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1-Proteinase Inhibitor Generated by Matrix Metalloproteinases
From the Second Department of Pathology*
and
First Department of Surgery,
Miyazaki Medical
College, Miyazaki, Japan, and the Department of Cancer Cell
Research,
The Institute of Medical Science,
The University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-proteinase inhibitor (PI)-degrading activity generating a
carboxyl-terminal fragment of ~5 kd (PI-C). This study reports
that overexpression of PI-C in S2–020, a cloned subline
derived from the human pancreas adenocarcinoma cell line
SUIT-2, potentiates the growth capability of the cells in nude
mice. After stable transfection of a vector containing a chimeric cDNA
encoding a signal peptide sequence of tissue inhibitor of
metalloproteinase-1 followed by cDNA for PI-C into S2–020
cells, three clones that stably secrete PI-C were obtained.
The ectopic expression of PI-C did not alter in vitro
cellular growth. However, subcutaneous injection of the
PI-C-secreting clones resulted in tumors that were 1.5 to 3-fold
larger than those of control clones with an increased tendency to
invasiveness and lymph node metastasis. These effects could be a result
of modulation of natural killer (NK) cell-mediated control of tumor
growth in nude mice, as the growth advantage of
PI-C-secreting clones was not observed in NK-depleted mice,
and PI-C-secreting clones showed decreased NK sensitivity in
vitro. In addition, production of PI and generation
of the cleaved form of PI by MMP were observed in various human
tumor cell lines and in a highly metastatic subline of SUIT-2 in
vitro. These results provide experimental evidence that the
PI-degrading activity of MMPs may play a role in tumor progression
not only via the inactivation of PI but also
via the generation of PI-C.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-proteinase
inhibitor (PI).7
PI, usually called 1-antitrypsin,
is a major plasma serpin that has a broad inhibitory spectrum against
serine proteinases and is the primary physiological inhibitor of
leukocyte elastase. Among MMPs, interstitial collagenase (MMP-1, EC
3.4.24.7), neutrophil collagenase (MMP-8, EC 3.4.24.34), stromelysin-1
(MMP-3, EC 3.4.24.17), matrilysin (MMP-7, EC 3.4.24.23), and
stromelysin-3 (MMP-11) effectively cleave PI.2-6
Of
these, matrilysin exhibits the most efficient PI-degrading
activity,4, 5
and stromelysin-3 is more potent than
collagenases and stromelysin-1.6
Moreover, a matter of
importance is that the mature forms of human stromelysin-3 exhibit a
very limited substrate specificity and appear unable to degrade any
major ECM component.6
The cleavages of PI by these MMPs
occur at peptide bonds within PI's active-site
loop,2-6
resulting in the inactivation of the inhibitory
activity and release of a carboxyl-terminal (C-terminal) fragment
of ~5 kd (PI-C) that is thought to be largely concealed in a
native PI. We have found that the tumor-cell-derived PI and its
cleaved form, which is ~5 kd smaller than the noncleaved PI, were
concomitantly present in the serum-free culture conditioned media of
the tumor cell lines.7
These observations led to the
postulation that the MMP-dependent hydrolysis of PI and the
subsequent generation of PI-C may occur in a tumor-cell
microenvironment.
