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Introduction
Chondrosarcoma is a malignant primary bone tumor with a poor response to currently-used chemotherapy or radiation
treatment, making the management of chondrosarcomas a complicated
challenge[1]. Clinically, surgical resection remains the
primary mode of therapy for chondrosarcoma. Due to the absence of an effective adjuvant therapy, this mesenchymal
malignancy has a poor prognosis[2] and therefore, it is important to explore a novel and adequate remedy.
Since chondrosarcoma is a type of highly malignant
tumor with a potent capacity to invade locally and distant
metastasis[3,4]. An approach decreasing the ability of invasion and
metastasis may facilitate the development of effective adjuvant therapy. In the processing of chondrosarcoma invasion and
metastasis, the degradation of extracellular matrix (ECM) and
the components of the basement membrane caused by the
concerted action of proteinases, such as matrix metalloproteinases (MMP), cathepsins, and plasminogen
activator, is a critical step in tumor invasion and
metastasis[3,5_8]. Among these enzymes, MMP-2 and MMP-9 degrade
most components of the ECM directly and deeply involved
in cancer invasion and metastasis[7,9]. Therefore, the
inhibition of migration or invasion mediated by MMP-2 could be a
preventive method of cancer
metastasis[9].
Recently, there has been increasing evidence indicating
that bisphosphonates (BP) have direct antitumor effects
in vivo and in vitro, in addition to their therapeutic
antiresorp-tive properties[10_19]. For example, Senaratne
et al reported that BP could induce apoptosis in human breast cancer cell
lines[17]. Forsea et al also demonstrated that BP had an
inhibitory effect in cell cycle progression in human melanoma
cells[13]. Furthermore, several studies have demonstrated
that alendronate, a BP, could inhibit cell invasion in human
epidermoid carcinoma cells[11] and osteosarcoma
cells[12]. However, the inhibitory effect related to cell invasiveness of
alendronate has not been studied yet. Therefore, in this
study, the impact of alendronate on cell invasion and
migration were examined in vitro in human chondrosarcoma cells
(JJ012).
Materials and methods
Cell culture and alendronate treatment The human
chondrosarcoma cell line (JJ012) was kindly provided from the
laboratory of Dr Sean P SCULLY (University of Miami School
of Medicine, Miami, FL, USA)[20]. The JJ012 cells were
cultured in Dulbecco's modified Eagle's medium/modified
Eagle's medium-α/Ham's F12 supplemented with 10% Fetal
Bovine Serum and maintained at 37 oC in a
humidified atmosphere of 5% CO2. Alendronate
(4-amino-1-hydrocybutylidene) was a gift from Merck Sharp and Dohme (Tipperary,
Ireland). For the alendronate treatment, appropriate amounts
of stock solution (1 mmol/L in DMSO) were added into the
culture medium to achieve the indicated concentrations. The
cells were treated in the culture medium for 12, 24, and 48
h. The DMSO solution without alendronate was used as a blank
reagent.
Determination of cell viability The effects of alendronate
on the viability of the JJ012 cells were evaluated by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay[21]. Briefly, the JJ012 cells were seeded in 24-well plates
at a density of 3.5×104 cells/well and treated with an
indicated concentration of alendronate at 37
oC for 24 h. At the end of the indicated exposure period, the cells were washed
with phosphate buffered solution and then incubated with
MTT (0.5 mg/mL; Sigma Chemical Co, St. Louis, MO, USA)
for 4 h. The viable cell number was directly proportional to
the production of formazan following solubilization with
isopropanol, which can be measured spectrophotometrically
at 563 nm.
Cell invasion assay The cell invasion assay was
performed to study the effects of alendronate on the invasion
ability of the chondrosarcoma cell line, according to the
methods described by Yuan et
al[2]. Briefly, cell invasion was assayed in a modified Boyden chamber (Neuro Probe, Cabin
John, MD, USA) with 8 µm pore size polycarbonate
membrane filters. The filters were precoated with 10 µL matrigel
(25 mg/50 mL; BD Biosciences, Bedford, MA, USA). After
being treated with alendronate for 24 h, the surviving cells
were harvested and seeded in a Boyden chamber at a
density of 1×104 cells/well with serum-free medium, and then
incubated for 24 h at 37 oC. After the assay, the filters were
air-dried for 5 h in a laminar flow hood. The invaded cells
were fixed with methanol and stained with Giemsa. The cell
numbers were counted under a light microscope.
Cell migration assay Cell migration experiments were
performed using Bio-Coat cell migration chambers (Becton
Dickinson, Meylan, France), which consisted of a
24-well companion plate with cell culture inserts containing 8
µm pore size filters. Briefly, untreated and
alendronate-treated cells
(1×104/500 µL) were added to each insert (upper
chamber), and the chemoattractant (10% Fetal Calf Serum)
was placed in each well of a 24-well companion plate (lower
chamber). After incubation for 24 h at 37
oC in a 5% CO2 incubator, the upper surface of
the filter was wiped with a cotton-tipped applicator to remove
non-migratory cells. Cells that had migrated through the filter pores
and attached on the under surface of the filter were fixed and stained. The
cells from the microscopic fields were counted.
Determination of MMP-2 by gelatin zymography
The activities of MMP-2 in the medium were measured by gelatin
zymography protease assays as previously
described[22]. Briefly, collected media of an appropriate volume were
prepared with SDS sample buffer without boiling or reduction
and subjected to 0.1% gelatin-8% SDS-PAGE electrophoresis.
After electrophoresis, the gels were washed with 2.5%
Triton X-100 and then incubated in a reaction buffer [40
mmol/L Tris-HCl (pH 8.0), 10 mmol/L
CaCl2, and 0.01% NaN3] at 37
oC for 12 h. Then the gels were stained with Coomassie
brilliant blue R-250.
