close

Вход

Забыли?

вход по аккаунту

?

830

код для вставкиСкачать
Int. J. Cancer (Pred. Oncol.): 69,9-16 (1996)
0 1996 Wiley-Liss, Inc.
I
Publicationof the International Union Against Cancer
Publicationde I’Union Internationale Contre le Cancer
ENHANCED PRODUCTION OF MATRIX METALLOPROTEINASES
AND ACTIVATION OF MATRIX METALLOPROTEINASE 2
(GELATINASE A) IN HUMAN GASTRIC CARCINOMAS
Hidehiro NOMLJRA~,~,
Noboru FUJIMOTO~,
Motoharu SEIK13, Masayoshi M A Iand
~ Yasunori OKADA’,~
Departments of ’MolecularImmunology and Pathology, 2Surgery and 3MolecularVirology and Oncology, Cancer Research Institute,
Kanazawa University, Kanazawa, Ishikawa 920; and 4Biophamaceutical Department, Fuji Chemical Industries Ltd.,
Takaoka, Toyama 933, Japan.
We examined the production and tissue localization of matrix
metalloproteinases(MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in gastric carcinoma tissues. MMP- I (tissue
collagenase), MMP-9 (gelatinase B) and TIMP-2 were immunolocalized in carcinoma cells and MMP-2 (gelatinase A) on tumor
cell membranes,whereas no or little immunostainingfor MMP-3
(stromelysin- I) and TIMP- I was seen in carcinoma cells. Stroma1 cells in carcinoma tissue were also positively stained for
these MMPs and TIMPs. MMP-2 immunostainingwas observed
exclusively on advanced gastric carcinoma cells and correlated
with vascular invasion by tumor cells. Sandwich enzyme immunoassays revealed enhanced production of MMP- I, MMP-2,
MMP-3. MMP-9 and TIMP- I by carcinoma tissues. Gelatinolytic
activities were significantly higher in carcinoma samples than in
normal controls. Using gelatin zymography, active forms of
MMP-2 and MMP-9 were more frequently detected in carcinoma tissue, and the activation rate of the zymogen of MMP-2
(proMMP-2). but not that of proMMP-9, correlated well with
degree of local invasion and lymphatic permeation. Our data
indicate an enhanced production of 4 MMPs in gastric carcinoma tissue and sunnest that activation of pro-MMP-2 may be a
key step for spreadEg of gastric carcinoma cells.
0
1996 Wiley-Liss,Inc.
Matrix metalloproteinases (MMPs) are a gene family of
Zn2+ metalloproteinases composed of at least 11 different
gene products and involved in degradation of the extracellular
matrix macromolecules associated with tissue destruction in
various pathological conditions (Birkedal-Hansen et al., 1993;
Stetler-Stevenson et al., 1993; Sato et al., 1994). Previous
experimental and clinicopathological studies have shown good
correlations between expression of MMPs and the invasive
phenotype of tumor cells (Stetler-Stevenson et al., 1993).
There are several studies on MMP expression and production
by gastric cancer (McDonnell et al., 1991; David et al., 1994;
Grigioni et al., 1994; Otani et al., 1994), which is the most
common cancer in Japan and some other countries. Gastric
carcinoma tissues have been reported to exhibit mRNA
expression of MMP-1 (tissue collagenase) and MMP-7 (matrilysin) but not MMP-3 (stromelysin-1) or MMP-10 (stromelysin-2) (McDonnell et al., 1991; Otani et al., 1994). MMP-2
(gelatinase A) has also been immunolocalized within carcinoma cells in stomach cancers (David et al., 1994; Grigioni et
al., 1994). However, there are few studies examining the tissue
localization of several MMPs in the same specimens of gastric
carcinoma.
The activity of MMPs is regulated by tissue inhibitors of
metalloproteinases (TIMPs), which comprise 3 different molecules, i.e., TIMP-l, TIMP-2 and TIMP-3 (Birkedal-Hansen et
al., 1993; Stetler-Stevenson et al., 1993). It is also known that
over-expression of TIMPs suppresses invasion and metastasis
by tumor cell lines (Stetler-Stevenson et al., 1993). In stomach
cancers, the percentage of TIMP-2-immunoreactive carcinoma cells is reported to decrease in advanced carcinomas,
showing an inverse correlation of TIMP-2 expression with
invasive properties of carcinoma cells (Grigioni et aZ., 1994).
This suggests that production of TIMPs at the local tissue level
also modulates invasion and metastasis of cancer cells. Although Lokeshwar et al. (1993) have shown that prostatic
cancer tissue produces less TIMP activity than controls, there
are no other studies describing the amounts of MMPs and
TIMPs produced by cancer tissues. Since MMPs are produced
as zymogen forms (pro-MMPs) and activated extracellularly,
activation is another key step for each MMP to function in vivo.
The occurrence of active MMP-2 species correlates well with
an invasive and metastatic phenotype in lung (Brown et al.,
1993) and breast (Davies et al., 1993) cancers. We have
identified an activator metalloproteinase for pro-MMP-2, i.e.,
membrane type-MMP (MT-MMP; Sato et al., 1994), and
demonstrated MT-MMP-associated activation of pro-MMP-2
in human gastric carcinomas (Nomura et al., 1995). However,
no studies are available that correlate activation of proMMP-2 with progression of human gastric carcinomas.
In the present work we have examined immunolocalization
and production of 4 different MMPs (MMP-1, MMP-2, MMP-3
and MMP-9) and 2 TIMPs (TIMP-1 and TIMP-2) in human
gastric carcinomas. The results suggest that overexpression of
these MMPs and activation of proMMP-2 are important in the
malignant behavior of stomach cancers.
