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,25-Dihydroxyvitamin D3 and Its Analogues Down-Regulate Cell Invasion-associated Proteases in Cultured Malignant Cells1
Departments of Virology [K. K., J. K-O.] and Pathology [J. K-O.], the Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland
Abstract
Vitamin D and its derivatives (deltanoids) are potent regulators
of cell proliferation and differentiation. Targeted production of
proteolytic enzymes like serine proteases and metalloproteinases is an
important part of the invasive process of cancer cells. Treatment with
1
25-dihydroxyvitamin D3
[1,25(OH)2D3] decreases the invasive
properties of breast carcinoma cells. Here we have analyzed the effects
of 1,25(OH)2D3 and its synthetic analogues
on the secretion and cell surface association of the components of the
plasminogen activator (PA) system and on the secretion of certain
matrix metalloproteinases (MMPs) and their inhibitors in MDA-MB-231
breast carcinoma cells. Deltanoids were able to decrease the secretion
of urokinase PA and tissue-type PA activity in a dose-dependent manner
and to increase PA inhibitor 1 secretion, leading to reduced total PA
activity. CB1093 was the most potent analogue, effective at
concentrations several logarithms lower than
1,25(OH)2D3. Transient transfection of
different urokinase PA promoter reporter constructs to HT-1080
fibrosarcoma indicator cells indicated that vitamin D-responsive
sequences were located between nucleotides -2350 and -1870 in the 5'
region of the promoter. Treatment of MDA-MB-231 cells with
1,25(OH)2D3 or other deltanoids also
resulted in decreased MMP-9 levels in association with increased tissue
inhibitor of MMP 1 activity. Membrane-type 1-MMP expression or
proteolytic processing were not appreciably affected by deltanoids.
Vitamin D and its analogues caused a decrease in Matrigel invasion
assays of MDA-MB-231 cells. Cancer cell invasion is associated with
coordinated secretion of proteolytic enzymes and their inhibitors.
Vitamin D and its derivatives can evidently influence invasive
processes by two means: (a) decreasing the expression and
activity of cell invasion-associated serine proteases and
metalloproteinases; and (b) inducing their inhibitors.
Introduction
Accumulating evidence indicates that deltanoids can suppress breast tumor progression in animal models, and this is due to a reduction of tumor growth as well as a reduction of the development of metastases (1, 2, 3) . The steps in the metastatic cascade that are regulated by deltanoids are not well understood. The metastatic process comprises sequential steps including the escape of cells from the primary tumor, survival and transport in the circulation, arrest in a distant organ, extravasation, and growth of cells in the new site (4) . The production of proteolytic enzymes is an important event in various steps of the metastatic process, especially in the degradation of basement membranes and cancer cell invasion into the surrounding normal tissue (5 , 6) .
There are three main groups of proteolytic enzymes, namely the serine proteases, the metalloproteinases, and the cysteine proteinases. The serine proteases uPA3 and tPA can convert plasminogen to plasmin, which is a wide-spectrum protease capable of degrading extracellular and basement membrane proteins as well as activating latent forms of metalloproteinases (7) . Plasmin production is negatively regulated by efficient inhibition of PAs by PAIs. Dysregulation of the PA system in various types of cancers is a frequent observation, and high levels of uPA, PAI-1, and uPAR in tumors correlate with poor patient prognosis (8 , 9) . MMPs are a family of proteases capable of degrading extracellular matrix and basement membrane components including collagens under physiological conditions (10) . These secreted or transmembrane proteases need zinc for catalytic function and are produced as inactive zymogens requiring extracellular activation. MMPs are susceptible to inhibition by TIMPs, which may also have other functions in MMP-mediated processes. The tight regulation of MMPs and TIMPs under normal physiological conditions is often disrupted in malignant disease, leading to increased invasion and tissue destruction (11) . Elevated activities of cysteine proteases have also been suggested to contribute to invasion and metastasis (12 , 13) .
Many hormones, growth factors, and cytokines regulate the PA system.