To date, little is known about the biological significance of PI and
the PI-degrading activity of MMPs such as matrilysin and
stromelysin-3 in tumors, and rather surprisingly, patients with
immunohistochemically PI-positive adenocarcinomas had worse
prognosis than the negative ones.8-10
On the other hand,
the ectopic expression of human stromelysin-3 in MCF-7 breast carcinoma
cells resulted in enhanced tumorigenicity of the cells in nude
mice.11
Similarly, overexpression of matrilysin in a colon
carcinoma cell line was found to increase its tumorigenicity in nude
mice without modulation of the invasive property in
vitro,12
and intestinal tumorigenesis was suppressed
in mice lacking matrilysin.13
This evidence suggests that
MMPs such as stromelysin-3 and matrilysin somehow favor the survival
and growth of the cancer cells in a tissue microenvironment in
vivo, possibly by functioning in an undefined capability
independent of ECM degradation.14
One of the key events in the survival and growth of cancer cells
in vivo is a resistance to the host immune system. Natural
cytotoxicity, mediated by natural killer (NK) cells and
lymphokine-activated killer (LAK) cells, plays an important role in the
host defense mechanism against cancer cells.15
As NK
activity within tumors of patients with cancer is lower than that found
in the peripheral blood, the presence of tumor-derived suppressor
factors has been suggested.15
Cercek et al16,17
have purified a peptide that could have immunosuppressive effects from
sera of patients bearing various solid cancers. This peptide,
designated as CRISPP (cancer recognition, immune defense suppression,
and serine protease protection peptide), was reported to have unique
reversible suppressive effects on NK and LAK cells in
vitro.17
Of particular interest is that the amino acid
sequence of the CRISPP is highly homologous (83% to 100%) to that of
the C-terminal part of PI
(Met358-Gln393).16
As this
C-terminal part overlaps in PI-C generated by the MMP-dependent
hydrolysis of PI, it can be hypothesized that PI-C may have
suppressive activity against NK and LAK cells and that MMPs,
particularly matrilysin and stromelysin-3, may contribute to the
survival of cancer cells in vivo through the generation of
PI-C from the tumor-cell-derived and/or host-derived PI. However,
the biological significance of PI-C in vivo is yet to be
clarified. To confirm the above hypothesis in vivo, we
have attempted to construct an PI-C expression/secretion vector to
examine the effects of PI-C in vivo. The generation
of PI-C by tumor cells enhanced the growth and invasiveness of the
tumor cells in nude mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All cultured cells are derived from cells of human origin. Cloned sublines S2–020, S2–007 and S2–028 were derived from a single pancreas adenocarcinoma cell line, SUIT-2.18 S2–020 formed poorly differentiated adenocarcinoma in the nude mouse.18 It was low metastatic in a spontaneous metastasis assay in nude mice but was highly invasive in vitro.19,20 S2–007 formed moderately differentiated adenocarcinoma and was highly metastatic in nude mice but was low invasive in vitro.18-20 S2–028 formed well differentiated adenocarcinoma and was nonmetastatic in nude mice and noninvasive in vitro.19,20 These clones were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS). S2–020 was used for the transfection study described below. In addition to the above-described SUIT-2 subclones, a normal fetal intestinal cell line (FHs74 int), a fibroblast cell line (Flow 2000), 7 colorectal adenocarcinoma cell lines (RCM-1, -2, and -3, CoCM-1, SW837, WiDr, and Colo 205), and 2 gastric adenocarcinoma cell lines (MKN-45 and -28), another SUIT-2 subline (S2–013), a renal cell carcinoma line (MRT-1), lung adenocarcinoma and squamous-cell carcinoma lines (LC-2/ad and LC-1/sq), 3 urinary bladder transitional-cell carcinoma lines (UMK-1 and -2 and T-24), a fibrosarcoma cell line (HT1080), an osteosarcoma cell line (OST), a primary culture of chordoma, and a glioblastoma cell line (MGM-1) were used. FHs74 int, WiDr, SW837, and Colo 205 were obtained from Dainihon Seiyaku (Osaka, Japan). MKN-45 and -28 were from IBL (Fujioka, Japan). Flow 2000 was from Japanese Cancer Research Resources Bank (Osaka, Japan). T-24 and OST were from RIKEN Cell Bank (Tsukuba, Japan). HT1080 was a kind gift from Dr. J. Suzumiya, Fukuoka University, Fukuoka, Japan. The other cell lines were established in our laboratory. To obtain serum-free conditioned medium (SFCM), confluent cells were washed three times with serum-free DMEM and cultured in it for 24 hours. To determine effects of tissue inhibitor of metalloproteinase-1 (TIMP-1), 2 µg/ml recombinant human TIMP-1 (rTIMP-1, Fuji Chemical Industries, Toyama, Japan) was added into the serum-free culture medium. Alternatively, the cells were transiently transfected with an expression plasmid pSG5 (Stratagene, La Jolla, CA) harboring a full-length cDNA for human TIMP-1 (pSG-TIMP), using lipofectamine reagent (Gibco-BRL, Gaithersburg, MD). Twenty-four hours after the transfection procedure, the cells were washed three times with serum-free medium, and SFCM was collected as described above. The enhanced secretion of TIMP-1 was confirmed by immunoblot analysis with anti-human TIMP-1 antibody (Fuji Chemical Industries) and by a TIMP-1 ELISA system (Amersham, Little Chalfont, UK). To examine in vitro growth characteristics, replicate 35-mm dishes were seeded at 2 x 105 cells/3 ml of growth medium. The number of viable cells was counted daily, and doubling time was determined during the log-phase of growth.
