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Experimental Therapeutics |
Institut National de la Santé et de la Recherche Médicale Research Unit 403, Faculté de Médecine Laënnec, 69372 Lyon, France [S. B., O. P., S. M., M. C., P. D., P. C.]; Center for Clinical & Basic Research, DK 2750 Ballerup, Denmark [M. F, J-M. D.]; Procter & Gamble Pharmaceuticals, Mason, Ohio 45040-8006 [F. H. E.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interestingly, BPs also induce apoptosis in human myeloma cells (7) and inhibit MMP production by human PC3 ML prostate carcinoma cells (8 , 9) . Moreover, adjuvant treatment of breast cancer patients with the BP clodronate in combination with standard hormonal therapy or chemotherapy reduces the incidence and number of bone and non-bone metastases (10) . These very important observations (3, 4, 5, 6, 7, 8, 9, 10) suggest, therefore, that BPs not only act on osteoclast-mediated bone resorption but may also affect the invasive behavior of metastatic cells in bone. In the present study, we investigated the effect of BPs on tumor cell invasion. Tumor cell invasion in vivo is prerequisite for breast cancer colonization in bone (4) . We have found that BPs inhibit breast and prostate carcinoma cell invasion through a specific action on tumor cells. In addition, BPs inhibited the proteolytic activity of MMPs, which is obligatory for successful tumor cell invasion. These results suggest that BPs may be useful agents for the prophylactic treatment of patients with cancers that are known to preferentially metastasize to bone.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor Cell Lines.
Human breast carcinoma cell line MDA-MB-231 was purchased from the
American Type Culture Collection (Rockville, MD). Cell line PmPC3 is a
highly metastatic variant of human prostate carcinoma cell line PC3
(6)
. Both the MDA-MB-231 and the PmPC3 cell lines are
highly metastatic to bone (3
, 4)
.4
Tumor cells were routinely cultured in RPMI 1640 supplemented with 10%
fetal bovine serum and 1% penicillin/streptomycin at 37°C in a
humidified atmosphere containing 5% CO2.
Drug Treatment of Tumor Cell Lines.
Tumor cell monolayers reaching 90% confluency were washed with
serum-free RPMI 1640 and treated for 24 h at 37°C with BPs
diluted in complete medium. Alternatively, cell monolayers were treated
for 1 h at 37°C with Taxol in complete medium. At the end of the
incubation, cell monolayers were washed with serum-free RPMI to remove
drugs, and cells were harvested with trypsin-EDTA. After a 90-min
incubation at 37°C to allow cells to recover from trypsin-EDTA
treatment, untreated and drug-treated cells in RPMI containing 0.1%
(w/v) BSA were used for cell migration and invasion assays.
Cell Migration Assay.
Cell migration experiments were performed using Bio-Coat cell migration
chambers (Becton Dickinson), which consist of a 24-well companion plate
with cell culture inserts containing 8 µm pore size filters. The
experimental procedure was as described previously (11)
.
Briefly, untreated and drug-treated tumor cells (5 x 104
/500 µl) were added to each insert (upper
chamber), and the chemoattractant (10% FCS) was placed in each well of
a 24-well companion plate (lower chamber). After a 6-h incubation at
37°C in a 5% CO2 incubator, the upper surface
of the filter was wiped with a cotton-tipped applicator to remove
nonmigratory cells. Cells that had migrated through the filter pores
and attached on the under surface of the filter were fixed and stained.
The membranes were mounted on glass slides, and the cells from 10
random microscopic fields (x400 magnification) were counted. All
experiments were run in duplicate, and migration was expressed in terms
of cells/mm2.
Matrigel Invasion Assay.
Cell invasion experiments were performed using Bio-Coat cell migration
chambers (Becton Dickinson) as described above. Filters (8 µm pore
size) were coated with the basement membrane Matrigel (37 µg/filter).
