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-NH2 and was associated with: rapid MMP-
Trang 1Inhibition of human MDA-MB-231 breast cancer cell invasion
by matrix metalloproteinase 3 involves degradation of plasminogen
Antonietta R Farina1, Antonella Tacconelli1, Lucia Cappabianca1, Alberto Gulino2and Andrew R Mackay1 1
Section of Molecular Pathology, Department of Experimental Medicine, University of L’Aquila, Italy;2Department 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-NH2 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 epro-vidence 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: mackay@univaq.it
Abbreviations: BM, basement membrane; t-PA, tissue-type
plasmi-nogen activator; scuPA, single-chain urokinase plasmiplasmi-nogen 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)
Trang 2breast 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
M A T E R I A L S A N D M E T H O D S
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-NH2), 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), 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/Pi,
resuspended in (EMEM/0.1% BSA with antibiotics) and
used in invasion assays at a final concentration of 0.5· 106
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
37C 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)1plasminogen
Following electrophoresis, gels were washed in 2% triton
X-100, rinsed in water and incubated in incubation buffer
(50 mMTris, 200 mMNaCl, 5 mMCaCl2pH 8.0)
Plasmi-nogen activator zymograms were incubated in incubation
buffer without CaCl2in the presence of 15 mMEDTA, 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 0.15M NaCl, 20 mM Hepes,
2 mMCaCl2, 100 lgÆmL)1leupeptin, 2.5 mgÆmL)1 pepsta-tin A and 1 mM phenyl methyl sulfonylflouride (pH 8.0) Cells were disrupted by cycles of freezing and thawing in liquid nitrogen and at 42C Nuclei were removed by centrifugation at 7000 g for 20 min at 4C and membranes obtained by ultracentrifugation at 100 000 g for 1 h at 4C; 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 mM Tris, 200 mMNaCl, 5 mMCaCl2 for enzyme assays Cell membranes were resuspended in nonreducing SDS/PAGE sample buffer for zymograms or
in 100 mMTris/0.5% Triton X-100 (pH 8.8) for plasmino-gen activator assays
Caseinase assays Caseinolytic metalloproteinase activity in supernatants was assessed using 14C-labelled a-casein Briefly, substrates (30 000 c.p.m.) were mixed with samples in a buffer containing 20 mM Tris, 200 mM NaCl and 5 mM CaCl2 (pH 8.0) in a final volume of 200 lL with or without the MMP activator APMA (1 mM) or with or without EDTA (15 mM) Assays were incubated at 37C 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 mM), peptide MMP-3 inhibitor (10 lM) or peptide MMP inhibitor-1 (30 lM) Undegraded proteins were precipitated with 10% trichloroacetic acid/0.5% tannic acid (TA) and precipitated material was removed by centrifuged
at 5000 g for 15 min or subjected directly to centrifugation (12 000 g for 10 min for type Icollagen) Radioactivity in the supernatants was counted in a Beta liquid scintillation counter (Beckman model LS 5000TD) Characterization of
Trang 3activity as MMP was determined by inhibition with EDTA
(15 mM)
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-ted for 0–3 h at 37C, in 96-well microplates in a buffer
containing 100 mM 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
R E S U L T S
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
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 lM) by
60% (significant at P < 0.001) but was not impaired by the peptide MMP inhibitor-1 (30 lM) (Fig 1A) At the concentrations used, peptide MMP-3 inhibitor (10 lM) 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 ) peptides on MMP-9 (100 ng) and MMP-3 (100 ng) activity.
Trang 4zymograms (Fig 1C), whereas peptide MMP inhibitor-1 at
a concentration of 30 lM 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 lM) but not by
MMP inhibitor-1 (30 lM), 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 mM APMA for 3 h at
37C) 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 37C, 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 lg lane)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.
Trang 5completely 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 mM),
peptide MMP-3 inhibitor and peptide MMP inhibitor-1
(100 lM) 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 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 37C 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 mM 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 37C 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 37C), 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 catalytic anti-plasminogen antibody but not by EDTA (15 mM) (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; lane 4).
Trang 6PBL-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 37C at an MMP-3 : plasminogen ratio of 1 : 100),
stimulated MDA-MB-231 invasion in the presence of
peptide MMP-3 inhibitor (10 lM) 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)
D I S C U S S I O N
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) of PLB-1 (3 lg) Results
are expressed as mean (± SD) absorbance at 405 nm of duplicate
experiments performed in quadruplet.
Trang 7of 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
A C K N O W L E D G E M E N T S
This work was partially supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), the National Research Council, Oncology Project, the Ministry of University and Research (MURST 40% and ex 60%), the Ministry of Health, MURST-CNR
Biomolecole per la Salute Umana Program and Associazione Italiana per la Lotta al Neuroblastoma and Progetto Speciale Ministero della Sanita`.
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