. Results are expressed as the
percentage (as compared to control) of the mean±s.e.m. of five independent experiments.
Significant difference from nontreatment control (*); from ZOL-treated cells (#) (*,#
P<0.05, **
P<0.01).
). As control, this SDF-1-mediated invasion could be abrogated by the addition of
SDF-1 to both the upper and lower compartments of Boyden's chamber, confirming the
specificity of the chemotactic response induced by this chemokine. Moreover, the addition
of CXCR-4 neutralising antibody to the top of the culture chamber, but not an isotype
control, decreased MDA-MB-231 cell invasion. This provides verification that the chemotactic
effect induced by SDF-1 is mostly dependent on CXCR-4 receptor binding (Figure 6).
Next, the ability of ZOL to influence SDF-1-induced breast carcinoma chemotactic effect
was studied. The number of cells that invade Matrigel was roughly the same in untreated
cells and cells treated with both SDF-1 and ZOL. Therefore, ZOL inhibited both invasion
and SDF-1 chemotactic effect of breast-cancer cells. However, in contrast to cell
invasion, the inhibition of chemotactic effect by ZOL was only rescued by 60% by GGOH
and partially mimicked by GGTI-298. In addition, the reduction of SDF-1-induced invasion
by C3 exoenzyme is much lower than that observed with ZOL-treated cells (30 vs 70%)
(Figure 6). These results indicate that the decrease of cell motility induced by RhoA
inhibition was not the only mechanism responsible for this inhibition. Recently, a
strong cell-surface expression of CXCR-4, the SDF-1 receptor, was described on the
aggressive MDA-MB-231 breast-cancer cell line (Müller et al, 2001). We confirmed these
data on these cells (59.8% of positive cells) by flow cytometry (Table 3
Table 3
Effect of zoledronic acid (ZOL) on the expression of chemokine receptor CXCR-4 on
MDA-MB-231 cells
ZOL (1 μ
M)
Control
ZOL (1 μ
M)
+GGOH
+FOH
C3 Exo.
59.8±3.1
15.7
**±2.3
18.6±4.7
13.1±1.5
47.5±6.1
MDA-MB-231 cells were treated or not by 1 μ
M ZOL with or without geranylgeraniol (GGOH) and farnesol (FOH). The effect of ZOL
was compared with that of C3 exoenzyme (C3 Exo). The chemokine receptor of SDF-1,
CXCR-4, was detected by flow cytometry by indirect immunofluorescence using an FITC-conjugated
anti-mouse Ab. Results are the mean±s.e.m. of four independent experiments and are
expressed as the percentage of positive cells (Bold indicates significance;
**
P<0.01).
). Subsequently, the effect of ZOL was tested on CXCR-4 expression. As indicated in
Table 3, an 18 h treatment with 1 μ
M ZOL reduced the CXCR-4 expression from 59.8 to 15.7% of positive cells. This decrease
of CXCR-4 expression by ZOL was not reversed by GGOH and FOH and was not induced by
C3 exoenzyme.
Inhibition of Cox-2 expression and PGE2 release by ZOL on MDA-MB-231 cells
One mechanism that contributes to osteoclast activation and metastasis-induced osteolysis
is the release of PGE2 by cancer cells (Ono et al, 2002). Therefore, we evaluated
the action of ZOL on Cox-2 mRNA and protein expression in MDA-MB-231 cells. According
to previous studies (Liu and Rose, 1996), MDA-MB-231 cells expressed a high constitutive
level of inducible Cox-2, which was observed at both mRNA and protein levels. Interestingly,
the Cox-2 transcript was greatly decreased from 6 h after 1 μ
M ZOL treatment (Figure 7A
Figure 7
Effect of zoledronic acid (ZOL) on Cox-2 mRNA, protein levels and activity in MDA-MB-231
cells – Comparison with C3 exoenzyme. (A) Time course of ZOL on Cox-2 mRNA expression.
MDA-MB-231 cells were treated for 3, 6 or 12 h with or without 1 μ
M ZOL. RNA was extracted and analysed by RT–PCR. Sizes of PCR products for Cox-2 and
GAPDH were, respectively, 300 and 222 bp. (B) Effect of ZOL on Cox-2 mRNA expression.