Epidermal growth factor and fibroblast growth factor as well as the
tumor promoter PMA can up-regulate uPA in different cell systems
(14)
. We have previously found that
1,25(OH)2D3 down-regulates the production of
uPA activity in skin keratinocytes and fibroblasts independent of their
antiproliferative responses (15
, 16)
. In the presence of
uPA stimulators epidermal growth factor or PMA,
1,25(OH)2D3 was still able to reduce
secreted uPA activity in keratinocytes, suggesting an important role in
the regulation of the PA system in these cells. MMPs are also under
tight regulation at both the level of activation of zymogens and the
level of transcription. These processes are under growth factor
regulation, with PMA being the most extensively studied regulator of
MMP activity. In human prostate cancer cells as well as in chondrocyte
matrix vesicles, vitamin D metabolites have been suggested to regulate
MMP-2 and MMP-9 activity (17
, 18)
.
The breast cancer cell line MDA-MB-231 is very invasive and
produces high amounts of uPA, PAI-1, and uPAR (9)
. These
cells also produce high constitutive levels of MMP-9 but undetectable
levels of MMP-2. Expression of MMP-1, MMP-3 (stromelysin-1), TIMP-1,
and TIMP-2 as well as MT1-MMP has been described previously (19
, 20) . The in vitro invasiveness of MDA-MB-231 cells
can be reduced by treatment with
1,25(OH)2D3, but the mechanisms of this
process are still unclear (21)
. Using zymography and
reverse zymography, we analyzed the effects of
1,25(OH)2D3 and its synthetic analogues on
the production of the components of the PA and MMP systems. To
characterize the mechanisms of regulation of uPA by
1,25(OH)2D3, we used uPA promoter constructs
that were transiently expressed in HT-1080 fibrosarcoma cells. In
addition, MT1-MMP expression and activity were analyzed in MDA-MB-231
cells. We report here that deltanoids are potent down-regulators of MMP
and PA activity in MDA-MB-231 cells, most likely influencing their
invasive behavior.
Results
Regulation of the PA System by
1,25(OH)2D3
Deltanoids are considered relatively new antitumor agents that
can influence tumor growth and progression by several mechanisms. They
are potent inhibitors of growth for many cancer cells and can also
decrease invasion and metastasis in in vitro models as well
as in mouse tumor models. Treatment with
1,25(OH)2D3 of estrogen receptor-negative,
highly invasive MDA-MB-231 breast carcinoma cells results in a
prominent decrease in their in vitro invasive potential.
This was shown not to be based exclusively on its antiproliferative and
antimigratory effects (21)
. Later it was found that the
in vitro invasive capacity of this cell line, as measured by
Matrigel invasion assay, was largely dependent on the cell surface uPA
system (9)
.
A caseinolysis in agar assay indicated that treatment of
MDA-MB-231 with 10-7 M
1,25(OH)2D3 cells under serum-free
conditions for 48 h decreased the secreted PA activity to 40% of
control values (Fig. 1A).
We then analyzed by zymography and reverse zymography the molecular
forms of PAs responsible for this effect. MDA-MB-231 cells secreted
both uPA and tPA into the culture medium (see also Fig. 2).
The amounts of PAs and the uPA:tPA ratio varied somewhat between
experiments, but uPA was found to be the prominent PA secreted into the
culture medium. 1,25(OH)2D3 down-regulated
secreted uPA activity in a concentration-dependent manner, as observed
after a 48-h incubation (Fig. 1B)
. The decrease in PA
activity was associated with a concomitant increase in secreted PAI-1
activity (Fig. 1B)
, leading to a prominent decrease in total
PA activity. Time dependence analysis of the effect indicated that a
decrease in uPA activity was already detectable after a 6-h treatment
and that the difference increased during the 48-h incubation (data not
shown).
|
|
,25(OH)2D3 induce
very modest (10–15%) growth inhibition in MDA-MB-231 cells (data not
shown; Ref. 16
), indicating that this breast carcinoma
cell line is insensitive to the antiproliferative effects of
deltanoids. This does not result from the lack of VDR, although
MDA-MB-231 cells have been shown to express low levels of VDR
(23)
.