Preparation of RNA and RT-PCR Total RNA were
extracted from the JJ012 cells by the Qiagen RNeasy Mini Kit
(Valencia, CA, USA). For reverse transcription, 4 µg of total
cellular RNA were used as the template in a 20 µL reaction
containing 4 µL dNTP (2.5 mmol/L), 2.5 µL oligo dT (10
pmol/mL), and RTase (200 U/mL); the reaction was performed
at 42 oC for 1 h. Afterwards, 5 µL cDNA product was used as
the template in PCR amplifications together with appropriate
primers (sense of MMP-2, 5'-GGCCCTGTCACTCCTGAGAT-3'; antisense of MMP-2,
5'-GGCATCCAGGTTATCGGGGA-3'; sense of GAPDH,
5'-CGGAGTCAACGGATTTGGTCGT-AT-3'; antisense of GAPDH,
5'-AGCCTTCTCCATGGTTGG-TGAAGAC-3'). The final products were subjected to
electrophoresis with 2% agarose gel and detected by ethidium
bromide staining.
Statistical analysis In a set of experiments, each assay
was performed in duplicate. Each experiment was repeated
independently at least 3 times. All data are presented as
mean±SD. Statistical significance was determined by the
Student's t-test. A P-value <0.05 was considered to be
statistically significant.
Results
Effect of alendronate on the viability of JJ012 cells
The cytotoxic effect of various concentrations of alendronate
(0_100 µmol/L) on JJ012 cells by MTT assay was assessed
as shown in Figure 1. It was clear that 24 h treatment of
alendronate, even at a concentration as high as 100
µmol/L, had no cytotoxicity to the JJ012 cells. This concentration
range was then applied in all subsequent experiments.
Inhibition on invasion and migration of JJ012 cells by
alendronate Using a cell invasion assay with a Boyden
chamber, the number of invading cells was significantly
reduced in JJ012 cells following alendronate treatment.
Furthermore, alendronate inhibited the invasive and
migration ability in a dose- and time-dependent manner (Figure
2A_2D). At a dose of 100 µmol/L, alendronate reduced the
invasion ability of the JJ012 cells to 21.3% of that of the
controls (=untreated cells, set to 100%).
Inhibitory effects of alendronate on MMP-2
By the use of gelatin zymography assays, it was shown that alendronate
prevented a time-dependent increase in MMP-2 enzyme
activity (Figure 3A,3B). There was only 23.4% of MMP-2
activity left after 100 µmol/L alendronate treatment, as
compared with that of non-alendronate-treated cells.
Inhibitory effects of alendronate on the mRNA levels of
MMP-2 To further evaluate if the significant regulatory
effects of alendronate on MMP-2 in the JJ012 cells are on the
mRNA level, a semiquantitative RT-PCR analysis was
performed. With GADPH as an internal control, the mRNA
levels of MMP-2 were significantly reduced by alendronate
in a concentration- and time-dependent manner (Figure 4). It
was therefore clear that the regulation of MMP-2
expressions by alendronate was, at least in part, on the
transcriptional level.
Discussion
Our study demonstrates that alendronate could
significantly reduce MMP-2 secretion in JJ012 cells in a time- and
dose-dependent manner and also inhibit invasion ability in a
time- and dose-dependent manner, without showing any
cytotoxity or apoptosis induction. Although the beneficial
effect of BP in the treatment of metastatic bone disease could
be due to their anti-osteoclastic
properties[23], preclinical and clinical trials and
several in vitro and in vivo reports have
suggested that BP might also have specific anticancer
action on several types of tumors, such as osteosarcoma,
breast, prostate, and melanoma
cancer[12_16]. They act either by
inhibiting cell viability and/or inducing apoptosis,
inhibiting cell adhesion and/or invasiveness, or anti-angiogenic
potential[24_26].
BPs are well tolerated by patients of all ages and
cate-gories. Alendronate is a relatively less potent class of BP
with minimal side effects[27]. However, to our knowledge, it
is the first time that the inhibitory effects of alendronate
related to cell invasiveness have been investigated and
analyzed in human chondrosarcoma cell (JJ012). In the present
study, alendronate was shown to inhibit in vitro
tumor cell invasion in the chondrosarcoma cell line. It is therefore highly
likely that the inhibitory effect of alendronate on
chondrosarcoma cell invasion may primarily be due to MMP-2
inhibi-tion. One of the key steps in the metastatic cascade
involves the disruption of ECM and basement membranes,
permitting tumor cells to access a distant metastatic site.
Furthermore, the different inhibitory effects on MMP-2
secretion and the mRNA level may indicate that the
regulation of MMP-2 expression by alendronate in JJ012 cells was
more involved at the transcriptional level than the
translational or post-translational levels. However, it warrants
further study to elucidate the underlying mechanism.
Tissue invasion requires the expression of proteinases
that are specific for interstitial ECM. In particular, the 72 kDa
gelatinase A/Type IV collagenase (MMP-2) is a well-studied
member of the MMP family, which plays a critical role in
tumor invasion, metastasis, and
angiogenesis[4,28]. Therefore, the inhibition of chondrosarcoma cell-derived MMP-2
production may significantly reduce local spread and invasion.
In the present study, our findings identified that
alendro-nate may reduce the activity and mRNA levels of MMP-2
and inhibit cell invasion in the chondrosarcoma cell line.
Therefore, alendronate may be a potential candidate for the
systemic therapy of chondrosarcomas, as well as other
malignant diseases.
Acknowledgement
The authors thank Ms Chun-ya LIANG and Ms Chia-li
LU for help with the MTT assay and gelatin zymography
analysis.
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