MATERIAL AND METHODS
Histology
Fresh tissue samples were obtained from 46 patients with
gastric carcinoma who underwent surgery in the Cancer
Research Hospital of Kanazawa University. Blocks of tissue
were excised from the margins of carcinoma and normal
gastric wall remote from the tumor. Each sample was fixed
with periodate-lysine-paraformaldehydefixative for 18-24 hr
at 4°C and embedded in paraffin wax. Surgical specimens were
stained with hematoxylin and eosin and classified according to
“The General Rules for the Gastric Cancer Study” (Japanese
Research Society for Gastric Cancer, 1981).
Imrnunohistochemistry
Monoclonal antibodies (MAbs) against MMP-1 (41-1E5),
MMP-2 (75-7F7), MMP-3 (55-2A4), MMP-9 (56-2A4), TIMP-1
(50-3D2) and TIMP-2 (67-4H11) have been characterized and
previously used for immunolocalization studies (Zhang et al.,
1993; Fujimoto et al., 1993a, b, 1994; Obata et al., 1992). All
antibodies for MMPs recognize both zymogen and active
forms. For immunohistochemistry, deparaffinized sections were
reacted with primary antibodies or non-immune mouse IgG for
15-18 hr at 4°C after blocking endogenous peroxidase by
treatment with 0.3% H202 and non-specific binding with 10%
normal horse serum. Some sections were treated with 0.04%
trypsin in 50 mM Tris-HC1 buffer, pH 7.6, for 10 min at 37°C
and then reacted with the antibody against TIMP-1. Concentrations of primary antibodies used for immunostaining were 1
5To whom correspondence and reprint requests should be sent, at
Department of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University. 13-1 Takara-machi, Kanazawa,
Ishikawa 920, Japan. Fax: (81) 0762-34-4508.
Received: August 9, 1995 and in revised form October 26, 1995
10
NOMURA ETAL
pg/ml for MMP-1,4 pg/ml for MMP-2,8.3 pg/ml for MMP-3,
1 kg/ml for MMP-9, 10 pg/ml for TIMP-1, 1 pg/ml for
TIMP-2 and 4 p,g/ml for non-immune mouse IgG. For absorption studies, the antibody against MMP-2 was first subjected to
pro-MMP-2-Sepharose or BSA-Sepharose column and the
effluent was used for immunostaining. Sections reacted with
antibodies were incubated with biotinylated horse IgG to
mouse IgG (Vector, Burlingame, CA, diluted 1:200) for 30 min
and then with an avidin-biotin-peroxidase complex (Dako,
Glostrup, Denmark) for 30 min at room temperature. Color
was developed with 0.03% 3,3’-diaminobenzidine tetrahydrochloride in 50 mM Tris-HCI buffer, pH 7.6, containing 0.006%
Hz02. Counter-staining was performed with hematoxylin. The
ratio (%) of immunoreactive cells to total carcinoma cells was
measured by counting cells in 3 different fields at a magnification of ~ 2 0 0 .
Tissue cultures
Tissue samples of carcinoma and normal gastric mucosa
were excised from samples obtained for immunostaining. After
rinsing in 0.01 M phosphate buffer, pH 7.5, containing 0.15 M
NaCI, they were cut into small blocks (approx. 2 x 2 x 2 mm),
and 18-20 blocks were cultured for 3 hr in 2 ml of serum-free
RPMI (GIBCO, Grand Island, NY) containing 0.2% lactalbumin hydrolysate (GIBCO). Culture media were stored at
-20°C until used for assays and Zymography, and tissue blocks
were weighed after lyophilization.
stained with 0.1% Coomassie brilliant blue R250. The intensity
of the gelatinolytic bands corresponding to MMP-2 and
MMP-9 was measured using computer-assisted image analysis
according to the method of Davies et al. (1993). The volume of
the media loaded was adjusted acclording to the weight of the
tissue used for the culture. Authentic pro-MMP-2 from rheumatoid synovial fibroblasts and pro-MMP-9 from HT-1080
fibrosarcoma cells were also subjected to gelatin zymography
for comparison. For inhibition studies, representative culture
media were subjected to gelatin qmography and sliced gels
were incubated at 37°C in SO mM Tris-HC1, pH 7.4, 0.15 M
NaCI, 10 mM CaC12 and 0.02% NaN3 in the presence and
absence of 20 mM EDTA, 1 mM o-phenanthroline, 2 mM
phenylmethane sulfonyl fluoride, 2 mM diisopropylfluorophosphate, 5 mMN-ethylmaleimide or 0.5 mM pepstatin A.
Statistical analysis
Statistical analysis was performed using 2-tailed MannWhitney U and x2 tests; p values below 0.05 were considered
significant.
RESULTS
Histology and immunohistochemistly
Forty-six gastric carcinomas were examined for histology
and immunohistochemistry. They were classified into early and
advanced gastric carcinoma groups according to wall penetration:
early carcinoma is defined as carcinoma confined to
Sandwich enzyme immunoassays
mucosa and submucosa and advanced carcinoma as more
Concentrations of MMP-1, MMP-2, MMP-3, MMP-9, invasive carcinoma (Japanese Research Society for Gastric
TIMP-1 and TIMP-2 in culture media were measured in the Cancer, 1981). Other clinicopathological parameters, includcorresponding sandwich enzyme immunoassay (EIA) systems ing lymphatic permeation, vascular invasion, lymph node
for these MMPs and TIMPs as described previously (Zhang et metastasis and peritoneal dissemination, were also examined.
al., 1993; Fujimoto et al., 1993a, b, 1994; Obata et al., 1992; Normal control tissues showed various degrees of inflammaKodama et al., 1990). The EIA systems for MMP-1 and tory cell infiltration and foci of intestinal metaplasia of the
MMP-3 measure both precursor and active forms of the epithelium.