MDA-MB-231 cells were incubated with increasing concentrations of
the analogues under serum-free conditions for 48 h. Zymographic
analysis of the conditioned media indicated that all three analogues
reduced the levels of both secreted uPA and tPA activity with a potency
at least 100 times higher than that of
1,25(OH)2D3 (Fig. 2)
. CB1093 was found to be
the most potent analogue in this respect. Even 10-10
M concentrations of CB1093 resulted in a prominent decrease
in secreted PA activity (Fig. 2A
, media). CB1093
was also a potent inducer of PAI-1 activity. The concentration-response
curve was different from that of uPA; PAI-1 was prominently induced in
a bell-shaped manner within a concentration range from
10-10 to 10-8 M, but not with
higher concentrations (Fig. 2A
, bottom panel).
Cell surface-associated uPA activity was analyzed by eluting
receptor-bound uPA with acidic buffer, followed by neutralization and
analysis by zymography (acid eluates). A decrease in cell surface-bound
uPA by CB1093 was evident and probably resulted from decreased
secretion of uPA as well as increased PAI-1 levels. At high
concentrations of CB1093, the cell surface-associated uPA activity
increased again, probably reflecting lower PAI-1 activity. Higher
molecular weight lysis zones in the zymogram represent the uPA/PAI-1
and tPA/PAI-1 complexes. Fig. 2B
illustrates the
down-regulation of secreted uPA and tPA by increasing concentrations of
EB1089 and KH1060.
Regulation of Invasion through Matrigel by Vitamin D Analogues
The effects of vitamin D analogues on the in vitro invasive
capacity of MDA-MB-231 cells were analyzed using Matrigel-coated
membranes (see "Materials and Methods"). In accordance with
previous reports, we found that the cells needed a preincubation with
vitamin D compounds to observe a decrease in their invasion through
Matrigel. Cells were treated with 10-7 M
concentrations of vitamin D analogues for 3 days before a 20-h cell
invasion assay. MDA-MB-231 cells are highly invasive, and
1,25(OH)2D3 and the analogues decreased cell
invasion to approximately half of the control level (Fig. 3).
Concentrations lower than 10-7 M of the
vitamin D compounds did not decrease the invasive capacity of
MDA-MB-231 cells. Unexpectedly, CB1093, EB1089, and KH1060 were not
found to be superior to 1,25(OH)2D3 in this
experimental design. Variability in the length of the experiment,
vitamin D concentration, and composition of Matrigel as well as effects
of vitamin D compounds on cell migration and cell adhesion probably add
to the variability of results in in vitro invasion assays.
Prostate cancer metastasis in vivo has been found to be
markedly and equivalently inhibited by
1,25(OH)2D3 and EB1089 (24)
.
However, EB1089 was significantly less calcemic, which makes it a
better candidate for cancer treatment.
|
,25(OH)2D3
and CB1093,25(OH)2D3 was
rather rapid and mediated through mRNA regulation (15)
.
MDA-MB-231 cells were treated with increasing concentrations of
1,25(OH)2D3 or CB1093 under serum-free
conditions for 4 or 18 h, and then uPA mRNA levels were analyzed
by Northern blotting (Fig. 4).
Short treatment with vitamin D analogues did not change uPA mRNA
levels, but after an 18-h incubation, a clear concentration-dependent
decrease was observed. CB1093 was clearly more potent in the regulation
of uPA mRNA, as suggested by data from activity assays. These results
suggest that vitamin D can regulate uPA at the mRNA level in MDA-MB-231
cells and that the kinetics of regulation depends on the cell type.
|
,25(OH)2D3-mediated regulation of
transcription (25)
. We analyzed the interaction of these
two interactive receptor systems on PA activity secreted by MDA-MB-231
cells. Treatment of cells with 9-cis-RA (10-6
M) in combination with CB1093 (10-9
M) did not affect the deltanoid-induced down-regulation of
uPA activity (Fig. 5).