The rest of the experimental procedure was as described for the cell
migration assay. After a 48-h incubation at 37°C in a 5%
CO2 incubator, noninvading cells were removed,
and the invading cells on the under surface of the filter were fixed
and stained. The membranes were mounted on glass slides, and the cells
from 10 random microscopic fields (x400 magnification) were counted.
All experiments were run in duplicate, and invasion was expressed in
terms of cells/mm2.
Cell Cycle Analysis.
Untreated, BP-treated and Taxol-treated tumor cells harvested with
trypsin-EDTA (106/500 µl for each experimental
condition) were washed in RPMI, and then fixed in 50% (v/v)
cold ethanol for 15 min at 4°C. After incubation, fixed cells were
stained with propidium iodide (Becton Dickinson) for 10 min at room
temperature in the dark. Cell cycle distribution was determined by flow
cytometric analysis using the red fluorescence of excited propidium
iodide-stained nuclei as a measure of DNA content. Linear displays of
fluorescence emissions were used to compare cell cycle phases and
quantitate the cells with the degraded sub-G1 DNA
content characteristic of apoptotic cells. Analysis of the data was
performed using software Lysis II (Becton Dickinson).
Flow Cytometry-based Apoptosis Assay.
Untreated, BP-treated, and Taxol-treated tumor cells were harvested
with trypsin-EDTA, and then resuspended in PBS containing 2% (m/v) BSA
(106 cells/ml for each experimental condition).
Detection of apoptosis in tumor cells in suspension was performed using
the TACS Annexin V apoptosis detection kit (Genzyme). The
staining was analyzed on a FACScan flow cytometer (Becton Dickinson) as
described previously (12)
.
Analysis of Cell Death in Adherent Tumor Cells.
Tumor cells were plated on chamber slides (3 x 104
cells/chamber) and cultured in complete
medium for 24 h at 37°C in a 5% CO2
incubator. Tumor cells were treated with BPs or Taxol as described
above in the drug treatment section. After incubation, monolayers were
washed in RPMI, and adherent cells were fixed in PBS containing 3%
paraformaldehyde. After a 30-min incubation, cells were stained with
Hoechst 33258 (10 µg/ml; Sigma) for 30–60 min in the dark and
examined under fluorescence microscopy.
Measurement of MMP Activities by Fluorometric Analysis.
Purified recombinant mouse pro-MMP-9 was activated with 1
mM p-aminophenylmercuric acetate for
4 h at 37°C. Human pro-MMP-2 from fibrosarcoma cells (Boehringer
Mannheim, Mannheim, Germany) was activated with 1
mM p-aminophenylmercuric acetate for
45 min at 37°C. The active form of rabbit MMP-12 was a gift of Dr. P.
Hou (Center for Clinical & Basic Research, Ballerup, Denmark). Enzyme
concentrations in the final preparations were estimated by active site
titration using the MMP inhibitor BB94 (British Biotech). Purified
activate MMP-2 (1 nM), MMP-9 (2
nM), and MMP-12 (10 nM) in
the presence or absence of increasing concentrations of BPs were
preincubated for 3 h at 37°C in 50 mM
Tris, 1 mM CaCl2, 150
mM NaCl, 0.05% (v/v) Brij 35 (pH 7.5) with or
without ZnSO4 (50 µM).
MMP activities were then measured by evaluating the proteolytic
cleavage of the fluorogenic peptide substrate
Mca-Pro-Leu-Gly-Leu-Dnp-Ala-Arg-NH2 (Bachem,
Bubendorf, Switzerland) following a previously described method
(13)
. After addition of the fluorogenic peptide substrate
(4 µM) to the preincubation mixture, aliquots
were taken at time points up to 45 min into 0.1 M
sodium acetate (pH 4.0) to stop the reaction before measurement of the
increase in fluorescence. This increase in fluorescence corresponding
to the release of Mca was measured using a SFM25 Kontron
spectrofluorometer with an excitation wavelength of 320 nm and an
emission wavelength of 387 nm. The rate of cleavage of the fluorogenic
peptide substrate over 45 min obtained with MMPs that were not
preincubated with BPs was used as a positive control and was set at
100%. Results obtained in the presence of BPs were then expressed as a
percentage of each positive control.
