Báo cáo khoa học: Inhibition of human MDA-MB-231 breast cancer cell invasion by matrix metalloproteinase 3 involves degradation of plasminogen docx

Inhibition of human MDA-MB-231 breast cancer cell invasion
by matrix metalloproteinase 3 involves degradation of plasminogen
Antonietta R. Farina
1
, Antonella Tacconelli
1
, Lucia Cappabianca
1
, Alberto Gulino
2
and Andrew R. Mackay
1
1
Section of Molecular Pathology, Department of Experimental Medicine, University of L’Aquila, Italy;
2
Department of Experimental
Medicine and Pathology, University of Rome ‘La Sapienza’, Italy
Matrix metalloproteinase (MMP)-3 inhibited human
MDA-MB-231 breast cancer cell invasion through recon-
stituted basement membrane in vitro. Inhibition of invasion
was dependent upon plasminogen and MMP-3 activation,
was impaired by the peptide MMP-3 inhibitor Ac-Arg-Cys-
Gly-Val-Pro-Asp-NH
2
and was associated with: rapid
MMP-3-mediated plasminogen degradation to microplas-
minogen and angiostatin-like fragments; the removal of
single-chain urokinase plasminogen activator from MDA-
MB-231 cell membranes; impaired membrane plasminogen
association; reduced rate of tissue plasminogen activator
(t-PA) and membrane-mediated plasminogen activation;

and reduced laminin-degrading capacity. Purified human
plasminogen lysine binding site-1 (kringles 1–3) exhibited a
similar capacity to inhibit MDA-MB-231 invasion, impair
t-PA and cell membrane-mediated plasminogen activation
and impair laminin degradation by plasmin. Our data pro-
vide evidence that MMP-3 can inhibit breast tumour cell
invasion in vitro by a mechanism involving plasminogen
degradation to fragments that limit plasminogen activation
and the degradation of laminin. This supports the hypothesis
that MMP-3, under certain conditions, may protect against
tumour invasion, which would help to explain why MMP-3
expression, associated with benign and early stage breast
tumours, is frequently lost in advanced stage, aggressive,
breast disease.
Keywords: angiostatin-like; invasion; laminin; matrix
metalloproteinase-3; plasminogen.
The transition from carcinoma in situ to invasive adeno-
carcinoma of the breast is a relevant index of malignant
behaviour and is characterized by loss and fragmentation
of the ductal basement membrane (BM) [1,2]. Invasive
breast tumour behaviour is associated with matrix-degra-
ding enzyme over-expression, considered to be responsible
for the promotion of the proteolytic environment required
for destabilization and fragmentation of the ductal BM
[3–9]. The invasive process has been effectively modelled
in vitro using a reconstituted BM matrix prepared from
mouse Engelbreath-Holm-Schwarm (EHS) sarcoma
[10–16].
A general concept is that a proteolytic cascade involving
matrix metalloproteinases (MMPs) and the plasmin system

degrades BM structures as a prerequisite for tumour cell
invasion [17–19]. Plasmin activated from plasminogen by
urokinase and tissue type plasminogen activators (uPA and
t-PA, respectively) amplified at the tumour cell or matrix
surface, degrades BM components and activates MMPs
resulting in further amplification of BM degradation
[17–21]. Amongst the MMP family, MMP-3 (stromelysin-1)
is considered pivotal to the integration of plasmin and
MMP systems, as it is highly sensitive to activation by
plasmin and activates several MMPs. In addition, MMP-3
exhibits substrate specificity for BM components and has
been implicated directly in both tumorigenesis and tumour
invasion in vivo [19,22–24].
Recently, however, there has been a change in opinion
about the potential roles played by MMPs in tumour
progression [25]. This has followed observations that
patterns of MMP over-expression do not always predict
aggressive tumour behaviour in either human [19,26,27] or
animal tumours [28], that enhanced expression of the MMP
inhibitors TIMP-1 and TIMP-2 frequently associates with
poor outcome or tumour recurrence [29,30] and that MMPs
can degrade components of the plasmin generating system
and produce angiostatin, suggesting potential roles for
MMPs in the regulation of cellular fibrinolytic activity and
in the down-regulation of tumour-associated angiogenesis
[31–38].
Human breast tumours exhibit some of these anomalies.
MMP-3 is expressed by benign and low stage tumours but
its expression is frequently lost in association with aggres-
sive, advanced stage, disease [26,27]. TIMP-1 and TIMP-2

over-expression associates with malignant breast tumour
behaviour in vivo [29,30]. Although MMP-3 expression by
benign breast tumours is consistent with a potential role in
tumorigenesis [23], the loss of its expression associated with
progressive disease raises an important question concerning
its role in breast tumour progression. In the present study,
we address this point by assessing the role of MMP-3 in an
established model of malignant human MDA-MB-231
Correspondence to A. R. Mackay, Section of Molecular Pathology,
Department of Experimental Medicine, University of L’Aquila,
67100, L’Aquila, Italy.
Fax: +39 862 433523, Tel.: +39 862 433542,
E-mail:
Abbreviations: BM, basement membrane; t-PA, tissue-type plasmi-
nogen activator; scuPA, single-chain urokinase plasminogen activator;
tcuPA, two-chain urokinase; MMP, matrix metalloproteinase;
APMA, amino-phenyl-mercuric acetate; EHS, Engelbreth-
Holm-Schwarm; PBL1, plasminogen lysine binding site-1;
EMEM, minimal essential medium with Earl’s salts.
(Received 6 June 2002, accepted 25 July 2002)
Eur. J. Biochem. 269, 4476–4483 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03142.x
breast cancer cell invasion in vitro [10–16]. We report that
conditions exist under which MMP-3 inhibits breast tumour
cell BM invasive capacity in vitro and we discuss the
potential mechanism involved.
MATERIALS AND METHODS
Cells and reagents
Human MDA-MB-231 breast carcinoma cells were obtained
from the American Tissue Culture Collection and grown in
ATCC recommended media supplemented with 10% foetal