Construction of PI-C Expression/ Secretion Vector
In an attempt to generate an PI-C expression/secretion vector,
a chimeric cDNA encoding a signal peptide sequence of TIMP-1 followed
by cDNA for PI-C, which is the C-terminal region of PI
(Met358-Arg394), with a stop codon (TAA) at the
3' terminus was constructed. First, cDNA corresponding to PI-C
including the stop codon was obtained by a reverse transcription
polymerase chain reaction (RT-PCR) using poly A+ RNA
obtained from cultured RCM-1 human rectal adenocarcinoma cell line that
synthesizes and secretes PI in vitro.21
A
37-mer forward primer (Atim 1), composed of a 17-mer that corresponded
to the 3' terminus of the signal sequence of TIMP-1 followed by a
20-mer corresponding to the 5' terminus of PI-C, and a 29-mer
reverse primer (Atim 2), composed of a 9-mer carrying a SalI
site followed by a 20-mer corresponding to the 3' terminus of PI-C,
including the stop codon, were used. Sequences of Atim 1 and Atim 2 are
5'-ATAGCCCCAGCAGGGCCATGTCTATCCCCCCAGAGGT-3' and
5'-GCGGTCGACTTATTTTTGGGTGGGATTCA-3', respectively. Second, cDNA
corresponding to the signal peptide sequence of TIMP-1 was amplified. A
plasmid, pSG-TIMP, was used as the template. A 29-mer forward primer
(Atim 3), composed of a 9-mer carrying an XhoI site followed
by a 4-mer corresponding to the untranslated region upstream from the
translational start site compatible with Kozak's rule and a 16-mer
corresponding to the 5' terminus of the signal peptide sequence of
TIMP, and a 20-mer reverse primer (Atim 4) corresponding to the 3'
terminus of the signal peptide sequence of TIMP. Sequences of Atim 3
and Atim 4 are 5'-CCGCTCGAGCCACCATGGCCCCCTTTGAG-3' and
5'-GGCCCTGCTGGGGGCTATCA-3', respectively. Third, the gel-purified
products from both of the above reactions were used as templates for
fusion by overlap extension using PCR. For this reaction, Atim 3 and
Atim 2 were used. The 206-bp DNA product, designated as ATIM, that was
generated in this PCR was ethanol precipitated and agarose gel
purified. In the fourth and final reaction, the product was double
digested by SalI and XhoI, ethanol precipitated,
agarose gel purified, and subcloned into pCI-neo (Promega, Madison,
WI) expression vector to create plasmid pCI-neoATIM. The inserted
sequence was confirmed by a double-strand DNA sequencing of the plasmid
(see Figure 3B
).
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PI-C-Secreting Stable Transfectants
S2–020 cells were transfected with pCI-neoATIM linearized by
BamHI using lipofectamine reagent. Stable transfectants were
selected with geneticin (0.5 mg/ml; Gibco-BRL), and isolated clones
were obtained by ring cloning. For control, S2–020 cells were
similarly transfected with linearized pCI-neo carrying no exogenous
DNA, and the stable transfectants were cloned. Twenty clones of
pCI-neoATIM-transfected (ATIM 1 to 20), and 10 clones of the
mock-transfected control (pCI 1 to 10) were isolated. Each clone was
cultured, and SFCM was harvested. The amount of PI-C peptide in SFCM
was measured by ELISA as described below. Three clones (ATIM 1, 8, and
11) secreted a notable amount of PI-C and were used in the
subsequent experiment.