Gelatin Zymography.
The serum-free conditioned media of untreated and BP-treated MDA-MB-231
and PmPC3 cells that had been cultured for 48 h on Matrigel-coated
plates were lyophilized. Lyophilized conditioned media were resuspended
in 0.5 ml of distilled water, and the protein concentrations were
measured with a Bradford protein assay kit (Bio-Rad). Conditioned media
(20 µg/lane) were run under nondenaturating conditions on 10%
SDS-polyacrylamide gels containing 1 mg/ml gelatin in the presence or
absence of the BP zoledronate for 4 h at 60 V. After
electrophoresis, the gels were incubated in 2.5% (v/v) Triton X-100
for 2 h to remove SDS, washed briefly in distilled water, and then
incubated in 50 mM Tris-HCl, 10 mM
CaCl2 (pH 7.5) in the presence or absence of the
BP zoledronate for overnight at 37°C. The gels were then stained with
0.25% (w/v) Coomassie brilliant blue and destained in methanol-acetic
acid-water (20:7:73, v/v/v). White bands indicated gelatinase
activity. The conditioned medium from human HT-1080 fibrosarcoma cells
treated for 48 h with
12-o-tetradecanoylphorbol-13-acetate (10 ng/ml) was
used as a positive control to visualize the gelatinolytic activity of
MMP-2 and -9.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The order of potency for four BPs in inhibiting tumor cell invasion
was: zoledronate > ibandronate > NE-10244 (active pyridinium analogue of risedronate) > clodronate. Zoledronate and ibandronate inhibited tumor cell invasion
with a half-maximal inhibition (IC50) of
10-12 M, whereas the
IC50s for NE-10244 and clodronate were
5 x 10-10 and 5 x 10-5 M, respectively. In
addition, NE-58051 (the inactive pyridylpropylidene analogue of
risedronate) had no inhibitory effect, whereas NE-10790 (a
phosphonocarboxylate analogue of risedronate in which one of the
phosphonate groups is substituted by a carboxyl group) inhibited tumor
cell invasion to an extent similar to that observed with NE-10244 (Fig. 1
). BP pretreatment of PmPC3 prostate carcinoma cells also inhibited
tumor cell invasion with the same magnitude (results not shown). The
order of potency for zoledronate, ibandronate, NE-10244, NE-10790, and
clodronate in inhibiting PmPC3 cell invasion was similar to that
observed with MDA-MB-231, and the BP NE-58051 did not exert any
inhibitory effect.
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). The proportion of Taxol-treated tumor cells within the
sub-G1 peak involved 40% of the cell population.
Unlike Taxol, zoledronate (1000 nM) did not
induce apoptosis in MDA-MB-231 and PmPC3 cells (Fig. 2A
, and
results not shown). Apoptosis was then assessed by a flow
cytometry-based assay using annexin V conjugated to fluorescein as an
early indicator of apoptosis (annexin V preferentially binds to
phosphatidylserine exposed on the surface of cells undergoing
apoptosis). As shown in Fig. 2B
, histograms of cell number
versus fluorescence intensity showed a rightward shift in
the histograms when MDA-MB-231 cells were treated with Taxol then
exposed to annexin V, whereas zoledronate did not induce annexin V
binding to the tumor cell surface. Similar results were obtained with
PmPC3 cells (results not shown). Because both the cell cycle and
annexin V binding assays used cells in suspension, analysis of cell
death was also performed in adherent tumor cells using Hoechst
staining. As shown in Fig. 2C
, many of the MDA-MB-231 cells
treated with Taxol had nuclei with separate globular structures
(apoptotic bodies), whereas nuclei of untreated and zoledronate-treated
cells had no such apoptotic bodies. Similar results were obtained with
PmPC3 cells (results not shown).