calf serun (Hyclone, defined) glutamine, penecillin and
streptomycin. EHS Matrigel, EHS laminin and EHS type
IV collagen-coated microtitre plates were from Becton
Dickinson. Purified human recombinant pro-MMP-3 was
kindly provided by PoliFarma (Rome, Italy). Glu-plasmi-
nogen, t-PA, two-chain urokinase plasminogen activator
(tcuPA), peptide MMP-3 inhibitor (Ac-Arg-Cys-Gly-Val-
Pro-Asd-NH
2
), peptide MMP inhibitor-1 (4-Abz-Gly-
Pro-D-Leu-D-Ala-NHOH) and recombinant human
pro-MMP-9 were from Calbiochem. Plasmin substrate
(Val-Leu-Lys-pNA), purified plasminogen lysine binding
site-1 (PBL-1; kringle domains 1–3), anti-catalytic anti-
plasminogen antibody (P5276) [14], amino-phenyl-mercuric
acetate (APMA) and EDTA were from Sigma. The anti-
human MMP-3 antibody has been previously described [14].
Matrigel invasion assays
Invasion through reconstituted BM Matrigel was per-
formed as described previously [13,14]. Briefly, 8 lm
Transwell membranes (Costar) coated with Matrigel
30 lg per 6-mm filter diluted in serum-free minimal
essential medium with Earl’s salts (EMEM), were air dried
and reconstituted for 2 h in EMEM prior to invasion
assay. MDA-MB-231 cells grown to subconfluence were
detached by trypsinization, washed three times in NaCl/P
i
,
resuspended in (EMEM/0.1% BSA with antibiotics) and
used in invasion assays at a final concentration of 0.5 · 10

6
cellsÆml
)1
in the presence or absence of plasminogen
(5 lgÆmL
)1
). Cell suspensions were added to the upper
well of invasion chambers and assays were incubated at
37 °C for 16 h. Cells that had traversed the filter were
counted by light microscopy, following removal of surface
adherent cells and staining with Hematoxylin. Exogenous
enzymes and inhibitors were added directly to invasion
medium and added to the upper well at the concentrations
stipulated.
Substrate gel electrophoresis
Regular gelatin, casein and plasminogen activator zymo-
grams were prepared as described previously [14]. Briefly,
samples were subjected to SDS/PAGE, under nonreducing
conditions, in gels copolymerized with either 0.1% gelatin,
0.1% casein or 0.1% casein plus 5 lgÆmL
)1
plasminogen.
Following electrophoresis, gels were washed in 2% triton
X-100, rinsed in water and incubated in incubation buffer
(50 m
M
Tris, 200 m
M
NaCl, 5 m
M

CaCl
2
pH 8.0). Plasmi-
nogen activator zymograms were incubated in incubation
buffer without CaCl
2
in the presence of 15 m
M
EDTA, to
inhibit endogenous metalloproteinase activity. Enzyme and
inhibitor activities were visualized by staining with Coo-
massie blue in a mixture of methanol : acetic acid : water
(4/1/5, v/v/v) and destaining in the same mixture without
Coomassie blue.
Western blots
Samples, separated by SDS/PAGE under reducing and
nonreducing conditions were electrophoretically transferred
to nitrocellulose (Hybond C-extra, Amersham Interna-
tional). Nonspecific protein binding sites were blocked by a
solution of 5% nonfat dried milk in NaCl/Pi. Membranes
were incubated with primary antibody diluted in blocking
solution and with horse radish peroxidase conjugated
secondary antibody diluted in blocking solution (Bio-Rad).
Antigen reactivity was demonstrated by chemiluminescence
reaction (Amersham International). Immunoreactive bands
were visualized on XAR-5 film (Kodak). Molecular weights
were approximated by comparison to prestained molecular
weight markers (Bio-Rad) using Molecular AnalystTM/PC
for the Bio-Rad Model GS-670 Imaging Densitometer.
Specificity of antibodies was assessed using preimmune IgG

preparations.
Purification of plasma membranes
Washed cell membrane fractions were prepared by scraping
cells into buffer containing 0.15
M
NaCl, 20 m
M
Hepes,
2m
M
CaCl
2
, 100 lgÆmL
)1
leupeptin, 2.5 mgÆmL
)1
pepsta-
tin A and 1 m
M
phenyl methyl sulfonylflouride (pH 8.0).
Cells were disrupted by cycles of freezing and thawing in
liquid nitrogen and at 42 °C. Nuclei were removed by
centrifugation at 7000 g for 20 min at 4 °C and membranes
obtained by ultracentrifugation at 100 000 g for 1 h at 4 °C;
membranes were then washed three times in 3 mL buffer,
the last wash without inhibitors, before resuspension in
either 1 · SDS/PAGE sample buffer for analysis in zymo-
grams or in 50 m
M
Tris, 200 m

M
NaCl, 5 m
M
CaCl
2
for
enzyme assays. Cell membranes were resuspended in
nonreducing SDS/PAGE sample buffer for zymograms or
in 100 m
M
Tris/0.5% Triton X-100 (pH 8.8) for plasmino-
gen activator assays.
Caseinase assays
Caseinolytic metalloproteinase activity in supernatants was
assessed using
14
C-labelled a-casein. Briefly, substrates
(30 000 c.p.m.) were mixed with samples in a buffer
containing 20 m
M
Tris, 200 m
M
NaCl and 5 m
M
CaCl
2
(pH 8.0) in a final volume of 200 lL with or without the
MMP activator APMA (1 m
M
) or with or without EDTA