Preparation of PI-C Antiserum and Immunoassay
Polyclonal anti-PI-C rabbit serum was obtained by immunizing
synthetic peptide corresponding to
Met358-Ile375 of PI synthesized as multiple
antigen peptide resin (Sawady Technology, Tokyo, Japan). The
immunoglobulin fraction was purified (E-Z-SEP purification kit,
Pharmacia, Uppsala, Sweden), and the antibody was further affinity
purified. A 13-mer synthetic peptide corresponding to 12 amino acid
residues of the amino terminus of PI-C with an additional cystein
residue at the C terminus (PI-C13) was conjugated to activated
2-fluoro-1-methylpyridinium toluene-4-sulfonate cellulofine (Seikagaku
Kogyo, Tokyo, Japan) and used for the affinity column preparation. The
secreted PI-C in the conditioned medium was measured by an antibody
capture immunoassay. Briefly, 96-well microtiter plates (MaxiSorp,
Nunc, Naperville, IL) were coated with samples or standard peptide
(PI-C13) solution in 20 mmol/L sodium carbonate buffer (pH 9.6) at
4°C overnight. The presence of adsorbed PI-C was detected with an
enzyme immunoassay using the rabbit anti-PI-C IgG prepared as
described above and peroxidase-conjugated swine anti-rabbit IgG
(Dakopatts, Glostrup, Denmark). O-Phenylenediamine was used
as the color reagent according to the standard technique.7
Absorbance at 492 nm was measured using the microplate
spectrophotometer, and values were analyzed using SOFT max PRO
software (Molecular Devices, Sunnyvale, CA). Sandwich ELISA for
human PI was done according to the method described
previously.7
With this system, bovine PI did not
cross-react.
RNA Blot Analysis, Gelatin Zymography, and Immunoblot Analysis
Poly(A)+-enriched RNA was extracted using the Fast
Track mRNA isolation kit (Invitrogen, San Diego, CA) from the cultured
cells. Two micrograms of poly(A)+-enriched RNA was
electrophoresed on 1% formaldehyde agarose gel and transblotted onto
Hybond-N+ nylon membrane (Amersham), and RNA was
ultraviolet cross-linked onto the membrane. Hybridization was performed
in a mixed solution of 50% formamide, 5X Denhardt's solution, 25
mmol/L phosphate buffer (pH 6.5), 0.1% SDS, 100 µg/ml sonicated and
heat-denatured salmon sperm DNA, and 5X standard saline citrate (SSC)
at 42°C for 16 hours. The blots were washed as follows: three times
in 0.1% SDS in 1X SSC for 15 minutes at room temperature and twice in
the same solution for 20 minutes at 65°C. The membranes were
autoradiographed with Kodak XR-5 film at -80°C for 6 hours or 24
hours. The PI-C cDNA corresponding to the C-terminal region of PI
(Met358-Lys394) was synthesized by PCR using
the pCI-neoATIM plasmid as a template. The 5' end of the reverse primer
was radiolabeled with [
-32P]ATP, and the corresponding
PCR product was gel purified and used as a probe. For internal control
of loading, the blots were subsequently hybridized to
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (Clontech, Palo
Alto, CA). The GAPDH probes were radiolabeled by random priming with
[-32P]CTP.
For gelatin zymography, each SFCM sample derived from the same cell number (3.6 x 104 cells) was applied to SDS-PAGE (10% separating gel) containing 0.1% gelatin as a substrate under nonreducing conditions without boiling. After electrophoresis, the gel was washed in 2.5% (v/v) Triton X-100 at room temperature for 60 minutes with two changes of the detergent solution to remove SDS. The gel was rinsed once with incubation buffer (5 mmol/L CaCl2 and 0.02% NaN3 in 50 mmol/L Tris/HCl, pH 7.6), incubated in the same buffer overnight at 37°C, and then stained with 2.5% Coomassie brilliant blue in 30% methanol and 10% acetate. Enzyme activity was detected as a clear band on the resulting blue background of undigested gelatin.
Immunoblot analyses for matrilysin and PI were done according to the
method described previously.7,22
For the detection of
PI, samples were pretreated with or without peptide N-glycosidase F
(Boehringer Mannheim, Mannheim, Germany) according to the
manufacturer's instructions.7
In addition, purified human
PI (12 µg/ml; Sigma Chemical Co., St. Louis, MO) treated with or
without human recombinant matrilysin (0.3 µg/ml; Oriental Yeast Co.,
Shiga, Japan) was also subjected to the immunoblot analysis using
rabbit anti-human PI IgG (Dakopatts) to confirm the cleavage of
PI by matrilysin.