MDA-MB-231 cells were incubated for 12 h with C3 exoenzyme (5 μg ml−1) or with 1 μ
M ZOL with or without geranylgeraniol (GGOH) (10 μ
M) and farnesol (FOH) (10 μ
M) and compared with untreated cells. RNA was extracted and analysed by RT–PCR. Sizes
of PCR products for Cox-2 and GAPDH were, respectively, 300 and 222 bp. (C) Effect
of ZOL on Cox-2 protein level. MDA-MB-231 cells were incubated with or without ZOL
for 18 h and Cox-2 protein expression was examined by Western blot, using a monoclonal
anti-Cox-2 antibody. Blots were developed with the enhanced chemoluminescence reagent
(ECL). The membrane was also probed with β-actin to confirm equal loading. (D) Effect
of ZOL on the secretion of PGE2 in the supernatant of MDA-MB-231 cells. The levels
of PGE2 were measured by ELISA assay on supernatants of control and ZOL (1 μ
M)-treated MDA-MB-231 cells in the presence or absence of GGOH and FOH. The effect
of ZOL was compared with C3 exoenzyme. Results are mean±s.e.m. of three independent
experiments and are expressed in picograms of PGE2 per microgram of protein (**
P<0.01).
). In addition, when MDA-MB-231 cells were treated with 1 μ
M ZOL for 18 h, a significant decrease of Cox-2 protein expression was noted (Figure
7B). As a consequence, it was found that treatment of cancer cells with ZOL induced
a decrease in Cox-2 enzyme activity: a high level of PGE2 (375.4±26.7 pg μg−1 protein
in 24 h) was observed in the medium of untreated cells whereas exposure of cells to
1 μ
M ZOL caused a clear decrease in PGE2 secretion (196.4±18.9 pg μg−1 protein in 24 h,
P<0.01) (Figure 7D). Importantly, the inhibition of Cox-2 expression by 1 μ
M ZOL was not reversed by GGOH and FOH. This indicates that RhoA inhibition seems
not to be involved in Cox-2 inhibition by ZOL. This RhoA-independent effect was also
confirmed by the absence of the effect of C3 exoenzyme on Cox-2 at transcript and
protein levels on these breast cancer cells (Figures 7B and C). This was also in good
agreement with the observations that GGOH did not reverse the effect of ZOL on PGE2
secretion (225.8±38.6 pg μg−1 protein in 24 h) and that C3 exoenzyme was also devoid
of effect on PGE2 secretion.
DISCUSSION
During the last years, many investigations have shown that BPs are targeted towards
osteoclasts and protected against metastasis-induced osteolysis. However, BPs can
also act directly on cancer cells. In the first part of this study, we analysed the
action of ZOL, a third-generation N-BP that is increasingly being used in the treatment
of bone metastases, on the proliferation and invasiveness of highly aggressive breast-cancer
cells MDA-MB-231.
In agreement with recent studies already published (Fromigue et al, 2000; Hiraga et
al, 2001; Jagdev et al, 2001), it was shown that ZOL inhibits MDA-MB-231 cell proliferation,
but only for high concentrations (>100 μ
M) which are certainly higher than the pharmacological concentrations obtained in
vivo. However, such elevations of BP concentrations cannot be excluded, for short
periods, within the bone metastases as the result of locally increased bone resorption
in patients. The decrease of cell proliferation induced by ZOL could be related to
the induction of apoptosis because it occurs in these cells at the same concentrations.
Next, the effect of ZOL was also studied on MDA-MB-231 cell invasion. After an 18 h
incubation time, ZOL, at low concentrations (from 100 nM), displayed a potent anti-invasive
property on MDA-MB-231 cells (62% decrease at 1 μ
M) in the in vitro invasion assay through Matrigel. As ZOL did not induce apoptosis
at these concentrations, the possibility that ZOL interfered with invasion by inducing
cell death was excluded. This is also in agreement with the reported observations
of Boissier et al (2000).