(15 m
M
). Assays were incubated at 37 °C for 24 h. All
assays were performed in the presence of plasmin inhibitory
concentrations of anti-catalytic anti-plasminogen antibody
(1 : 15 dilution) [14] and the presence or absence of EDTA
(15 m
M
), peptide MMP-3 inhibitor (10 l
M
)orpeptide
MMP inhibitor-1 (30 l
M
). Undegraded proteins were
precipitated with 10% trichloroacetic acid/0.5% tannic acid
(TA)andprecipitatedmaterialwasremovedbycentrifuged
at 5000 g for 15 min or subjected directly to centrifugation
(12 000 g for 10 min for type I collagen). Radioactivity in
the supernatants was counted in a Beta liquid scintillation
counter (Beckman model LS 5000TD). Characterization of
Ó FEBS 2002 MMP-3 inhibits MDA-MB-231 invasion (Eur. J. Biochem. 269) 4477
activity as MMP was determined by inhibition with EDTA
(15 m
M
).
Plasminogen activator assay
PA activity was assayed using the synthetic plasmin
substrate Val-Leu-Lys-pNA by a modification of a previ-
ously described method [14]. Briefly, samples were incuba-
tedfor0–3hat37°C, in 96-well microplates in a buffer

containing 100 m
M
Tris and 0.5% triton X-100 (pH 8.8),
3 lg of plasminogen and 25 lg of plasmin substrate in
a final volume of 100 lL. Reactions were monitored at
15-min intervals at 405 nm in a spectrophotometer.
Statistical analysis
Analyses were performed on mean values from replicate
experiments, as indicated, using the Student’s t-test. Results
were considered significant at P £ 0.05.
RESULTS
Inhibition of MDA-MB-231 invasion by MMP-3
Human MDA-MB-231 breast cancer cells constitutively
express, MMP-9, low level MMP-3, TIMP-1, TIMP-2, uPA
and t-PA but do not express MMP-2 [14].
In Matrigel invasion assays, exogenous plasminogen
augmented MDA-MB-231 invasivity by approximately 3.5-
fold (P < 0.001), at a physiologically relevant concentra-
tion of 5 lgÆmL
)1
(Fig. 1A), confirming our previous report
[14]. Percentages represent mean levels of invasion in 10
random high power microscopic field (magnification ·40)
per filter. To provide perspective to percentage values,
invasion in the absence of plasminogen exhibited a mean of
120 cells per filter whereas a mean of 410 cells per filter
invaded in the presence of plasminogen.
Exogenous recombinant pro-MMP-3 (0.1 and
1 lgÆmL
)1

) inhibited MDA-MB-231 invasion in the pres-
ence of plasminogen by 24% and 49% (significant at
P < 0.001), respectively (Fig. 1A) but did not modulate
MDA-MB-231 invasion in the absence of plasminogen
(data not shown). Recombinant pro-MMP-9 (1 lgÆmL
)1
)
augmented MDA-MB-231 invasion in the presence of
plasminogen by  30% (significant at P < 0.005)
(Fig. 1A), but did not significantly stimulate invasion in
the absence of plasminogen (data not shown).
Inhibition of MDA-MB-231 invasion is impaired
by a peptide MMP-3 inhibitor and is associated
with MMP-3 activation
We have previously shown that inhibitors of plasmin
activity, including the anti-catalytic anti-plasmin antibody
used in this study, abrogate the invasion stimulating effects
of plasminogen upon MDA-MB-231 cells, confirming
dependence upon plasmin activity [14]. MMP-3 inhibition
of MDA-MB-231 invasion in the presence of plasminogen
was impaired by the peptide MMP-3 inhibitor (10 l
M
)by
 60% (significant at P < 0.001) but was not impaired by
the peptide MMP inhibitor-1 (30 l
M
) (Fig. 1A). At the
concentrations used, peptide MMP-3 inhibitor (10 l
M
)

inhibited solution phase caseinolytic MMP activity
(Fig. 1B) and MMP-3 but not MMP-9 activity in
Fig. 1. Inhibition of MDA-MB-231 invasion by MMP-3. (A) Histogram depicting levels of MDA-MB-231 cell invasion through reconstituted
Matrigel in the presence (shaded bars) or absence (open bars) of plasminogen (5 lgÆmL
)1
), pro-MMP-3 or pro-MMP-9, at the concentrations
stipulated and peptide MMP-3 inhibitor (10 l
M
) or peptide MMP inhibitor-1 (30 l
M
). Results are expressed as a percentage difference with respect
to plasminogen independent invasion (± SD) of three independent experiments performed in quadruplet. (B) Western blot demonstrating MMP-3
species in conditioned media from invasion assays performed in the presence (+) or absence (–) of plasminogen and a histogram depicting solution
phase caseinolytic activity, assayed in the presence of EDTA (15 m
M
), peptide MMP-3 inhibitor (10 l
M
) or peptide MMP inhibitor-1 (30 l
M
), in
conditioned media from invasion assays performed in the presence of pro-MMP-3 (1 lgÆmL
)1
) and in the presence (+) or absence (–) of
plasminogen (Pg) (5 lgÆmL
)1
). (C) Gelatin and casein zymograms demonstrating the effect of peptide MMP-3 inhibitor (10 l
M
) and MMP
inhibitor-1 (30 l
M

) peptides on MMP-9 (100 ng) and MMP-3 (100 ng) activity.
4478 A. R. Farina et al. (Eur. J. Biochem. 269) Ó FEBS 2002
zymograms (Fig. 1C), whereas peptide MMP inhibitor-1 at
a concentration of 30 l
M
inhibited partial MMP-9 inhib-
itory activity in gelatin zymograms but did not inhibit
caseinolytic MMP activity in solution nor MMP-3 activity
in zymograms (Fig. 1C).
MMP-3 inhibition of plasminogen-dependent MDA-
MB-231 invasion was associated with the induction of
solution phase caseinolytic MMP activity, inhibited by
EDTA and peptide MMP-3 inhibitor (10 l
M
) but not by
MMP inhibitor-1 (30 l
M
), and was associated with the
conversion of 57 kDa pro-MMP-3 into doublet species of
approximately 45 kDa and 28 kDa, consistent with activa-
tion, as determined by Western blotting (Fig. 1B).
MMP-3 rapidly degrades plasminogen
Zymogram analysis of non-EDTA inhibitable (non-MMP)
caseinolytic activity in conditioned media from MDA-MB-
231 cells incubated for 6 h in the presence of plasminogen
(5 lgÆmL
)1
), pro-MMP-9 (1 lgÆmL
)1
) or pro-MMP-3