In Vitro Invasion Assay
The In vitro matrigel invasion assay was done according to the method described previously.20 Briefly, a Chemotaxicell containing an 8-µm pore size polyvinylpyrrolidone-free polycarbonate filter (Kurabo, Osaka, Japan) was coated with Matrigel (Collaborative Research, Bedford, MA; 63.3 µg/cm2) and allowed to dry. The assay was done in the serum-free culture medium containing 0.1% bovine serum albumin, and 50 µg/ml fibronectin (Collaborative Research) was used as a chemoattractant. The cells (2 x 105 cells/well) were placed in the Chemotaxicell and cultured at 37°C for 24 hours. After the incubation, the cells were fixed, and the cells on the upper surface of the filter were wiped off. Then the cells on the lower surface were stained with hematoxylin and counted.
In Vivo Tumorigenicity, Growth, and Invasion/Metastasis Assays
A total volume of 0.15 ml containing 5 x 106
cells was subcutaneously injected into 6-week-old male nude mice
(BALB/c-nu/nu). Tumor volume was estimated by the formula
V = L x W2
x
0.5, where V is volume, L is length, and
W is width. Mice were sacrificed 40 days after the
injection. For an intraperitoneal injection, 1 x 106
cells/0.15 ml were injected, and the body weight of each injected mouse
was examined daily. Mice were sacrificed 28 days after the injection,
and mean gain of the body weight (g)/day of each mouse during the last
2 weeks was calculated. All mice were necropsied, and each organ and
tumor was examined histologically after fixation in phosphate-buffered
formalin (4%) followed by embedding in paraffin. Immunohistochemical
localization of PI-C was performed using anti-PI-C rabbit IgG
prepared as described above using the avidin-biotin complex method.
Anti-Asialo GM1 Antibody Treatment
Mice were given intraperitoneally a 100-µl aliquot (500 µg) of anti-asialo GM1 antibody (Wako Pure Chemical Industries, Osaka, Japan) twice a week from 3 days before until 28 days after the subcutaneous injection of the tumor cells (5 x 105 cells/mouse). Tumor volume was estimated as described above. The mice were sacrificed 28 days after the injection.
NK Sensitivity Assay
Tumor cell targets in the subconfluent culture in 96-well
microtiter plates (1 x 104
cells/well) were labeled
with Na51CrO4 (400 µCi/107
cells)
for 3 hours at 37°C in DMEM with 5% FBS. Then they were washed twice
in serum-free RPMI 1640 medium and cultured in this medium for 30
minutes. They were washed once again and cultured in 100 µl/well of
the serum-free medium for 2 hours to ensure the secretion of PI-C
before addition of effector cells. The effector cells were prepared
from nude mouse spleen cells and added into the targets at different
concentrations. The total volume per well was 200 µl. The assays were
performed in quadruplicate at effector to target ratios (E/T) ranging
from 25:1 to 200:1. Controls included labeled target cells incubated in
culture medium alone (spontaneous release) and labeled target cells
incubated in 1% HCl (maximal release). After 5 hours of incubation at
37°C, the plates were centrifuged for 10 minutes at 700 x
g, and 100 µl of sample was harvested from each well and
counted in a gamma counter. The percentage of specific chromium release
was calculated as follows: (experimental release - spontaneous
release)/(maximal release - spontaneous release) x 100. Results
were expressed as percentage of specific chromium release at different
E/T ratios.
Statistical Analysis
Comparison between groups was determined by one-way ANOVA or Mann-Whitney U test. The P values lower than 0.05 were considered as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PI by SUIT-2 Subclones with
Different Invasive/Metastatic Properties
Before we started our study, the in vivo effects of
PI-C, synthesis and the properties of PI were analyzed in the
subclones with different metastatic and invasive potentials, derived
from a single pancreatic adenocarcinoma cell line, SUIT-2. A metastatic
subclone, S2–007, secreted larger (20-fold or more) amounts of PI
than low (S2–020) or nonmetastatic (S2–028) clones (Table 1
and Figure 1A
). Expression of the PI gene was
also confirmed by RT-PCR in S2–007 (data not shown). With immunoblot
analysis, the S2–007-derived PI showed a very broad band compared
with the normal serum PI. S2–028-derived PI showed a little
higher molecular weight (MW) than the normal serum form, which may
represent an altered heavy N-glycosylation of the tumor-cell-derived
PI as we have reported previously.7
In fact, after
N-glycosidase treatment, S2–028-derived PI showed the same
molecular size (45 kd) as the normal deglycosylated form (Figure 1A)
.