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, inset). BPs inhibited the proteolytic activity of MMP-2,
-9, and -12 in a dose-dependent manner with IC50s
of 
40, 160, and 80 µM, respectively (Fig. 3
). Surprisingly, despite structural differences in their bioactive
moieties, all of the BPs used in this study (including the inactive
analogue of risedronate, NE-58051) were equipotent in inhibiting the
proteolytic activity of MMP-2, -9, and -12. These BPs have in common a
structural motif, the so-called bone hook, which consists of the two
phosphonate groups that provide binding to bone mineral
(15)
. This bone hook is also responsible for the chelation
of divalent cations and the growth inhibition of calcium crystals
(15)
. Because MMPs are zinc-dependent endopeptidases
(16)
, we hypothesized that the phosphonate groups of BPs
could inhibit the proteolytic activity of MMP-2, -9, and -12 through
zinc chelation. To address this question, we first examined the effect
of BPs on the proteolytic activity of MMP-2, -9, and -12 in the
presence of an excess of zinc (50 µM). The
inhibitory effect of BPs on MMP activity was completely reversed in the
presence of zinc (results not shown). We also examined the properties
of a risedronate analogue (NE-10790), which belongs to the group of
phosphonocarboxylates in which one of the phosphonate groups is
substituted by a carboxyl group (Fig. 1
, inset). The
phosphonocarboxylate NE-10790 has a markedly reduced affinity for bone
mineral compared with that observed with BPs (17)
.
Increasing concentrations of NE-10790 in the absence of an excess of
zinc did not inhibit the proteolytic activity of MMP-9 and -12, whereas
a modest inhibitory effect was observed on MMP-2 activity (Fig. 3
).
Thus, the bone hook of BPs is responsible for the inhibition of MMP
activity.
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, pretreatment of MDA-MB-231 cells with zoledronate
(10-6 M) did not inhibit production
of MMP-2 and -9 (Fig. 4
, Lane 3) when compared with that
observed with untreated cells (Fig. 4
, Lane 2). Similarly,
the addition of zoledronate (10-6
M) in the gels and
Ca2+-containing buffers used for gelatin
zymography did not significantly inhibit MMP activity (Fig. 4
,
Lane 4). However, the use of zoledronate at a higher
concentration (10-4 M)
caused a drastic inhibition of gelatinolytic activity of MMP-2 and -9
(Fig. 4
, Lane 5). Such an inhibition was also observed when
zoledronate was replaced by 20 mM EDTA in the
Ca2+-containing buffer (results not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(inset), minor changes in the
R2 chain length of NE-10244 (the active analogue
of risedronate) drastically affected its inhibitory activity on tumor
cell invasion. Indeed, the inactive analogue of risedronate (NE-58051)
is 10,000-fold less potent than NE-10244 as an antiresorptive agent
in vivo (18)
, and we previously reported
similar large potency variations between NE-10244 and NE-58051 on
inhibition of tumor cell adhesion (6)
. Interestingly, the
use of a phosphonocarboxylate analogue of risedronate (NE-10790), in
which one of the phosphonate groups is substituted by a carboxyl group,
inhibited tumor cell invasion to an extent similar to that observed
with NE-10244. These data obtained with NE-58051 and NE-10790 strongly
suggested that the inhibitory effect of BPs on tumor cells involves the
R2 chain (but not the so-called bone hook) of the
molecule. Such an inhibitory effect of BPs on tumor cells was
independent of apoptosis induction. Zoledronate (the most potent BP
used in this study) did not induce apoptosis at concentrations
(10-12–10-6
M) that did inhibit tumor cell invasion (Fig. 2
)
nor at a higher concentration (10-4
M).4
This is in
contrast to the observation that human myeloma cells exposed to high
concentrations (up to 5 x 10-4
M) of different BPs (including clodronate,
pamidronate, and zoledronate) undergo apoptosis (7
, 19)
. However, it must be emphasized that in our study, BP
treatment of tumor cells was performed in the presence of serum, which
is a survival factor for these cells. It is therefore most conceivable