(1 lgÆmL
)1
), revealed marked differences in caseinolytic
activity in the presence of MMP-3, with the appearance of
caseinolytic species between 30 and 40 kDa; these caseino-
lytic species were present to a far lesser extent in samples
incubated in the absence of exogenous MMPs or in the
presence of pro-MMP-9 (Fig. 2A).
APMA activated MMP-3 (1 m
M
APMA for 3 h at
37 °C) rapidly degraded glu-plasminogen, at an
enzyme : substrate ratio of 1 : 100, to microplasminogen
( 30 kDa) and various nonenzymatically active kringle
fragments ranging from 15 to 55 kDa, within 6 h (Fig. 2B).
APMA activated MMP-9 did not degrade plasminogen
under the same conditions (Fig. 2B). However, limited
degradation of plasminogen by MMP-9 was detected at
96 h confirming a previous report [36] (data not shown).
MMP-3 degradation of plasminogen was not associated
with plasminogen activation, determined by a solution phase
plasmin substrate degradation assay (data not shown).
Plasminogen degraded by MMP-3 exhibits reduced
laminin-degrading capacity
Plasminogen degraded by MMP-3 for 6 h at 37 °C, prior to
being activated by tcuPA, exhibited a reduced capacity to
degrade EHS laminin, when compared to tcuPA-activated
intact plasminogen assayed under identical conditions. As
demonstrated in Fig. 2C, intact tcuPA-activated plasmino-
gen, at an enzyme substrate ratio of 1 : 100, almost

Fig. 2. MMP-3 degrades plasminogen, impairs plasminogen activation and impairs plasmin-mediated laminin degradation and laminin. (A) Repre-
sentative casein zymogram demonstrating non-MMP caseinolytic activity of serum-free conditioned medium from MDA-MB-231 cells incubated
with plasminogen (5 lgÆmL
)1
) in the presence or absence of MMP-9 or MMP-3 (100 ng mL
)1
) for 6 h. (B) Representative silver stained SDS/
PAGE gel and casein zymogram demonstrating species and associated caseinolytic activity of plasminogen incubated for 6 h in the presence of
APMA-activated (1 m
M
for 1 h at 37 °C) MMP-3 or MMP-9 (enzyme : substrate ratio of 1 : 100). (C) Representative Coomassie blue-stained
SDS/PAGE gel run under reducing conditions depicting molecular species present in preparations of laminin (30 lglane
)1
) incubated for 12 h in
the presence or absence of intact plasminogen (100 ng), plasminogen previously degraded for 6 h with MMP-3 (100 ng), tcuPA (1 ng), PBL-1
(10 lgÆmL
)1
) or anti-catalytic anti-plasminogen antibody (1 : 15 dilution). (D) Line graph demonstrating VLLpNA-degrading activity associated
with intact glu-plasminogen (1 lgÆmL
)1
)(s) and MMP-3-degraded plasminogen (1 lgÆmL
)1
; d) in the presence of t-PA (10 ng), type IV collagen
(coated plates), purified 100 000 g MDA-MB-231 cell membrane preparations containing the equivalent of 10 ng uPA activity and tcuPA (10 ng).
Results are expressed as mean (± SD) absorbance at 405 nm of duplicate experiments performed in quadruplet.
Ó FEBS 2002 MMP-3 inhibits MDA-MB-231 invasion (Eur. J. Biochem. 269) 4479
completely degraded 400 kDa and 200 kDa laminin species
within 12 h generating lower molecular mass species ranging
from 100 kDa to 150 kDa, as determined by Coomassie blue
stained SDS/PAGE. A marked reduction in the loss of both

200 kDa and 400 kDa laminin species followed incubation
for 12 h with tcuPA-activated plasminogen, previously
degraded by MMP-3. Assays were performed in the presence
of MMP inhibitory concentrations of EDTA (15 m
M
),
peptide MMP-3 inhibitor and peptide MMP inhibitor-1
(100 l
M
). Similar inhibition of laminin degradation was
observed when tcuPA-activated intact plasminogen was
assayed in the presence of PBL-1 (10 lgÆmL
)1
). Laminin
degradation was completely inhibited by anti-catalytic anti-
plasminogen antibody (1 : 15 dilution) (Fig. 2C).
Plasminogen degraded by MMP-3 exhibits reduced t-PA
and membrane PA-mediated activation
In solution phase plasmin substrate degradation assays,
plasminogen (3 lg) that had been previously degraded by
MMP-3, then activated by t-PA (100 ng) in the presence of
type IV collagen cofactor, or by partially purified MDA-
MB-231 cell membranes containing single-chain urokinase
plasminogen activator (scuPA) and t-PA activity (see Fig. 3;
activity equivalent to 100 ng uPA, approximated by casein
zymograms) exhibited a marked reduced rate of activation,
when compared with intact plasminogen activated by the
same PAs, over a 3-h time course. In contrast, tcuPA-
activated plasminogen, previously degraded by MMP-3, did
not exhibit reduced activity in plasminogen activation

assays when compared to tcuPA-activated intact plasmino-
gen (Fig. 2D).
MMP-3 removes scuPA from MDA-MB-231 cell
membranes, impairs membrane-associated plasminogen
activation and the plasminogen association with
the cell surface
Incubation of partially purified, 100 000 g,MDA-MB-231
cell membrane fractions, containing both t-PA and scuPA
activity, for 6 h with APMA-activated MMP-3 (1 lgÆmL
)1
)
at 37 °C resulted in the relative loss of 45 kDa scuPA
activity and the appearance of 30 kDa tcuPA activity in
subsequent membrane wash supernatants, assayed by casein
zymogram under MMP inhibitory conditions (15 m
M
EDTA) (Fig. 3A). Membranes treated with MMP-3 exhib-
ited significantly reduced plasmin substrate-degrading
activity upon incubation with intact plasminogen (3 lg)
assayed over a 3-h time course, when compared with
non-MMP-3-treated membranes processed in an identical
manner (Fig. 3B).
Incubation of MDA-MB-231 cells for 1 h at 37 °C
with intact plasminogen (10 lgÆmL
)1
) in the presence of
BSA (10 lgÆmL
)1
) resulted in cell–plasminogen associ-
ation, as determined by casein zymogram of washed cell