S2–020-derived PI also showed a very weak signal similar to that of
S2–028 (data not shown).
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PI in
Vitro
Peptide N-glycosidase F treatment of SFCM from S2–007 revealed
the presence of a cleaved PI of 40 kd as well as a normal 45-kd
deglycosylated PI (Figure 1B)
. Therefore, the very broad band of
S2–007-derived PI observed in Figure 1A
seems to represent not only
the altered N-glycosylation but also a co-presence of the cleaved form.
The generation of the cleaved form was inhibited by 30% in the
presence of exogenously added rTIMP-1 (2 µg/ml) in the medium, or was
significantly inhibited (85% inhibition) in S2–007 transfected with
human TIMP-1 cDNA in the expression vector pSG5 (Figure 1B)
. The
transfection of TIMP-1 cDNA resulted in markedly increased (~10-fold:
5.65 ± 0.31 µg/ml, mean ± SD) TIMP-1 secretion by S2–007
cells. Endogenous TIMP-1 levels of untreated S2–007, S2–020, and
S2–028 sublines were 0.60 ± 0.27, 0.52 ± 0.01, and
0.42 ± 0.04 µg/ml, respectively. By contrast, addition of 1
µg/ml aprotinin did not cause detectable inhibition (data not shown).
These observations suggested that MMPs were involved, at least partly,
in the generation of the cleaved PI. Indeed, S2–007 secreted high
levels of both latent and active forms of matrilysin (Figure 2A)
, which has a potent PI-degrading
activity (Figure 2B)
.4,5
Moreover, an active form of
interstitial collagenase, which also exhibits the PI-degrading
activity, was also secreted by S2–007.19
As the cleavages
of PI by these MMPs occur at peptide bonds within PI's
active-site loop (Figure 3A)
, resulting
in a release of the C-terminal fragment of ~5 kd (PI-C), it is
reasonable to hypothesize that PI-C can be generated in pericellular
microenvironments of certain tumor cells, through the cleavages of
tumor-cell-derived or host-derived PI. In addition to the 40-kd
PI fragment, a 34-kd fragment was observed in the TIMP-1
transfection study. However, the generation of the 34-kd fragment was
not affected by the expression of TIMP-1 (Figure 1B)
, and the nature of
this fragment is uncertain at present.
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, suggesting that additional phenotypes were
lacking in this subline to sufficiently exhibit its aggressive
potential in nude mice. For instance, S2–020 was most sensitive to
nude mouse NK cells (Table 1)
and secreted very low levels of
matrilysin without detectable active forms (Figure 2A)
as compared with
S2–007. Therefore, we decided to use the S2–020 subline for the
following transfection study to search for a possible role of PI-C
in vivo.
Generation of PI-C Secreting Clones of S2–020
S2–020 cells were transfected with a mammalian expression vector
pCI-neo carrying a chimeric cDNA encoding of a signal peptide sequence
of TIMP-1 followed by cDNA for PI-C, ie, the C-terminal region of
PI (Met358-Lys394) that can be generated by
MMP-dependent hydrolysis of PI (pCI-neoATIM, Figure 3, A and B
).
Among 20 clones of the stable transfectant, 3 clones (ATIM 1, 8, and
11) secreted notable amounts of PI-C into SFCM compared with the
parent and mock-transfected clone (pCI; Figure 3C
). Low levels of
immunoreactivity observed in SFCMs of the control clones may represent
a cross-reactivity of the antibody to the endogenous PI, as S2–020
secreted low levels of PI (Table 1)
, and the immunoreactivity was
decreased when ultrafiltrates (cutoff of 10 kd) of SFCM were used. The
ectopic expression of PI-C mRNA in the transfected clones was also
confirmed by RNA blot analysis (Figure 3D)
.
Effects of Ectopic PI-C Expression on Cellular Characteristics
in Vitro
The secretion of PI-C did not alter the growth of the cells
in vitro significantly, and only ATIM 8 grew a little more
slowly than other clones. In vitro doubling time of S2–020
(parent), pCI 1, pCI 2, ATIM 11, ATIM 1, and ATIM 8 were 35.8, 35.2,
37.3, 38.5, 35.0, and 45.0 hours, respectively. Culture morphology of
the cells was not altered also as judged by a phase-contrast microscopy
(data not shown). To confirm that the transfection procedure did not
affect the secretion of MMPs, gelatin zymography of SFCM derived from
each clone was done. As shown in Figure 4A
, patterns of the gelatinolytic bands
were similar between the clones, and several bands of between 45 and 55
kd were observed as the main activities. In addition, secretion of
matrilysin was not significantly altered by the transfection (Figure 4B)
. Both parent S2–020 cells and transfectants secreted low levels of
matrilysin, and the active forms were not detectable.