This suggests that RXR ligand is not involved in the regulation of uPA
by CB1093. RA (10-6 M) alone and in
combination with CB1093 decreased both secreted and cell
surface-associated uPA levels.
|
,25(OH)2D3 Regulation of uPA Promoter
Activity,25(OH)2D3, followed by CAT activity
measurements. The corrected CAT activity (see "Materials and
Methods") results are expressed as a percentage of untreated control.
The activity of the full-length uPA promoter construct was
down-regulated by 1,25(OH)2D3 treatment
(Fig. 6).
The -1870 and -818 constructs were not responsive to
1,25(OH)2D3, but the basal level of activity
was decreased or increased, respectively. Similar results were obtained
in transfected MK-2 mouse keratinocytes (data not shown). These results
suggest that the 1,25(OH)2D3-responsive
regulatory regions in the uPA promoter are located between nucleotides
-2350 and -1870.
|
,25(OH)2D3 and CB1093 Regulation of
Gelatinolytic MMP Activity
Treatment of cells with 10-7 M
1,25(OH)2D3 under serum-free conditions for
48 h decreased the secreted MMP-9 activity consistently to
one-third of the control levels (Fig. 7)
. CB1093 decreased MMP-9 levels
at concentrations less than 1% of those of
1,25(OH)2D3. Maximal effect was seen at
10-9 M concentration of CB1093. CB1093 was
also able to strongly induce the secretion of TIMP-1 activity, with a
bell-shaped response curve similar to the PAI-1 response compare (Fig. 7
with Fig. 2
). TIMP-2 was also induced, but to a much lower extent.
These results suggest that deltanoids can influence the MMP/TIMP
balance in breast carcinoma cells in addition to modulating the PA
system.
|
MDA-MB-231 cells express large amounts of MT1-MMP mRNA as well as
protein. We analyzed whether 1,25(OH)2D3
could alter MT1-MMP production or activation in these cells. A 24-h
treatment of cells with increasing concentrations of
1,25(OH)2D3 under serum-free conditions did
not affect MT1-MMP mRNA levels (data not shown). PMA (4
nM), alone or in combination with
1,25(OH)2D3 (10-7
M), did not affect MT1-MMP protein levels when total cell
lysates were analyzed by immunoblotting after a 24-h treatment. The
Mr 60,000 MT1-MMP was the predominant form in
MDA-MB-231 cells, and PMA did not induce the appearance of the known
prominent cleavage product (Mr 43,000 form), as
seen in control HT-1080 cells (Fig. 8A).
1,25(OH)2D3, when added in combination with
PMA, did not affect the formation of the Mr
43,000 form in HT-1080 cells (data not shown). The band of
Mr 50,000 is nonspecific because blotting with
preimmune rabbit serum resulted in the detection of the same band
(27)
. Immunoblotting analysis of purified membrane
fractions of MDA-MB-231 cells revealed low levels of the inactive
Mr 63,000 form in untreated cells (Fig. 8B)
, suggesting that although MDA-MB-231 cells produce
considerable amounts of the Mr 60,000 form, it
does not remain membrane associated during purification from untreated
cells. PMA induced the appearance of very low levels of the
Mr 60,000 active MT1-MMP, whereas ConA induced a
clear increase in membrane-associated Mr 60,000
MT1-MMP. 1,25(OH)2D3 did not have any
detectable effects on MT1-MMP production or activation. These results
are in accordance with the results of Yu et al.
(20)
, which suggest that ConA but not PMA can induce
MT1-MMP-dependent MMP-2 processing in MDA-MB-231 cells.