MMPs, but those for MMP-2 and MMP-9 detect only their
Immunohistochemically, carcinoma cells showed intracytolatent forms (Zhang et al., 1993; Fujimoto et al., 1993a, 1994; plasmic
staining for MMP-1 (Fig. la), MMP-9 (Fig. Id) and
Obata et al., 1992). The EIA for TIMP-1 determines the whole
(Fig. 2b),and percentages of positive cases were 83%,
amount of TIMP-1, including free TIMP-1 and the complexed TIMP-2
35%
and
43%, respectively (Table I). However, MMP-2 was
forms with MMPs and pro-MMP-9 (Kodama et al., 1990). immunolocalized on the cell membranes of carcinoma cells in
However, the EIA for TIMP-2 detects free TIMP-2 and 14 cases (30%) (Fig. lb), though 41 of the 14 cases exhibited
TIMP-2 complexed with active MMPs but not the complex intracytoplasmic staining as well. Ratios of immunoreactive
with pro-MMP-2 (Fujimoto et al., 19936).Values are expressed cells to total carcinoma cells in the positively stained cases for
as nmol/g dry weight tissue. The following m.w. were used for MMP-1, MMP-2, MMP-9 and TIMP-2 were 45 c 26%, 18 ?
calculation: 51,929 for MMP-1, 70,952 for MMP-2, 52,220 for 16%, 28 c 24% and 32 ? 22% (mean 2 SD), respectively.
MMP-3,78,426 for MMP-9, 20,685 for TIMP-1 and 21,755 for These MMPs showed no special1 distribution patterns of
TIMP-2 (see references in Birkedal-Hansen et al., 1993).
immunoreactive carcinoma cells in tumor cell nests, but
staining of TIMP-2 had a tendency to be stronger in the
Enzyme assays
Gelatinolytic activities in culture media were measured in an marginal carcinoma cells than in the tumor center. Immunoassay using [I4C] acetylated type I gelatin in the presence and staining of MMP-3 (Fig. lc) and TIIMP-1 (Fig. 2a) was weak or
absence of 1 mM p-aminophenylmercuric acetate (APMA). negative in carcinoma cells. Stromal and inflammatory cells in
Collagenolytic and carboxymethylated transferrin (Cm-Tf)- the tumors, including fibroblasts, endothelial cells, macrodegrading activities in media were also measured using [l4C] phages and neutrophils, were positive for MMP-1 (Fig. l a ) and
type I collagen and [3H] Cm-Tf, respectively. Samples were MMP-9 (Fig. Id). In most cases, jibroblasts and endothelial
incubated prior to assays with 1mM APMA at 37°C for 18 hr to cells in the tumors were also immuinostained for MMP-2 (Fig.
activate pro-MMP-1 and pro-MMP-3. All of these assays were lb), TIMP-1 (Fig. 2a) and TIMP-2 (Fig. 2b),and MMP-3 was
performed in the presence of 2 mM phenylmethane sulfonyl occasionally positive in stromal macrophages (Fig. lc). Stainfluoride and 5 mM N-ethylmaleimide to inhibit serine and ing of stromal cells was intensely positive in cancer tissue, but it
cysteine proteinases. One unit of activity is defined as the was weak or almost negative in normal tissue remote from the
carcinoma. No specimens were positively stained in cancer
amount of enzyme that degrades 1 pg substrate/min at 37°C.
tissue when reacted with non-immune mouse IgG (Fig. le). In
addition, MMP-2 staining on the carcinoma cell membranes
Gelatin zymography
Zymography in SDS-PAGE containing 0.2% gelatin was was abolished when the primary antibody was absorbed with
performed. Culture media were incubated at 37°C for 20 min pro-MMP-2 (Fig. I f ) .
When the immunostain in the carcinoma cells was compared
in SDS sample buffer without reducing agent and then
electrophoresed on 8.5% polyacrylamide gels at 4°C. After between the early and advanced carcinoma groups, MMP-2
electrophoresis, gels were washed in 2.5% Triton-X 100 to was localized exclusively in the advanced carcinomas (42% vs.
remove SDS, incubated for 16 hr at 37°C in 50 mM Tris-HCI, 0% of early carcinomas; Table I). MMP-9 was also immunopH 7.4,0.15 M NaCI, 10 mM CaC12 and 0.02% NaN3 and then stained in more cases of advanced (42%) than early (15%)
MMPs AND TIMPs IN GASTRIC CANCER
11
FIGURE
1 - Immunolocalization of MMP-1, MMP-2, MMP-3 and MMP-9 in gastric carcinoma tissues. Immunostaining was performed
with murine MAbs to MMP-1 (a), MMP-2 (b), MMP-3 (c), MMP-9 (d), non-immune mouse IgG (e) or antibody to MMP-2 absorbed
with pro-MMP-2 (f), as described in “Material and Methods”. Note that MMP-1 and MMP-9 are immunostained within the
adenocarcinoma cells, whereas MMP-2 is localized on carcinoma cell membranes, and that staining was abolished by absorption.
Positively stained stromal fibroblasts (arrows) and inflammatory cells (arrowheads) are indicated. Hematoxylin counter-stain. Bar:
50 pm.
N O M U R A ETAL.
12
FIGURE2 - Immunolocalization of TIMP-1 and TIMP-2 in gastric carcinoma tissues. Tissues were inimunostained with antibodies
against TIMP-1 (a) and TIMP-2 (b), as described in “Material and Methods”. Note positive staining for TIMP-1 in stromal cells and for
TIMP-2 in both stromal and carcinoma cells. Hematoxylin counter-stain. Bar: 50 pm.