lysates. Cells incubated with plasminogen, previously
degraded by MMP-3 (12 h at 37 °C), did not bind
plasminogen fragments with caseinolytic activity
(Fig. 3C). MDA-MB-231 cells incubated with intact
plasminogen (10 lgÆmL
)1
), in the presence of PLB1
(10 lgÆmL
)1
), exhibited a marked reduction in plasmino-
gen-cell association as assessed by casein zymogram.
Caseinolytic activity was inhibited by anti-catalytic anti-
plasminogen antibody but not by EDTA (15 m
M
)(data
not shown).
Fig. 3. MMP-3 removes plasminogen activators from MDA-MB-231 cell membranes and impairs membrane plasminogen-activating capacity. (A)
Representative plasminogen activator zymograms depicting plasminogen activator activity associated with purified washed MDA-MB-231 cell
membranes (1 · 10
6
cells) incubated for 3 h at 37 °C with (+) or without (–) APMA-activated MMP-3 (1 lg) and in 10-fold concentrated washes
from purified MDA-MB-231 membranes preincubated for 3 h with (+) or without (–) APMA-activated MMP-3. (B) Line graph of VLL-pNA-
degrading activity associated with plasminogen activated by purified washed MDA-MB-231 membranes (1 · 10
6
cells) either preincubated (3 h at
37 °C) with (d) or without (s) APMA-activated MMP-3 (1 lg). Results are expressed as mean (± SD) absorbance at 405 nm of two independent
experiments performed in triplicate. (C) Representative casein zymogram demonstrating non-MMP caseinolytic activity associated with cell lysates
of MDA-MB-231 cells incubated for 1 h in the presence (lanes 2 and 4) or absence (lane 1) of intact plasminogen (10 lgÆmL
)1
; lane 2), plasminogen

predegraded by MMP-3 (10 lgÆmL
)1
; lane 3) and PBL-1 (10 lgÆmL
)1
;lane4).
4480 A. R. Farina et al. (Eur. J. Biochem. 269) Ó FEBS 2002
PBL-1 inhibits MDA-MB-231 invasion, impairs laminin
degradation, plasminogen activation and plasminogen
association with cell membranes
Plasminogen (5 lgÆmL
)1
), previously degraded by MMP-3
(6 h at 37 °C at an MMP-3 : plasminogen ratio of 1 : 100),
stimulated MDA-MB-231 invasion in the presence of
peptide MMP-3 inhibitor (10 l
M
) by 86% (significant at
P < 0.001) (Fig. 4A).
Purified human PBL-1 (kringles 1–3) did not contain
plasmin substrate-degrading activity, as determined by both
casein zymogram and plasmin substrate degradation assay
(data not shown). PLB-1 (1 and 10 lgÆmL
)1
) inhibited
MDA-MB-231 invasion, in the presence of plasminogen, by
 26% and  46% (P < 0.001), respectively (Fig. 4A) but
did not modulate invasion in the absence of plasminogen
(data not shown).
PLB1 (3 lg) inhibited plasmin substrate-degrading capa-
city of intact plasminogen (3 lg) activated by t-PA (100 ng)

in the presence of collagen type IV cofactor, or by cell
membrane-associated PAs (equivalent of 100 ng scuPA) but
did not inhibit the activity of soluble tcuPA-activated
plasminogen, assayed over a 3-h time course (Fig. 4B). As
stated above, PLB-1 impaired the laminin-degrading capa-
city of tcuPA-activated intact plasminogen (Fig. 2C) and
impaired the capacity of intact plasminogen to associate
with MDA-MB-231 cells (Fig. 3C).
DISCUSSION
In this study we report conditions in which MMP-3 can
inhibit tumour cell BM invasivity in an established model
of human breast cancer cell invasion in vitro. MMP-3
inhibition of invasion was dependent upon plasminogen,
involved MMP-3 activity and was associated with rapid
MMP-3 degradation of plasminogen to fragments that
exhibited reduced activation by t-PA and membrane
scuPA, reduced substrate specificity for laminin and
reduced capacity to associate with the MDA-MB-231 cell
surface. Furthermore, MMP-3 eliminated scuPA from
MDA-MB-231 cell membranes and reduced membrane-
mediated plasminogen activation. We highlight a poten-
tial role for PBL-1 in the mediation of several of these
effects.
Exogenous plasminogen augmented MDA-MB-231 BM
invasion in vitro, confirming our previous report [14].
Exogenous pro-MMP-3 exhibited dose-dependent inhibi-
tion of plasminogen-stimulated MDA-MB-231 invasion.
Invasion inhibition was associated with the activation of
both plasminogen and MMP-3, as determined by plasmin
substrate assay, solution phase caseinolytic MMP assay and

the conversion of pro-MMP-3 to molecular species of 45
and 28 kDa, consistent with activation [19]. As MMP-3
inhibition of MDA-MB-231 invasion was impaired by
peptide MMP-3 inhibitor at an MMP-3 inhibitory concen-
tration (this study and [39]) but not by peptide MMP
inhibitor-1 at an MMP-1 inhibitory concentration (this
study and [40]) and as invasion was stimulated rather than
inhibited by recombinant pro-MMP-9, we conclude that
inhibitory effects were specific to MMP-3.
Co-incubation of MDA-MB-231 cells with plasminogen
and pro-MMP-3 resulted in the appearance of plasmin
species that were not present following MDA-MB-231
incubation with plasminogen alone or with plasminogen in
the presence of pro-MMP-9. In the absence of MDA-MB-
231 cells APMA-activated MMP-3 rapidly degraded
plasminogen to microplasminogen and des-serine protease
kringle fragments, confirming a previous report [31,32].
Plasminogen previously degraded by MMP-3 exhibited
reduced capacity to stimulate MDA-MB-231 invasion,
suggesting that MMP-3 invasion inhibitory effects were
dependent upon plasminogen fragmentation. APMA-acti-
vated pro-MMP-9, in contrast, did not degrade plasmino-
gen under the same conditions, providing further evidence
Fig. 4. Human PBL-1 (angiostatin) inhibits invasion and impairs
plasminogen activation. (A) Histogram depicting levels of MDA-MB-
231 cell invasion through reconstituted Matrigel in the presence (sha-
ded bars) or absence (open bars) of plasminogen (5 lgÆmL
)1
),
plasminogen predegraded by MMP-3 (5 lgÆmL