|
There are several steps in tumor progression that could possibly be regulated by deltanoids. They are potent growth inhibitors for cells of epithelial origin, and this is achieved by induction of cell cycle arrest as well as apoptosis (2 , 31) . They also induce differentiation, which results in a less motile and thus less malignant phenotype. Cell-matrix contacts mediated by integrins are often changed in cancer cells (32 , 33) . Deltanoids have been shown to regulate the expression of certain integrins and their ligands. At least laminin and its receptor, fibronectin, and tenascin-C are regulated by vitamin D, with modification of expression depending on the cell type (34, 35, 36, 37) . In addition, deltanoids regulate the expression and activity of various growth factors that modulate the proliferative and angiogenic properties of cells. Induction of transforming growth factor ß and its receptor mediates at least in part the growth-inhibitory response in human keratinocytes and in breast carcinoma cells (38, 39, 40) . Recently, an interplay between transforming growth factor ß and vitamin D signaling pathways through SMAD transcriptional coactivators has been found (41) . Vitamin D has also been suggested to inhibit the motility and metastasis of Lewis lung carcinoma tumors via inhibition of granulocyte-macrophage colony-stimulating factor production (42) . In addition, induction of the expression of insulin-like growth factor-binding protein 3 is associated with growth-inhibitory response in some breast cancer cell lines (43) .
We report here that vitamin D and its analogues are potent regulators of the activity of invasion-associated proteases. Involvement of vitamin D in the regulation of the PA system has been reported in human keratinocytes, rat osteogenic sarcoma cells, and U-937 mononuclear phagocytes (15 , 44 , 45) . Like growth factor regulation of the PA system in general, the response to vitamin D is cell-type specific. In keratinocytes, vitamin D down-regulates both uPA and PAI-1, whereas in sarcoma cells and phagocytes, enhanced PA activity is associated with down-regulation of PAI-1 or PAI-2, respectively. Our new data indicate that in MDA-MB-231 breast carcinoma cells, vitamin D down-regulates uPA and up-regulates PAI-1, leading to a decrease in the secreted PA activity. Reduced PA activity is probably the main mechanism by which vitamin D inhibits invasiveness of this particular cell line because the results of Holst-Hansen et al. (9) suggest that the in vitro invasiveness of MDA-MB-231 cells is dependent on uPA activity.
Loss of cell-cell contacts through loss of E-cadherin expression is typical for an invasive cancer cell. It has been shown in breast carcinoma cells that blockade of E-cadherin-dependent adhesion induces the production of uPA, which could facilitate tumor cell invasion (46) . High levels of uPA in patients with breast tumors correlate with shorter disease-free interval and overall survival (47 , 48) . The therapeutic down-regulation of uPA levels might be beneficial because studies using monoclonal antibody against urokinase or antisense oligonucleotides show reduced invasion and metastasis of tumor cells in mice (49 , 50) . The role of the uPA system in the metastatic cascade is not clear, but obviously it provides localized extracellular matrix degrading activity as well as increased migratory capacity for tumor cells. The presence of uPAR at focal contacts serves to localize plasmin production to special sites at cell surfaces. Immunohistochemical studies suggest that components of the uPA system are located at the invasive edge of cancers and also in the associated stromal cells (51) . Production of plasmin may also release and activate growth factors important for tumor growth and angiogenesis. uPA levels in breast carcinomas have been shown to correlate with microvascular density, suggesting the involvement of the uPA system in neovascularization of tumors (52) .
As in human keratinocytes (15)
,
1,25(OH)2D3 regulation of uPA was found to
be at the transcriptional level in HT-1080 cells used in the promoter
assays. HT-1080 fibrosarcoma cells were selected for the assays because
of good transfection efficiency. These cells are responsive to vitamin
D treatment, and their in vitro invasiveness is reduced by
vitamin D (37)
. More detailed analysis of the uPA promoter
activity suggested that the responsive regulatory region is between
nucleotides -2350 and -1870 in the 5' end of the promoter. No known
vitamin D-inhibitory sequences were found from this region. Therefore,
we do not know whether VDR binds directly to the uPA promoter. The 5'
end of the uPA promoter contains at least Sp-1, c-ets-1,
cAMP-responsive element-binding protein, CRE-BP1/c-jun, and two AP-1
sites, of which one is important for the basal expression of uPA
(26)
. Interference between AP-1 and VDR on osteocalcin
gene expression has been described in human osteosarcoma cells
(53)
.