TABLE 1- IMMUNOHISTOCHEMICAL DATA OF MMPs AND TlMPs IN GASTRIC CARCINOMA CELLS
Case
Total cases
Early carcinoma
Advanced carcinoma
Percentage of positive cases
number
MMP-1
MMP-2
MMP-3
MMP-9
TIMP-1
TIMP-2
46
83%
92%
79%
30%
0%
42%
0%
0%
0%
35%
15%
42%
4%
43%
23%
52%
13
33
carcinomas, but there was no statistical significance (Table I).
It is notable that MMP-Zpositive cases showed vascular
invasion much more frequently (86%) than MMP-2-negative
carcinomas (27%; p < 0.01, x2 test). However, there were no
correlations between immunolocalization of other MMPs and
TIMPs and clinicopathological parameters.
Sandwich enzyme immirnoassays
To measure the amounts of MMP-1, MMP-2, MMP-3,
MMP-9, TIMP-1 and TIMP-2 produced by the carcinoma
tissue, EIA systems were applied to culture media. The levels
of MMP-I, MMP-2, MMP-3 and MMP-9 in the culture media
from tumor tissue were 0.032, 0.197, 0.039 and 0.129 nmol/g
weight (median). respectively, and significantly higher than in
the control normal tissue ( p < 0.01); molar ratios of MMP-1,
MMP-2, MMP-3 and MMP-9 in the tumor samples compared
to controls were 5.4 ? 7.7, 3.3 f 3.0, 5.7 f 7.5 and 6.2 2 9.7
(mean 2 SD), respectively (Fig. 3). The TIMP-1 level in the
tumor samples (median = 0.179 nmol/g weight) was also
significantly higher than that in the normal (median = 0.064),
but there was no difference in the TIMP-2 level between the
tumor (median = 0.035 nmol/g weight) and control samples
(median = 0.033; Fig. 3). Since our EIA for TIMP-2 does not
measure TIMP-2 complexed with pro-MMP-2, TIMP-2
amounts were corrected by adding the values of MMP-2 to
those of TIMP-2, assuming that all of the MMP-2 detected by
the assay was the pro-MMP-2-TIMP-2 complex. When the
total amounts of MMP-1, MMP-2, MMP-3 and MMP-9 were
divided by the sum of TIMP-1 and corrected TIMP-2 in each
case, the following molar ratios were obtained: 1.25 f 1.00
(mean 2 SD) for carcinoma samples and 1.15 f 0.62 for
controls, which were not significantly different.
0%
6%
Enzyme activities
Proteinase activities in culture media were measured using
‘jC-gelatin, 14C-collagenand -?H-Cm-Tfsubstrates in the presence of serine and cysteine prol~einasesinhibitors. Figure 4
shows gelatinolytic activities in the media. The level of the total
activities detected in the presence of APMA was significantly
higher in the carcinoma samples (median = 33.0 mU/mg
weight) than in the control specimens (median = 8.5)
( p < 0.01). The level assayed in the absence of APMA showed
that tumor samples (median = 10.5 mU/mg weight) contained
more active enzymes than the normal tissue group (median = 4.0) ( p < 0.01) (Fig. 4). However, no definite correlations between activities and clinicopathological parameters
were found. Inhibition studies using 4 representative samples
from the carcinoma and control normal tissues revealed that
EDTA (20 mM) remarkably inhibits gelatinolytic activities
(82-100% of activity).
Cm-Tf-degrading activity was minimal in both carcinoma
(median = 1.4 mU/mg weight) and normal control samples
(median = 1.1, n = 33), and no significant difference was seen.
Collagenolytic activity was detectable in 3 cases only (n = 30).
Gelatin zymography
Gelatinolytic activities in culture media from 39 gastric
cancer cases were also analyzed by gelatin Zymography. ProMMP-9 of 92 kDa and pro-MMP-2 of 68 kDa were detected in
all carcinoma and normal tissue samples, but carcinoma tissue
produced remarkably larger amounts of pro-MMP-9 than
normal tissue (Fig. 5). Production of pro-MMP-2 appeared to
be also higher in most carcinoma specimens than in controls
(Fig. 5). Computer-assisted image analyses of the proteolytic
bands of pro-MMP-2 and pro-MIUP-9 showed that proteolytic
activities of pro-MMP-2 and pro-MMP-9 in carcinoma samples
were about 2-fold higher than in normal counterparts (data not
13
MMPs AND TIMPs IN GASTRIC CANCER
nmol I g weight
**
n
.
1.0
F-l
0.8
..
0.6
n
0.4
a
t
**
.
n
8
**
.
I,
n
.I
.
0
t
0.2
.
.
.
.
.
..
..
NS
n
8
.
4
- ..
0
i
at
0
T
N
MMP-I
T
N
MMP-2
T
N
MMP-3
T
N
MMP-9
(n=26)
(n=31)
(n=23)
(n=32)
T
N
TIMP-I
(n=27)
T
N
TIMP-2
(n=34)
FIGURE
3 - Amounts of MMPs and TIMPs in the culture media secreted by carcinoma and normal control tissues. Culture media were
prepared by culturing fragments from gastric carcinoma (T) and normal controls (N). MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and
TIMP-2 were measured by the corresponding EIA systems. Values (nmolig weight) were calculated as described in “Material and
Methods”. Bars indicate mean values. **p < 0.01; NS, not significant (Mann-Whitney U test).
shown). Since there was a direct correlation between the data
obtained by zymography and EIA systems, the assays are
considered mutually supportive.