)1
; cross-hatched bars),
BSA or PBL1, at the concentrations stipulated. Results are expressed
as a percentage of basal plasminogen independent invasion (± SD) of
three independent experiments performed in quadruplet. (B) Line
graphs demonstrating VLLpNA-degrading activity associated with
intact glu-plasminogen (3 lg) activated by t-PA (100 ng) in the pres-
ence of type IV collagen (coated plates), purified 100 000 g MDA-MB-
231 cell membrane preparations (equivalent of 100 ng uPA) or tcuPA
(100 ng) in the presence (d) or absence (s)ofPLB-1(3lg). Results
are expressed as mean (± SD) absorbance at 405 nm of duplicate
experiments performed in quadruplet.
Ó FEBS 2002 MMP-3 inhibits MDA-MB-231 invasion (Eur. J. Biochem. 269) 4481
of a unique degradative interaction between plasminogen
and MMP-3.
In plasmin substrate degradation assays, t-PA and
membrane-associated scuPA but not tcuPA exhibited
reduced capacity to activate plasminogen that had been
degraded by activated MMP-3, when compared with
nondegraded plasminogen. Because des-serine plasminogen
kringle domains are involved in t-PA-, membrane scuPA-
but not tcuPA-mediated plasminogen activation [41–43],
the data suggest a mechanism dependent upon MMP-3-
mediated des-serine plasminogen kringle domain generation
for reduced plasminogen activation. Furthermore, as
MDA-MB-231 cells utilize both t-PA and membrane-
associated uPA for invasivity in vitro [14,44], this may
represent a mechanism involved in MMP-3 inhibition of
MDA-MB-231 invasion.
In addition to generating fragments exhibiting impaired

scuPA and t-PA-mediated activation, MMP-3 also removed
scuPA from, and impaired plasminogen association with,
MDA-MB-231 cell membranes. This adds to previous
reports [31,33,34,45] and provides additional details for a
mechanism involving reduced cell-mediated plasminogen
activation, through which MMP-3 may inhibit MDA-MB-
231 invasion.
Plasminogen degraded by MMP-3 also exhibited reduced
capacity to degrade EHS laminin upon activation by tcuPA
when compared to tcuPA-activated intact plasminogen.
This is consistent with reports that plasminogen kringle
domains regulate macromolecular substrate specificity and
that microplasmin exhibits differences in substrate specifi-
city from plasmin [43,46]. Because laminin is the major
structural component of reconstituted Matrigel [10,11], the
data suggest an additional mechanism for invasion inhibi-
tion dependent upon reduced laminin substrate specificity.
Many of the phenomena observed using MMP-3 were
mimicked by purified human PBL-1 (kringle domains 1–3)
[47]. PBL-1 inhibited t-PA-, membrane scuPA- but not
tcuPA-mediated plasminogen activation, impaired laminin
degradation by plasmin, impaired plasminogen association
with MDA-MB-231 cells and inhibited plasminogen-
dependent MDA-MB-231 invasion in vitro, adding to
previous reports in other cell systems in vitro [41,43,46].
As MMP-3 degrades plasminogen to angiostatin-like frag-
ments (this study and [32]), these data implicate angiostatin
in MMP-3 inhibition of MDA-MB-231 invasion.
It is unlikely that inhibition of MDA-MB-231 invasion
by MMP-3 was dependent upon matrix over-digestion, as

plasminogen degraded by MMP-3 exhibited reduced lami-
nin substrate specificity and MMP-9, which also degrades
BM components [19], stimulated MDA-MB-231 invasivity
under the same conditions, confirming previous reports
[14,15]. The fact that MMP-9 stimulated, but MMP-3
impaired, plasminogen-dependent MDA-MB-231 invasion
suggests that changes in the MMP-3-MMP-9 equilibrium
may well decide whether invasion is stimulated or inhibited.
In conclusion, MMP-3 inhibition of MDA-MB-231 BM
invasion in vitro is likely to depend upon the degradation of
plasminogen by MMP-3 to fragments that exhibit impaired
macromolecular specificity for laminin, that exhibit
impaired activation in response to t-PA and membrane
scuPA and that interfere with cell membrane plasminogen
association further reducing cell-mediated plasminogen
activation. Therefore, conditions do indeed exist for
MMP-3 inhibition of breast cancer cell invasivity, which
may help to explain the association of MMP-3 over-
expression with benign and low stage but not advanced
stage, aggressive, breast disease.
ACKNOWLEDGEMENTS
This work was partially supported by grants from the Associazione
Italiana per la Ricerca sul Cancro (AIRC), the National Research
Council, ÔOncologyÕ Project, the Ministry of University and Research
(MURST 40% and ex 60%), the Ministry of Health, MURST-CNR
ÔBiomolecole per la Salute UmanaÕ Program and Associazione Italiana
per la Lotta al Neuroblastoma and Progetto Speciale Ministero della
Sanita
`
.