1,25(OH)2D3 has shown good promise for
treatment of cancer patients, but a major problem for use has been its
hypercalcemia-inducing capacity. Therefore, several vitamin D analogues
with lesser hypercalcemia-inducing properties have been synthesized.
The functional analyses of these analogues have resulted in the
discovery of several analogues with potent antiproliferative effects in
association with lower calcaemic capacity. Three analogues with side
chain modifications, CB1093, EB1089, and KH1060, were found here to be
at least 100 times more potent that
1,25(OH)2D3 in the regulation of uPA
activity. EB1089 and CB1093 are potent in the inhibition of breast
cancer cell proliferation and in producing regression of experimental
mammary tumors (22)
. CB1093 has been found to be 10 times
more potent than EB1089 in inducing apoptosis in MCF-7 breast carcinoma
cells. The potencies of the analogues in reducing uPA activity
correlate well with their potencies in inducing apo-ptosis. Results
from Danielsson et al. (22)
suggest that CB1093
shows a preference for the activation of DR-3-type VDREs, whereas
EB1089 selectively activates IP9-type VDREs. It will be interesting to
find whether uPA promoter has some kind of a VDRE.
Degradation of basement membrane collagen is essential for tumor cell invasion and spread. MMPs are capable of degrading collagen, and their expression is often increased during tumor progression. MMP-9 and MMP-2 can degrade type IV collagen, which is a major component of basement membranes. MDA-MB-231 cells were found to secrete MMP-9 activity, which was down-regulated by treatment with vitamin D or CB1093. In human prostate cancer cells as well as in human mononuclear phagocytes, vitamin D has been reported to reduce MMP-9 activity (18 , 54) . A concomitant increase in the secretion of TIMP-1 and, to a slightly lower extent, TIMP-2 was observed in MDA-MB-231 cells, indicating that vitamin D can change the MMP/TIMP balance in breast carcinoma cells, probably contributing to the less invasive phenotype. TIMP-1 levels have been shown to correlate with the invasive capacity of tumor cells (55 , 56) . Recent studies have suggested that MMPs have a role not only in the breakdown of physical barriers but also in the regulation of tumor growth (for review, see Ref. 6 ). The mechanisms may involve the generation of active matrix fragments as well as the release and activation of growth factors leading to modulation of the growth environment (57) .
Expression of MT1-MMP in breast carcinomas correlates with the presence
of lymph node and distant metastasis, clinical stage, and size of
tumors (30)
. MT1-MMP plays a key role in the activation of
pro-MMP-2. In human breast carcinoma tissues, MMP-2 mRNA is
predominantly localized to the stroma, possibly requiring a tumor
component for activation (58)
. Soluble factors produced by
tumor cells may also induce fibroblast production of MT1-MMP and
consequent MMP-2 activation, as suggested by Polette et al.
(59)
. MDA-MB-231 cells express high levels of MT1-MMP mRNA
and activate exogenously added MMP-2 in response to ConA treatment
(20)
. We found that MT1-MMP mRNA levels were not affected
by vitamin D in MDA-MB-231 cells. The predominant form produced by the
cells was the active Mr 60,000 form, but it was
not localized to the membrane fraction unless cells were treated with
ConA. 1,25(OH)2D3 had no detectable effect
on MT1-MMP production or activation, indicating that its interference
with MMP-2 activation was not probable.
Our studies indicate that deltanoids are potent regulators of invasion-associated proteases. In both the PA and the MMP systems, vitamin D decreased the production of proteases and increased the production of their inhibitors. Potent vitamin D analogues in combination with tamoxifen or cisplatin will probably prove to be a good choice for the treatment of breast cancer patients, as in vitro work from Koshizuka et al. (60) and Vink-van Wijngaarden et al. (61) suggests.
Materials and Methods
Reagents
1,25(OH)2D3 and its synthetic analogues
EB1089, KH1060 (62)
, and CB1093 (22)
were
obtained from Leo Pharmaceutical Products (Copenhagen, Denmark). The
uPA-CAT constructs -2350, -1870, and -818 (26)
were
kindly provided by Prof. Francesco Blasi (H. S. San Raffaele
Scientific Institute, Milan, Italy). Polyclonal rabbit antibodies
against MT1-MMP were used as described previously (27)
.