The active MMP-2 species of 62 kDa was found on gelatin
zymography in 59% of carcinomas (23/39 cases) but in only
23% of controls (9/39 cases) ( p < 0.01, x2 test). When the
intensity of the proteolytic bands of pro-MMP-2 and its active
species was compared by zymography, the ratio of the 62
kDa-active form to pro-MMP-2 plus active MMP-2 (activation
ratio of pro-MMP-2) was significantly higher in carcinoma
specimens than in normal controls ( p < 0.01) and in the
MMP-2 staining-positive carcinoma group (n = 13) than in the
negative group (n = 20) ( p < 0.05). In addition, analysis of
the relationships between zymography data and wall penetration of carcinoma cells demonstrated that the activation ratio
of pro-MMP-2 was significantly higher in advanced carcinomas
(n = 32) than in early carcinomas (n = 7 ; p < 0.05; Fig. 6a).
Appearance of active MMP-2 also correlated well with lymphatic permeation (ly): the activation ratio of pro-MMP-2 was
remarkably higher in ly-positive cases (n = 27) than in lynegative ones (n = 12;p < 0.05; Fig. 6b).However, no correlation was seen between activation and vascular invasion. The 83
kDa-form of MMP-9 was detected in 31% of carcinoma
samples (12/39 cases) but in only 8% of controls (3/39 cases)
( p < 0.01, x2 test), though the ratio of the 83 kDa-MMP-9 to
pro-MMP-9 plus MMP-9 exhibited no significant difference
between carcinoma and control samples and showed no
correlations with any clinicopathological factor.
Inhibition studies showed that all gelatinolytic activities of
92, 83,68 and 62 kDa, in addition to higher m.w. species, were
almost completely inhibited with 20 mM EDTA and 1 mM
o-phenanthroline but not with 2 mM phenylmethane sulfonyl
fluoride, 2 mM diisopropylfluorophosphate, 5 mM N-ethylmaleimide or 0.5 mM pepstatin A, indicating that all of the
activities belong to a class of metalloproteinases (data not
shown).
DISCUSSION
Our immunolocalization studies have shown that stomach
carcinoma cells stain positively for several MMPs including
MMP-I, MMP-2 and MMP-9 as well as TIMP-2. The staining
pattern of MMP-2 on the plasma membranes of carcinoma
cells was unique. Previous studies using in situ hybridization
and immunohistochemistry for MMP-2 in human carcinomas
(Autio-Harmainen et al., 1993; David et al., 1994; Davies et a[.,
1993; Grigioni et al., 1994; Poulsom et al., 1992; Pyke el al.,
1993; Wolf et al., 1993) have yielded contradictory datanamely, that stromal fibroblasts, but not carcinoma cells, give
strong signals for mRNA, whereas MMP-2 staining is present
in the cytoplasm of both carcinoma cells and fibroblasts. This
discrepancy has been explained by several possibilities: (i)
uptake of fibroblast-derived MMP-2 by carcinoma cells, (ii)
differences in the rates of mRNA translation and capacity for
intracellular storage of the protein or (iii) differences in the
threshold of detection by in situ hybridization and immunohistochemistry (Poulsom et al., 1992). However, the antibody used
in our study revealed a novel staining pattern: localization on
plasma membranes of the carcinoma cells and within stromal
fibroblasts. lmmunostaining was considered to be specific since
the antibody absorbed with pro-MMP-2 resulted in no staining. Similar membrane staining of carcinoma cells has been
reported in breast cancers (Visscher et al., 1994). A growing
body of evidence indicates that carcinoma cells possess binding
14
rnU I mg weight
-
NOMURA ETAL
**
m
400
**
300
I
1
m
0
m
200
m
m
100
m
0
T
N
APMA( +)
(n= 32)
T
N
APMA(-)
(n= 32)
FIGURE
4 - Gelatinolyticactivities in the culture media of tumor
(T) and normal control (N) samples. The assay was performed
using I4C-gelatinat 37°C in the presence and absence of APMA
[APMA (+) and APMA (-), respectively]. All assays were
performed in the presence of 2 mM phenylmethane sulfonyl
fluoride and 5 mM N-ethylmaleimide; activities were expressed on
the basis of dry weight of each sample. **p < 0.01 (Mann-Whitney
U test).
sites for pro-MMP-2 on the membranes (Emonard et a/., 1992).
In the present study, membrane localization of MMP-2 was
observed only in advanced gastric carcinomas, which give
tissue reactions with MMP-2-positive fibroblasts. Thus, our
data suggest that in gastric carcinoma tissue MMP-2 is produced mainly by stromal fibroblasts and captured on the
membranes of carcinoma cells.
Inconsistency between immunohistochemistry and in situ
hybridization results was also apparent for MMP-1 localization. Although our study showed that MMP-1 is immunolocalized in carcinoma cells and stromal fibroblasts in most cases of
stomach cancer, Otani et a/. (1994) have reported that mRNA
expression is present only in gastric cancer stromal fibroblasts.