REFERENCES
1. Gusterson, B.A., Warburton, M.J., Mitchell, D., Ellison, M.,
Munro Neville, A. & Rudland, P.S. (1982) Distribution of
myoepithelial cells and basement membrane proteins in the nor-
mal breast and in benign and malignant breast diseases. Cancer
Res. 42, 4763–4770.
2. Guelstein, V.I., Tchypysheva, T.A., Ermilova, V.D. & Ljubimov,
A.V. (1993) Myoepithelial and basement membrane antigens in
benign and malignant breast tumors. Int.J.Cancer53, 269–277.
3. Soini, Y., Hurskainen, T., Hoyta, M., Oikarinen, A. & Autio-
Harmainen, H. (1994) 72 kDa and 92 kDa type IV collagenase,
type IV collagen and laminin mRNAs in breast cancer: a study by
in situ hybridisation. J. Histochem. Cytochem. 42, 945–951.
4. Lebbeau, A., Nerlich, A.G., Sauer, U., Lichtinghagen, R. &
Lohrs, U. (1999) Tissue distribution of major matrix metallopro-
teinases and their transcripts in human breast carcinomas. Anti-
cancer Res. 19, 4257–4264.
5. Basset, P., Bellocq, J.P., Wolf, C., Stoll, I., Huntin, P., Limbacker,
J.M., Podhajcer, O.L., Chenard, M.P., Rio, M.C. & Chambon, P.
(1990) A novel metalloproteinase gene specifically expressed in
stromal cells of breast carcinomas. Nature 348, 699–704.
6. Remacle, A.G., Noel, A., Duggen, C., McDermott, E., O’Higgins,
N., Foidart, J.M. & Duffy, M.J. (1998) Assay of metalloprotei-
nases types 1, 2, 3 and 9 in breast cancer. Br.J.Cancer77,926–
931.
7. Heppner, K.J., Matrisian, L.M., Jensen, R.A. & Rogers, W.H.
(1996) Expression of most metalloproteinase family members in
breast cancer represents a tumor induced host response. Am. J.
Pathol. 149, 273–282.
8. Foekens, J.A., Schmitt, M., Van Putten, W.L.J., Peters, H.A.,

Bontenbal, M., Janicke, F. & Klijn, J.G.M. (1992) Prognostic
value of urokinase-type plasminogen activator in 671 primary
breast cancer patients. Cancer Res. 52, 6101–6105.
9. Janicke, F., Schmitt, M. & Graeff, H. (1991) Clinical relevance of
urokinase-type and tissue type plasminogen activators and their
type-1 inhibitor breast cancer. Semin. Thromb. Hemostasis 17,
303–332.
10. Grant, D.S., Kleinman, H.K., Leblond, C.P., Inoue, S., Chung,
A.E. & Martin, G.R. (1985) The basement membrane-like matrix
of the mouse EHS tumor. Immunohistochemical quantitation of
six of its components. Am.J.Anat.174, 387–398.
11. Kleinman, H.K., McGarvey, M.L., Hassell, J.R., Star, V.L.,
Cannon, F.B., Laurie, G.W. & Martin, G.R. (1986) Basement
membrane complexes with biological activities. Biochemistry 25,
312–318.
12. Albini, A., Iwamoto, Y., Kleinman, H.K., Martin, G.R., Aaron-
son, S.A., Kozlowski, J.M. & McEwan. T.N. (1987) A rapid
in vitro assay for quantitating the invasive potential of tumor cells.
Cancer Res. 47, 3239–3245.
13. Repesh, L.A. (1989) A new in vitro assay for quantitating tumor
cell invasion. Invasion Metastasis 9, 192–208.
4482 A. R. Farina et al. (Eur. J. Biochem. 269) Ó FEBS 2002
14. Farina, A.R., Coppa, A., Tiberio, A., Tacconelli, A., Turco, A.,
Colletta, G., Gulino, A. & Mackay, A.R. (1998) Transforming
growth factor beta-1 enhances the invasiveness of human MDA-
MB-231 breast cancer cells by up-regulating urokinase activity.
Int. J. Cancer 75, 721–730.
15. Ramos-DeSimone, N., Hahn-Dantona, E., Sipley, J., Nagase, H.,
French, D.L. & Quigley, J.P. (1999) Activation of matrix
metalloproteinase-9 (MMP-9) via a converging plasmin/strom-

elysin-1 cascade enhances tumor cell invasion. J. Biol. Chem. 274,
13066–13076.
16. Deryugina, E.I., Luo, G.X., Reisfeld, R.A., Bourdon, M.A. &
Strongin, A. (1997) Tumor cell invasion through Matrigel is
regulated by activated matrix metalloproteinase. Anticancer Res.
17, 3201–3210.
17. Mignatti, P. & Rifkin, D.B. (1993) Biology and biochemistry of
proteinases in tumor invasion. Physiol. Rev. 73, 161–195.
18. Andreasen, P.A., Egelund, R. & Petersen, H.H. (2000) The plas-
minogen system in tumor growth, invasion and metastasis. Cell.
Mol. Life Sci. 57, 25–40.
19. Woessner, F.J. & Nagase, H., eds (2000) Matrix Metalloprotei-
nases and TIMPs. Oxford University Press, Oxford.
20. Stahl, A. & Mueller, B.M. (1994) Binding of urokinase to its
receptor promotes migration and invasion of human melanoma
cells in vitro. Cancer Res. 54, 3066–3071.
21. Tiberio, A., Farina, A.R., Tacconelli, A., Cappabianca, L.,
Gulino, A. & Mackay, A.R. (1997) Retinoic acid enhanced
invasion through reconstituted basement membrane by human
SK-N-SH neuroblastoma cells involves membrane associated
tissue-type plasminogen activator. Int. J. Cancer 73, 740–748.
22. Bejarano, P.A., Noeo
`
ken, M.E., Suzuki, K., Hudson, B.G. &
Nagase, H. (1988) Degradation of basement membranes by
human matrix metalloproteinase-3 (stromelysin). Biochem. J. 256,
413–419.
23. Sternlicht, M.D., Lochter, A., Sympson, C.J., Huey, B., Rougier,
J P., Gray, J.W., Pinkel, D., Bissell, M.J. & Werb, Z. (1999) The
stromal proteinase MMP-3/stromelysin-1 promotes mammary