Cell Culture
MDA-MB-231 breast carcinoma cells were obtained from Prof. R. Vihko
(University of Oulu, Oulu, Finland) and cultured in RPMI 1640
containing 10% FCS (Life Technologies, Inc., Rockville, MD), 100
units/ml penicillin, and 50 µg/ml streptomycin. HT-1080 human
fibrosarcoma cells were obtained from American Type Culture Collection
(Manassas, VA) and cultured in MEM containing 10% heat-inactivated FCS
and the above-mentioned antibiotics.
Caseinolysis Assays
Caseinolysis in agarose assays were carried out as described previously
(63)
. The caseinolysis gels contain plasminogen
(Cromogenix, Mölndal, Sweden) and casein in 1.2% agarose (FMC
BioProducts, Rockland, MD). Plasminogen, when activated by the PA
present in the medium sample, degrades casein and forms a clear zone of
caseinolysis in the gel during the sample diffusion proportional to the
PA activity of the sample and time of diffusion. Different dilutions of
human high molecular weight uPA (Calbiochem, La Jolla, CA) were used to
draw standard plots in which PA activity (in international units) is
plotted against the diameter of the lytic sphere, which describes the
area of the zone of clearing.
Zymographic assays were used to identify the molecular forms of PAs and PAI-1 (64) . Medium samples or acid eluates [cells treated with 50 mM glycine-HCl buffer (pH 3.0) containing 0.1 mM NaCl at room temperature for 3 min] were first electrophoresed in 4–15% gradient polyacrylamide gels containing SDS (Bio-Rad, Hercules, CA) under reducing (for reverse zymography) or nonreducing (for zymography) conditions. SDS was removed by extensive washing with PBS/Triton X-100 (2.5%). For reverse zymography, uPA (2 IU/ml) was added to the final wash for 15 min. The gels were then placed on caseinolysis gels and incubated at 37°C for 24 h. The position of PAs in the SDS gel was shown as lysis of the indicator gels. The position of PAI-1 was shown as lysis-resistant opaque band.
Invasion Assay
MDA-MB-231 cells were incubated with vitamin D analogues for 48 h
in growth medium, after which serum-free medium supplemented with
vitamin D analogues was changed for 24 h. Cells were harvested and
counted, and 0.5 x 105 cells/chamber was used for
each invasion assay. Cell culture inserts in a 24-well plate (Becton
Dickinson Labware, Bedford, MA) were coated with 85
µg/cm2 growth factor-reduced Matrigel (Becton
Dickinson Labware) for 3 h at 37°C. Unbound material was
aspirated, and the chambers were rinsed gently with serum-free media
before the addition of cells. Cells were added to coated inserts in
serum-free media containing vitamin D analogues. The lower chambers
contained medium with 5% FCS and vitamin D analogues. The inserts were
incubated for 20 h at 37°C. The cells that had invaded to the
lower surface of the membranes were fixed and stained with Dade
Diff-Quick stain kit (Dade AG, Düdingen, Switzerland), and
random fields were counted under a light microscope. The decreased
number of invasive cells is expressed as a percentage of untreated
cells.
Gelatin Zymography and Reverse Zymography
To analyze the gelatinolytic proteins in cell-conditioned medium,
samples were run under nonreducing conditions in 10%
SDS-polyacrylamide gels containing 2 mg/ml gelatin. For reverse
zymography, samples were run under nonreducing conditions in 12%
SDS-polyacrylamide gels containing 2 mg/ml gelatin and 40 ng/ml
gelatinase A (Chemicon International Inc., Temecula, CA). After
electrophoresis, the gels were washed twice with 50 mM
Tris-HCl buffer (pH 7.6) containing 5 mM CaCl2,
1 µM ZnCl2, and 2.5% Triton X-100 (v/v) for
15 min to remove SDS, followed by a brief rinsing in washing buffer
without Triton X-100. The gels were then incubated at 37°C for 24–48
h in washing buffer containing 1% Triton X-100. After incubation, the
gels were stained with Coomassie Brilliant Blue R250 (Bio-Rad) and
destained with 10% acetic acid.