Additionally, we attempted to determine the mechanism involved in the ZOL-induced
anti-invasive effect on MDA-MB-231 cells. This mechanism did not imply proteases involved
in tumour invasion by inducing the degradation of the extracellular matrix (ECM).
Indeed, neither MMP secretion nor u-PA expression was modified at concentrations that
inhibit cell invasion. Only high concentrations were needed to reduce the secretion
of both MMP-2 and MMP-9 (Boissier et al, 2000) and u-PA expression in MDA-MB-231 cells
as shown in Table 2. In contrast, u-PAR expressed on the cell surface of MDA-MB-231
cells was dramatically reduced by ZOL at low concentrations (Table 2). u-PAR is a
ligand for vitronectin, which is a common protein in mature bone microenvironment
(Cooper et al, 2002). Consequently, the decrease of u-PAR by ZOL could contribute
to the previously reported prevention of breast-cancer cell attachment onto bone matrices
(van der Pluijm et al, 1996; Boissier et al, 1997).
Since the small GTPases of Ras and Rho families have to be prenylated to play an essential
role in carcinoma cell invasion (Oxford and Theodorescu, 2003), we attempted to assess
if the anti-invasive action of ZOL could be related to Ras and/or RhoA inactivation,
following the decreased formation of FPP and GGPP, respectively. In this study, it
was demonstrated that GGOH, which restores geranylgeranylation, but not FOH, which
restores farnesylation, reversed the effect of ZOL, suggesting that the inhibition
of protein(s) geranylgeranylation rather than farnesylation seems to account for ZOL
anti-invasive action. To test this hypothesis further, the effect of FTI-277 and GGTI-298
that potently and selectively inhibit FTase and GGTase, respectively, was compared
with the action of ZOL on MDA-MB-231 cell invasiveness. These inhibitors have been
widely used to identify the prenylated proteins involved in the biological functions
of various cell types (Lerner et al, 1995; Vogt et al, 1996). The incubation of MDA-MB-231
cells with GGTI-298 mimicked the anti-invasive effect of ZOL, whereas FTI-277 was
devoid of effect. Thus, inhibition of protein geranylgeranylation seems to be important
to explain the anti-invasive action of ZOL. This effect was also mimicked by C3 exoenzyme,
which is a specific inhibitor of RhoA, but not for other Rho subfamily members, Rac
and Cdc42 (Boquet, 1999). Therefore, it was suggested that the inhibition of cell
invasion by ZOL could be related to the inhibition of RhoA cell signalling. This was
also supported by our observation showing that ZOL at low concentrations prevents
the translocation of RhoA from cytoplasm to the cell membrane. Strikingly, there was
a parallel between ZOL inhibition of cell invasion, the decrease in membrane-associated
RhoA and the morphological changes characterised by a disorganisation of actin cytoskeleton,
as shown by confocal microscopy. Therefore, RhoA inhibition by ZOL could be responsible
for its anti-invasive effect on MDA-MB-231 cells by decreasing their motility as a
result of the disorganisation of actin cytoskeleton accompanied by a loss of stress
fibres. Additionally, Ras inactivation did not appear to be involved in the inhibition
of MDA-MB-231 cell invasion because, at the same low concentration, Ras is not inactivated
by ZOL, as clearly evidenced by its membrane localisation. Finally, the decrease in
cell invasion induced by ZOL was not modified by treatment with 10 μ
M SQUA, the late metabolite in the cholesterol synthesis pathway, suggesting that
regulation of cellular cholesterol level was not involved in this effect.
In contrast to the effect of ZOL on cell invasion, the inhibition of cell proliferation
seems independent of RhoA inactivation because at the concentration for which RhoA
is inhibited, cell proliferation and apoptosis are unaltered. This inhibition seems
more likely due to the inhibition of Ras prenylation as recently demonstrated by Senaratne
et al (2002).