However, some colorectal carcinoma cells (Hewitt et a/., 1991)
also express MMP-1 at the invasive edges. Our findings on
MMP-1 immunolocalization, in addition to previous studies,
suggest that MMP-1 is produced by both carcinoma cells and
stromal fibroblasts in gastric cancer. However, it is also
possible that our antibody recognizes collagenase-3 (MMP-13;
Freije et a/., 1994) as well as MMP-1. Our antibody was
prepared against the synthetic oligopeptide corresponding to
the COOH-terminal domain of pro-MMP-1 (residues 332350) (Zhang et a/., 1993). Although homology of the peptide to
the corresponding sequence of MMP-13 is 37%, we cannot
RGURE
5 - Gelatin zymography of culture media from stomach
tissues. Culture media were electrophoresed under non-reducing
conditions on 8.5% SDS-polyacrylamidegels containing gelatin at
4°C; gels were incubated in 50 mM Tris-HCI,pH 7.5,0.15 M NaCI,
10 mM CaCI, and 0.02% NaN, for 16 hr at 37°C after removal of
SDS, as described in “Material and “ethods”. Three representative samples of tumors (T) and their normal counterparts (N) are
shown. Protein standards include phosphorylase b (94 kDa ,
human transferrin (77 kDa), BSA (68 kDa), IgG-heavy chain ( 5
kDa), ovalburnin (43 kDa) and carbonic anhydrase (29 kDa).
Major gelatinolytic activities with 92 and 83 kDa corresponding to
MMP-9 (arrows) and with 68 and 62 kDa corresponding to
MMP-2 (arrowheads) are indicated. Authentic pro-MMP-9 (B)
and pro-MMP-2 samples (A), the latter of which contains active
MMP-2 species of 62 kDa, are also electrophoresed for comparison.
2
exclude the possibility that the epifope of the antibody has a
higher homology to some portions of MMP-13.
Carcinoma tissues produce MMP-9 (Brown et a/., 1993;
Davies et a/., 1993). Previous studies using in situ hybridization
have shown that carcinoma cells and/or stromal cells, such as
macrophages, express the mRNA for MMP-9 in carcinoma
tissues (Wolf et a/., 1993; Pyke et a/., 1993). However, very little
is known about the immunolocalization of MMP-9, except one
report showing localization in carcinoma cells of breast cancer
(Visscher et a/., 1994). Our study has shown that MMP-9 is
immunolocalized in gastric carcinoma cells as well as in
fibroblasts and macrophages infiltrading tumor tissue, suggesting production by these cells. In co’ntrast, MMP-3 was barely
immunostained in gastric carcinomas. Although MMP-3 is
expressed in esophageal carcinomas (Shima et a/., 1992),
MMP-3 mRNA expression is undetectable in stomach, colon
(McDonnell et al., 1991) and breast (Wolf et a/., 1993)
adenocarcinomas. These and our results suggest that MMP-3
expression in tumors is more specifiic of squamous cell carcinomas. TIMP-1 and TIMP-2 share biochemical properties as
inhibitors of MMPs (Birkedal-Hansen et a/., 1993; StetlerStevenson et a/., 1993), and both TIMPs tended to be expressed mainly in stromal cells of carcinoma tissues (Poulsom
et al., 1992; Visscher et a/., 1994; Hewitt et a/., 1991). In gastric
cancers, TIMP-2 is immunolocalized in carcinoma cells, inversely correlating with invasiveness and metastasis (Grigioni
et a/., 1994). In the present study, TIMP-1 was immunolocal-
15
MMPs AND TIMPs IN GASTRIC CANCER
(9)
Activation ratio
of proMMP-2
*
m
0
0.5
Activation ratio
of proMMP-2
*
I
0.5
A
A
0.4
y.
0
m
0.4
A
0
&
0.3
0.2
0
i,
0.3
0.2
A
A
Y
A
A
A
A
A
t?
6
0.1
0
0
0
t?
0. I
0
lY (-1
(n=12)
lY (+I
(n=27)
FIGURE6 - Correlations of MMP-2 activation with wall penetration (a) and lymphatic permeation (b) of carcinoma cells. Activation
rate of pro-MMP-2 was calculated by computer-assisted image analysis of the data from gelatin zymography, as described in “Material
and Methods”. Data on early carcinomas (early) and lymphatic permeation-negative (ly-) cases were compared with advanced
(advanced) and ly-positive (ly+) cases, respectively. *p < 0.05 (Mann-Whitney U test).
ized occasionally, but exclusively to stromal fibroblasts, and
TIMP-2 was detected in carcinoma cells as well as fibroblasts
without any definite correlation of expression with invasion
and metastasis.
To examine the quantitative balance between MMPs and
TIMPs, we have assayed enzymic activities. Gelatinolytic
activities were detected in culture media of carcinoma and
normal tissues, and the levels were significantly higher in
carcinoma samples than in controls. Our data indicate that the
amounts of gelatinases (MMP-2 and MMP-9) exceed those of
their inhibitors in the media, in agreement with previous
results showing predominance of MMPs over TIMPs in prostatic cancer (Lokeshwar et af., 1993). Actually, our EIA data
illustrate the imbalance between the total amounts of MMPs
and of TIMPs in favor of the proteinases. Our studies on EIA
and gelatin zymography demonstrated significant elevation of
MMP-2 and MMP-9 in carcinoma tissues. The enhanced
gelatinolytic activity is thus ascribed to over-production of
these MMPs. This is also supported by our immunohistochemical findings that many carcinoma cells were positively immunostained. However, discrepancies between EIA data and imrnunostaining were noted with MMP-1, TIMP-1 and TIMP-2.
Low levels of MMP-1 in culture media and high immunochemical expression may be due to intracellular storage of MMP-1 in
carcinoma cells and/or binding of the enzyme to collagen
fibers in tissues. When COS-1 cells were transfected with
cDNA for TIMP-1 or TIMP-2, TIMP-1 was recovered mainly
in culture media but very little in cell lysates. However,
TIMP-2 was present in cell lysates, with only small amounts in
media (data not shown). It is known that TIMP-2 can bind to
tumor cell membranes, probably via receptors (Hayakawa et
al., 1994). The discrepancies of TIMP results may thus be
explained by different secretion pathways and binding properties to carcinoma cells.