Transfection and CAT Assay
HT-1080 cells were transfected with different uPA-CAT constructs by
electroporation (65)
. Cotransfection with pCMVß-gal
plasmid (Clontech, Palo Alto, CA) was performed as an internal control.
Cells were transfected with 3 µg of pCMVß-gal + 16 µg of
promoter-CAT constructs using 400 V and 960 µF. The following day,
the transfected cells were treated with
1,25(OH)2D3 for 24 h, and then cells
were harvested, and CAT and ß-galactosidase activities were measured
according to standard protocols (65)
. CAT activities were
corrected for ß-galactosidase activity.
RNA Isolation and Northern Blotting
Total cellular RNA was purified from cultured cells using the RNeasy
total RNA kit (Qiagen, Hilden, Germany). For Northern blot analysis, 10
µg of RNA were fractionated on 1.2% agarose gels containing
formaldehyde and transferred to Hybond-N nylon membranes (Amersham
Pharmacia Biotech, Uppsala, Sweden) by capillary transfer.
Prehybridization and hybridization were performed at 68°C in
ExpressHyb hybridization solution (Clontech). cDNA probes for MT1-MMP,
uPA, and G3DPH were labeled with [32P]dCTP (>3000
Ci/mmol; Amersham Pharmacia Biotech) using a Rediprime Labeling Kit
(Amersham Pharmacia Biotech). Radioactivity levels were quantified with
a BAS-2500 bioimaging analyzer (Fuji, Tokyo, Japan).
Cell Lysis and Immunoblotting
The cells were lysed with Triton lysis buffer [50 mM
Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 1% (v/v)
Triton X-100, 10 mM EDTA, 10 µg/ml aprotinin, 1 µg/ml
pepstatin A, and 1 µg/ml aminoethylbenzenesulphonyl fluoride] on
ice, and the lysates were clarified by centrifugation. Cell membrane
extracts were prepared as described previously (27)
. For
immunoblotting, aliquots of cell lysates or membrane extracts were
electrophoresed in 4–15% gradient SDS-polyacrylamide gels, and
proteins were electrophoretically transferred to Protran nitrocellulose
membranes (Schleicher & Schuell, Dassel, Germany). Immunodetection of
MT1-MMP was performed as described previously (27)
.
Acknowledgments
We thank Dr. Lise Binderup (Leo Pharmaceutical Products, Copenhagen, Denmark) for providing the deltanoids and Sami Starast for fine technical assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by the Academy of Finland, the Finnish
Cancer Foundation, Sigrid Juselius Foundation, Novo Nordisk Foundation,
Helsinki University Hospital Fund, Biocentrum Helsinki, and the
University of Helsinki. 
2 To whom requests for reprints should be
addressed, at department of Virology, P. O. Box 21, Haartman
Institute, University of Helsinki, Haartmaninkatu 3, FIN-00014
Helsinki, Finland.
3 The abbreviations used are: uPA, urokinase
plasminogen activator; tPA, tissue-type plasminogen activator; PA,
plasminogen activator; PAI, PA inhibitor; MMP, matrix
metalloproteinase; TIMP, tissue inhibitors of metalloproteinases; RXR,
retinoid X receptor; RA, retinoic acid;
1,25(OH)2D3, 1,25-dihydroxyvitamin
D3; VDR, vitamin D receptor; uPAR, uPA receptor; MT-MMP,
membrane-type MMP; ConA, concanavalin A; PMA, phorbol 12-myristate
13-acetate; CAT, chloramphenicol acetyltransferase; VDRE, vitamin
D-responsive element; G3DPH, glyceraldehyde-3-phosphate
dehydrogenase.
Received for publication 9/ 7/99. Accepted for publication 3/ 3/00.
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Mr 50,000 band represents a nonspecific band