tumorigenesis. Cell 98, 137–146.
24. Lecheter, A., Srebrow, A., Sympson, C.J., Terracio, N., Werb, Z.
& Bissel, M.J. (1997) Misregulation of stromelysin-1 expression in
mouse mammary tumor cells accompanies acquisition of stro-
melysin-1-dependent invasive properties. J. Biol. Chem. 272, 5007–
5015.
25. Chambers, A.F. & Matrisian, L.M. (1997) Changing views of the
role of matrix metalloproteinases in metastasis. J. Natl Cancer
Inst. 89, 1260–1270.
26. Nakopoulou, L., Giannopoulou, J., Gakiopoulou, H., Liapis, H.,
Tzonou,A.&Davaris,P.S.(1999)Matrixmetalloproteinase-1
and -3 in breast cancer: correlation with progesterone receptors
and other clinico-pathologic features. Hum. Pathol. 30, 436–442.
27. Brummer,O.,Athar,S.,Riethdorf,L.,Loning,T.&Herbst,H.
(1999) Matrix-metalloproteinase, 1, 2, 3, their tissue inhibitors, 1, 2
in benign and malignant breast lesions: an in situ hybridisation
study. Virchows Arch. 435, 566–573.
28. Bisgaard, H.C., Mackay, A.R., Gomez, D.E., Ton, P.T., Thor-
geirsson, S.S. & Thorgeirsson, U.P. (1999) Spontaneous metastasis
of rat liver epithelial cells transformed with v-raf and v-raf/v-myc:
association with different phenotypic properties. Invasion Meta-
stasis 17, 240–250.
29. McCarthy, K., Maguire, T., McGreal, G., McDermott, E.,
O’Higgins, N. & Duffy, M.J. (1999) High levels of tissue inhibitor
of metalloproteinase-1 predict poor outcome in patients with
breast cancer. Int.J.Cancer84, 44–48.
30. Visscher, D.W., Hoyhtya, M., Ottosen, S.K., Liang, C.M., Sarcar,
F.H., Crissman, J.D. & Fridman, R. (1994) Enhanced expression
of tissue inhibitor of metalloproteinases 2 (TIMP2) in the stroma
of breast carcinomas correlates with tumour recurrence. Int. J.

Cancer Res. 58, 1–6.
31. Lijnen, H.R. (2002) Matrix metalloproteinases and cellular
fibrinolytic activity. Biochemistry (Moscow) 67, 92–98.
32. Lijnen, H.R., Ugwu, F., Bini, A. & Collen, D. (1998) Generation
of an angiostatin-like fragment from plasminogen by
stromelysin-1 (MMP-3). Biochemistry 37, 4699–4702.
33. Ugwu, F., Van Hoef, B., Bini, A., Collen, D. & Lijnen, H.R.
(1998) Proteolytic cleavage of urokinase-type plasminogen acti-
vator by stromelysin-1 (MMP-3). Biochemistry 37, 7231–7236.
34. Orgel, D., Schroder, W., Hecker-Kia, A., Weithmann, K.U.,
Kolkenbrock, H. & Ulbrich, N. (1998) The cleavage of pro-
urokinase type plasminogen activator by stromelysin-1. Clin.
Chem. Lab. Med. 36, 697–702.
35. Ugwu, F., Lemmens, G., Collen, D. & Lijnen, H.R. (1999)
Modulation of cell-associated plasminogen activation by strome-
lysin-1 (MMP-3). Thromb. Haemost. 82, 1127–1131.
36. Patterson, B.C. & Sang, Q.A. (1997) Angiostatin-converting
enzyme activities of human matrilysin (MMP-7) and gelatinase
B/type IV collagenase (MMP-9). J. Biol. Chem. 272, 28823–
28825.
37. O’Reilly, M.S., Wiederschain, D., Stetler-Stevenson, W.G.,
Folkman, J. & Moses, M.A. (1999) Regulation of angiostatin
production by matrix metalloproteinase-2 in a model of con-
comitant resistance. J. Biol. Chem. 274, 29568–29571.
38. O’Reilly, M.S., Holmgren, L., Shing, Y., Chen, C., Rosenthal,
R.A.,Moses,M.,Lane,W.S.,Cao,Y.,Sage,E.H.&Folkman,J.
(1994) Angiostatin: a novel angiogenesis inhibitor that mediates
the suppression of metastases by a Lewis lung carcinoma. Cell 79,
315–328.
39. Fotouhi, N., Lugo, A., Visnick, M., Lusch, L., Walsky, R., Coffey,

J.W. & Hanglow, A.C. (1994) Potent peptide inhibitors of stro-
melysinbasedonthepro-domainregionofmatrixmetallopro-
teinases. J. Biol. Chem. 269, 30227–30231.
40. Odake, S., Morita, Y., Morikawa, T., Yoshida, N., Hori, H. &
Nagai, Y. (1994) Inhibition of matrix metalloproteinases by pep-
tidyl hydroxamic acids. Biochem. Biophys. Res. Commun. 199,
1442–1446.
41. Stack, S., Gately, S., Bafetti, L.M., Enghild, J.J. & Soff, G.A.
(1999) Angiostatin inhibits endothelial and melanoma cellular
invasion by blocking matrix enhanced plasminogen activation.
Biochem. J. 340, 77–84.
42. Shi, G.Y., Wu, D.H. & Wu, H.L. (1991) Kringle domains and
plasmin denaturation. Biochem. Biophys. Res. Commun. 178, 360–
368.
43. Komorowicz, E., Kolev, K. & Machovich, R. (1998) Fibrinolysis
with des-kringle derivatives of plasmin and its modulation by
plasma protease inhibitors. Biochemistry 37, 9112–9118.
44. Arnoletti, J.P., Albo, D., Granick, M.S., Solomon, M.P., Casti-
glioni, A., Rothman, B.S. & Tuszynski, G.P. (1995) Thrombos-
pondin and transforming growth factor-beta 1 increase expression
of urokinase-type plasminogen activator and plasminogen acti-
vator inhibitor-1 in human MDA-MB-231 breast cancer cells.
Cancer 76, 998–1005.
45. Ranson, M., Andronicos, N.M., O’Mullane, M.J. & Baker, M.S.
(1998) Increased plasminogen binding is associated with meta-
static breast cancer cells: differential expression of plasminogen
binding proteins. Br. J. Cancer 77, 1586–1597.
46. Shi, G.Y. & Wu, H.L. (1988) Isolation and characterisation of
microplasminogen. A low molecular weight form of plasminogen.
J. Biol. Chem. 263, 17071–17075.

47. Wiman, B., Lijnen, H.R. & Collen, D. (1979) On the specific
interaction between the lysine-binding sites in plasmin and com-
plementary sites in alpha2-antiplasmin and in fibrinogen. Biochim.
Biophys. Acta. 579, 142–154.
Ó FEBS 2002 MMP-3 inhibits MDA-MB-231 invasion (Eur. J. Biochem. 269) 4483