Previous studies have indicated that appearance of active
MMP-2 correlates with tumor grade and/or spreading of
breast (Davies et al., 1993) and lung (Brown et al., 1993)
carcinomas. Our zymographical analyses also demonstrated
correlation between activation of pro-MMP-2 and tumor cell
invasion, including wall penetration and lymphatic permeation. Although no correlation was observed between activation in culture media and vascular invasion, the degree of
carcinoma cell membrane localization of MMP-2 directly
correlated with vascular invasion. Our data further support the
notion that activation of pro-MMP-2, in addition to its production, in local cancer tissues contributes to local and vascular
invasion by malignant tumor cells (Brown et af., 1993; Davies et
af., 1993). Our studies on pro-MMP-2 activation in gastric
cancer tissues have shown that activation is achieved in
carcinoma cell nests via the action of MT-MMP (Nomura et af.,
1995), stressing the importance of MT-MMP in the progression of gastric carcinoma through the activation of proMMP-2. Activation of pro-MMP-9 in some lung carcinoma
tissue samples was also observed in the present and previous
studies (Brown et af., 1993). However, our and other studies
(Brown et al., 1993) showed no correlations between proMMP-9 activation and clinicopathological factors.
ACKNOWLEDGEMENTS
We are grateful to Dr. I. Nakanishi, Department of Pathology, School of Medicine, Kanazawa University, for his useful
advice and critical reading of the manuscript. This work was
supported by the Hokkoku Foundation for Cancer Research
and a Grant-in-Aid (06281107) from the Ministry of Education, Science and Culture of Japan (Y.O.).
16
NOMURA ETAL.
REFERENCES
T., HOYHTYA,
AUTIO-HARMAINEN,
H., KARTNNEN,T.. HURSKAINEN,
M., KAUPPILA,
A. and TRYGGVASON,
K., Expression of 72 kilodalton
type IV collagenase (gelatinase A) in benign and malignant ovarian
tumors. Lab. Invest., 69,312-321 (1993).
BIRKEDAL-HANSEN,
H., MOORE,W.G.I., BODDEN,M.K., WINDSOR,
L.J., BIRKEDAL-HANSEN,
B., DECARLO,A. and ENGLER,
J.A., Matrix
metalloproteinases: a review. Crit. Rev. oral biol. Med., 4, 197-250
(1993).
BROWN,P.D., BLOXIDGE,
R.E., STUART,N.S.A., GAPER, K.C. and
CARMICHAEL,
J., Association between expression of activated 72kilodalton gelatinase and tumor spread in non-small-cell lung carcinoma. J. nut. Cancerhist.. 85,574-578 (1993).
J.M., HOLM,R. and SOBRINHO-SIMOES,
M.,
DAVID,L., NESLAND,
Expression of laminin, collagen IV, fibronectin, and type IV collagenase in gastric carcinoma. Cancer, 73,518-527 (1994).
DAVIES,B., MILES,D.W., HAPPERFIELD,
L.C., NAYLOR,M.S., BoBROW, L.G., RUBENS,
R.D. and BALKWILL,
F.R., Activity of type IV
collagenases in benign and malignant breast disease. Brit. J. Cancer, 67,
1126-1131 (1993).
EMONARD,
H.P., REMACLE,A.G., NOEL, A.C., GRIMAUD,J.-A.,
STETLER-STEVENSON,
W.G. and FOIDART,J.-M., Tumor cell surfaceassociated binding site for the M,72,000 type IV collagenase. Cancer
Res., 52,5845-5848 (1992).
FREIJE,
J.M.P., DiEZ-ITZA. I., BALB~N,
M., S ~ C H E L.M.,
Z , BLASCO,
R.,
TOLIVIA,
J. and LOPEZ-OT~N,
C.: Molecular cloning and expression of
collagenase-3, a novel human matrix metalloproteinase produced by
breast carcinomas. J. biol. Chem., 269,16766-16773 (1994).
N., IWATA,K., SHINYA,
T., OKADA,Y. and
FUJIMOTO,
N., HOSOKAWA,
HAYAKAWA,
T., A one-step sandwich enzyme immunoassay for inactive precursor and complexed forms of human matrix metalloproteinase 9 (92-kDa gelatinaseitype IV collagenase, gelatinase B) using
monoclonal antibodies. Clin. chim.Acfa, 231,79-88 (1994).
FUJIMOTO,
N., MOURI,N., IWATA, K., OHUCHI,
E., OKADA,Y. and
HAYAKAWA,
T., A one-step sandwich enzyme immunoassay for human
matrix metalloproteinase 2 (72-kDa gelatinaseitype IV collagenase)
using monoclonal antibodies. Clzn. chim. Acfa, 221,91-103 (19930).
T., OKADA,
Y. and
FUJIMOTO, N., ZHANG,J., IWATA,K., SHINYA,
HAYAKAWA,
T., A one-step sandwich enzyme immunoassay for tissue
inhibitor of metalloproteinases-2 using monoclonal antibodies. Clin.
chim. Acta, 220,31-45 (1993b).
GRIGIONI,
W.F., D'ERRICO, A,. FORTUNATO,
C., FIORENTINO, M.,
MANCINI,A.M., STETLER-STEVENSON,
W.G., SOBEL,M.E., LIOTTA,
L.A.. ONISTO,M. and GARBISA,S., Prognosis of gastric carcinoma
revealed by interactions between tumor cells and basement membrane. Mod. Pathol., 7,220-225 (1994).
T., YAMASHITA,
K., OHUCHI, E. and SHINAGAWA,
A,, Cell
HAYAKAWA,
growth-promoting activity of tissue inhibitor of rnetalloproteinases-2
(TIMP-2). J. CeIlSci., 107,2373-2379 (1994).