that serum counteracts the pro-apoptotic effect of high concentrations
of BPs. Because tumor cell invasion requires both cell migration and digestion of the basement membrane by MMPs (14) , we hypothesized that BPs could affect one or the other of these two mechanisms. In agreement with the observation that the BPs pamidronate and tiludronate do not inhibit the migration of preosteoclasts (20 , 21) , BPs used in this study failed to inhibit tumor cell migration.4 In contrast, we observed that BPs inhibited the proteolytic activity of MMP-2, -9, and -12. The BP clodronate also inhibits MMP-1 proteolytic activity (22) . These BPs have in common a structural motif, the so-called bone hook, which consists of the two phosphonate groups that provide binding to bone mineral (15) . Because MMPs are zinc-dependent endopeptidases (16) , we suggest that the bone hook of BPs inhibits the proteolytic activity of MMPs through chelation of divalent cations. This contention is based on a number of findings. (a) Despite structural differences in their bioactive moiety (i.e., the R2 chain), all of the BPs used here (including the inactive analogue of risedronate, NE-58051) were equipotent in inhibiting the proteolytic activity of MMP-2, -9, and -12. (b), An excess of zinc reversed the inhibitory effect of BPs on the proteolytic activity of MMP-1, -2, -9, and -12 (Ref. 22 , and this study). (c) The phosphonocarboxylate NE-10790, which has a strongly reduced affinity for divalent cations (17) , did not inhibit the proteolytic activity of MMP-2, -9, and -12. (d) Zoledronate inhibited the gelatinolytic activity (but not the production) of MMP-2 and -9. The observation that BPs inhibited the proteolytic activity of MMPs is of potential relevance to the treatment of bone metastases. BPs are selectively located on the bone surface in the resorption space where their local concentration can reach several hundred micromolar (15) . In bone metastases, it is conceivable that BPs released from resorbed bone inhibit the proteolytic activity of MMPs secreted from tumor cells. However, our results did not give a satisfactory explanation to understand how BPs directly interfere with tumor cell invasion. We have provided clear evidence that the inhibitory activity of BPs on tumor cell invasion required the R2 chain of the molecule (and was effective at low concentrations), whereas inhibition of MMP activity by BPs occurred through the so-called bone hook of the molecule (and was effective only at high concentrations). Thus, other mechanisms are involved. Recently, it has been demonstrated that nitrogen-containing BPs inhibit osteoclastic resorption by affecting enzymes of the cholesterol metabolic pathway involved in the geranylgeranylation of small GTP-binding proteins (23 , 24) . It is conceivable that BPs also affect the prenylation of some small GTP-binding proteins in breast and prostate carcinoma cells. This hypothesis warrants further investigation.
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FOOTNOTES |
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1 This study was supported by grants from Leiras
Oy (to P. C.), Procter & Gamble (to P. C.), Novartis Pharma
S.A. (to P. C.), and the Comité Départemental du
Rhône de la Ligue Nationale contre le Cancer (to P. C.).
S. B. is a recipient of an award from the Association pour la
Recherche sur les Tumeurs de la Prostate. S. M. is a recipient of
an award from the Association pour la Recherche sur le Cancer. 
2 To whom requests for reprints should be
addressed, at INSERM Research Unit 403, Faculté de Médecine
Laënnec, Rue Guillaume Paradin, 69372 Lyon Cedex 08, France.
Phone: 33-4 78 78 57 37; Fax: 33-4 78 77 86 63; E-mail: clezardin{at}lyon151.inserm.fr
3 The abbreviations used are: BP, bisphosphonate;
MMP, matrix metalloproteinase.
4 S. Boissier and P. Clézardin, unpublished
results.
Received 8/31/99. Accepted 3/29/00.
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), ibandronate (
), NE-10244 (
), NE-58051 (
),
NE-10790 (•), and clodronate (
); bars, SE.
Inset, structures of the antiresorptive active
(NE-10244), inactive (NE-58051), and phosphonocarboxylate (NE-10790)
analogues of risedronate.