In the second part of this study, it was shown that ZOL inhibits the chemotactic effect
induced by the chemokine SDF-1 on MDA-MB-231 cells. This observation constitutes an
important addition to the mechanistic understanding of how BPs, given in adjuvant
setting, could prevent the development of bone metastases as shown by two clinical
trials (Diel et al, 1998; Powles et al, 2002). Moreover, we attempted to elucidate
whether this effect of ZOL could be explained by the inhibition of the MVA pathway,
and more particularly by RhoA inactivation. However, this inhibition was incompletely
circumvented by GGOH and only partially mimicked by GGTI-298, suggesting that in contrast
to the inhibitory effect induced by ZOL on cell invasion, which could be mainly explained
by the disorganisation of cytoskeleton induced by RhoA inhibition, an additional mechanism
may occur. Indeed, we demonstrated that the ZOL induces also a potent inhibition of
CXCR-4 expression on MDA-MB-231 cells, which was not reversed by GGOH and not mimicked
by C3 exoenzyme. Consequently, at least two mechanisms cooperate to induce this inhibiting
effect induced by ZOL; one related to defective actin stress fibres formation responsible
for the loss of traction forces required for cell motility, which is RhoA-dependent,
and the other related to decreased CXCR-4 expression, which is RhoA-independent. This
effect of ZOL could be crucial for the inhibition of cancer cell metastasis in bone,
as it was reported that neutralising anti-human CXCR-4 monoclonal antibody suppresses
metastases in a breast-cancer model (Müller et al, 2001).
Finally, in bone, breast-cancer cells interact mainly with bone-resorbing osteoclasts,
supplying them with stimulatory factors, which lead to disruption of bone structure
and release of stroma-bound factors that in turn stimulate the growth of cancer cells
(Yin et al, 1999). Invasive breast cancers, which constitutively express inducible
Cox-2, enhance osteoclast formation through the production of high levels of PGE2
and subsequently an increase of osteolysis (Ono et al, 2002). As an important mechanism
involved in the effect of BPs in bone marrow metastasis is the reduction of osteoclast-induced
bone osteolysis, we also analysed whether ZOL could inhibit osteoclast activation
by cancer cells. In this study, it was demonstrated that Cox-2 mRNA synthesis is greatly
reduced by 1 μ
M ZOL treatment for 6 h. This was responsible for the decrease in Cox-2 protein level
and the consequent decrease in PGE2 secretion by MDA-MB-231 cells. These decreases
in Cox-2 expression and PGE2 secretion by ZOL are independent of RhoA inactivation
as they were not mimicked by C3 exoenzyme and were not reversed by GGOH. Whereas Ras
was reported to be involved in Cox-2 expression in these cancer cells (Gilhooly and
Rose, 1999), Ras inhibition does not appear to be involved in this Cox-2 inhibition
by ZOL, as it occurs at low concentrations that did not inhibit Ras activation. Although
the molecular mechanism of this inhibition was not yet elucidated, it is suggested
that it could contribute to the beneficial effect induced by ZOL on the inhibition
of metastatic breast-cancer-mediated osteolysis. Interestingly, since ZOL, with its
high affinity to hydroxyapatite, is concentrated on the bone surface, its effect could
be more important than that induced by other Cox-2 inhibitors, which although reducing
breast-cancer metastasis, have a reduced affinity for bone in comparison to ZOL (Kundu
and Fulton, 2002).
In summary, our results suggest that ZOL could be used not only to treat cancer metastasis-induced
osteolysis but also to prevent metastasis. The growth inhibition of osteoclasts has
been suggested as the main mechanism of the inhibitory effect of ZOL on bone metastases.
In this study, a new concept was proposed by which, in breast-cancer cells, ZOL inhibits
the production of PGE2, suggesting a decreased cooperation between osteoclasts and
cancer cells for inducing osteolysis. In addition, ZOL induces a potent inhibition
of both breast-cancer cell invasion and SDF-1-mediated chemotactic effect. It may
be useful to correlate these results with clinical trials to test the efficacy of
ZOL on the prevention of metastases in patients with highly aggressive breast cancer.
In addition, as both Cox-2 and CXCR-4 inhibitions are RhoA-independent, further investigations
are required to determine whether the invasion of cancer cells without RhoA activation
could be reduced by ZOL.