HEWITT,R E , LEACH,I.H., POWE,D.G., CLARK,
I.M., CAWSTON,
T.E.
and TURVER.
D.R.. Distribution of collagenase and tissue inhibitor of
metalloproteinases (TIMP) in colorecta tumors. Znt. J. Cancer, 49,
666-672 (1991).
JAPANESE
RESEARCHSOCIETYFOR GASTRICCANCER:The general
rules for the Gastric Cancer Study in Surgery and Pathology: 11.
Histological classification of gastric cancer. Jpn. J. Surg., 11, 140-145
(1981).
KODAMA,
S., IWATA,K.. IWATA,H., YAMASHITA,
K. and HAYAKAWA,
T., Rapid one-step sandwich enzyme innmunoassay for tissue inhibitor
of metalloproteinases. An application for rheumatoid arthritis serum
and p1asma.J. imnunol. Mefh., 127, 103'-108 (1990).
LOKESHWAR,
B.L., SELZER,M.G., BLOCK, N.L. and GUNJA-SMITH,
Z.,
Secretion of matrix metalloproteinases and their inhibitors (tissue
inhibitor of metalloproteinases) by human prostate in explant cultures:
reduced tissue inhibitor of metallo roteinases secretion by malignant
tissues. Cancer Res., 53,4493-4498 f1993).
S., NAVRE,M.: COFFEY,R.J., JR. and MATRISIAN,
L.M.,
MCDONNELL,
Expression and localization of the matrix metalloproteinase pump-1
(MMP-7) in human gastric and colon carcinomas. Mol. Carcinogenesis,
4,521-533 (1991).
NOMURA,
H., SATO,H., SEIKI,M., MAI,M. and OKALIA,
Y., Expression
of membrane type-matrix metalloproteinase in human gastric carcinomas. Cancer Res., 55,3263-3266 (1995).
OBATA,K., IWATA,K., OKADA,Y., KOHRIN, Y., OHUCHI,
E., YOSHIDA,
S., SHINMEI,
M. and HAYAKAWA,
T., A one-step sandwich enzyme
immunoassay for human matrix metalloproteinase 3 (stromelysin-1)
using monoclonal antibodies. Clirz. chinr. Acta, 211,59-72 (1992).
OTANI,Y., OKAZAKI,
I., ARAI, M., KAMEYAMA,
K., WADA, N.,
K., YOSHINO,K., KITPJIMA,M., HOSODA,Y. and
MARUYAMA,
TSUCHIYA,
M., Gene expression of iinterstitial collagenase (matrix
metalloproteinase 1) in gastrointestinal tract cancers. J. Gastroenterol.,
29,391-397 (1994).
POULSOM, R., PIGNATELLI, M., STETLER-STEVENSON, W.G., LIOTTA,
P.A.,JEFFERY,
R.E., LONGCROFT,
J.M., ROGERS,L. and
L.A., WRIGHT,
STAMP,G.W.H., Stromal expression of 72 kda type IV collagenase
(MMP-2) and TIMP-2 mRNAs in colorectal neoplasia. Anier. J.
Patho/., 141,389-396 (1992).
PYKE?C., RALFKIAER,
E., TRYGGVASON,
K. and DAN^, K.. Messenger
RNA for two type IV collagenases is located in stromal cells in human
colon cancer. Amer. J. Pathol., 142,359--365 (1993).
SATO,H., TAKINO,
T., OKADA,Y., CAO,J., SHINAGAWA,
A., YAMAMOTO,E. and SEIKI,M., A matrix metalloproteinase expressed on the
surface of invasive tumor cells. Nature (Lond.), 370,61-65 (1994).
SHIMA,I., SASAGURI,
Y., KUSUKAWA,
J., YAMANA,H., FUJITA,H.,
KAKEGAWA,
T. and MORIMATSU,
M., Production of matrix metalloproteinase-2 and metalloproteinase-3 related to malignant behavior of
esophageal carcinoma. Cancer, 70,2747-2753 (1992).
STETLER-STEVENSON,
W.G., AZNAVOORIAN,
S. and LIOTTA,L.A.,
Tumor cell interactions with the extracellular matrix during invasion
and metastasis. Annu. Rev. CellBioL, 9, 541-573 (1993).
VISSCHER,
D.W., HOYHTYA,
M., OPOSE~N,
S.K., LIANG,C.-M., SARKAR,
F.H.. CRISSMAN,
J.D. and FRIDMAN,
R., Enhanced expression of tissue
inhibitor of metalloproteinases-2 (TIhdP-2) in the stroma of breast
carcinomas correlates with tumor recurrence. Int. J. Cancer, 59,
339-344 (1994).
WOLF,C., ROUYER,
N., LUTZ,Y . , ADIDA,C., LORIOT, M., BELLOCQ,
J.-P., CHAMBON,
P. and BASSET,P., Stromelysin 3 belongs to a
subgroup of proteinases expressed in breast carcinoma fibroblastic
cells and possibly implicated in tumor progression. Proc. nut. Acad. Sci.
(Wash.), 90,1843-1847 (1993).
ZHANG,J., FUJIMOTO,
N., IWATA,K., SAKAI,T., OKADA,Y. and
HAYAKAWA,
T., A one-step sandwich enzyme immunoassay for human
matrix metalloproteinase 1 (interstitial collagenase) using monoclonal
antibodies. Clin. chirn. Acfa, 219, 1-14 (1993).
Документ
Категория
Без категории
Просмотров
3
Размер файла
831 Кб
Теги
830
1/--страниц
Пожаловаться на содержимое документа