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PI-C Expression in Nude Mice
In contrast to in vitro study, subcutaneous injection
of the PI-C-secreting clones in nude mice (5 x 106
cells/mouse) resulted in a tendency of enhanced tumor growth. The
enhancement was statistically significant in two clones (ATIM 1 and 8)
but not in ATIM 11 (Figure 5A)
. Forty
days after the injection, mice were sacrificed and autopsied.
Expression of the PI-C peptide in vivo was confirmed
immunohistochemically (Figure 5B)
. Although the anti-PI-C antibody
could cross-react to the endogenous human PI in the clones, the
amount of PI secreted by each clone was very low and almost the same
(data not shown), and thus the differences of immunoreactivity observed
would be derived from PI-C synthesized by the ATIM clone.
Histologically, the PI-C-secreting clones formed tumors having more
infiltrative margins than controls with a tendency of decreased
infiltration of mononuclear leukocytes (Figure 6)
. Neutrophils were variably
infiltrated, and it may be a reaction to necrotic changes. Metastases
to the axillary lymph nodes were present only in the PI-C-secreting
clones (Table 2)
. Moreover,
intraperitoneal or intrapleural dissemination, mostly due to direct
invasion into the peritoneal or pleural cavity, was observed in 6 of 21
mice in which the PI-C-secreting clones were injected, whereas
neither parent (0/6) nor controls (0/13) formed any dissemination
(Table 2)
. These aggressive behaviors were even noted in ATIM 11, which
did not show significant growth enhancement. Although the
PI-C-secreting clones may exhibit a vague tendency of increased lung
metastases, it was not a statistically significant level compared with
the parent or mock-transfected controls, and numbers of the metastatic
foci were low. In addition, when the mice (n =
3) carrying ATIM-1-derived tumor were sacrificed 18 days after the
injection (mean tumor size, 535 mm3, which is comparable
with the sizes of control tumors on the 40th day shown in Table 2
),
no lung metastasis could be found, whereas lymph node metastases
were present in one mouse (1/3).
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PI-C-secreting clones (Table 2)
, and these mice were
more cachexic. Invasion into the liver and/or pancreas was more evident
in the PI-C-secreting clones (data not shown), resulting in jaundice
in five mice. However, the lung metastases were observed in only one
mouse (ATIM 1) in this experiment, and this may be due to the shorter
experimental period and the lower number of the cells injected compared
with the subcutaneous injection study.
Effects of PI-C Expression on the Growth of S2–020 Cells in
NK-Depleted Nude Mice
As in vitro analysis of cell proliferation did
not show obvious differences between the PI-C secreting clones and
the control clones, one possible explanation for the enhanced growth of
the PI-C secreting clones in vivo is that PI-C may
modulate host-tumor interaction in favor of the tumor cells to survive
and to grow in the tissue microenvironment. To obtain more information
on the mechanism of the PI-C-induced tumor growth and/or
aggressiveness in nude mice, we injected the cells into nude mice
treated with or without anti-asialo GM-1 antibody. This antibody causes
marked depletion of NK activity in the mice.23
In nude
mice, NK-cell-mediated control of tumor growth is an important defense
mechanism against the tumor, and in fact, anti-asialo GM-1 treatment
enhances the growth of tumor cells in nude mice.23,24
When
5 x 105
cells (1/10 of the first experiment) were
injected subcutaneously, the PI-C-secreting clone again showed
enhanced tumor growth compared with the control clone in untreated
mice. This effect was particularly evident at the early stage of the
tumor development (Figure 7A)
. However,
this growth advantage of the PI-C-secreting clone over the control
clone was abolished by anti-asialo GM-1 treatment. The treatment
resulted in an enhanced growth of S2–020 cells in vivo
(Figure 7A)
accompanied by a tendency of reduced infiltration of
mononuclear leukocytes (data not shown), indicating that
NK-cell-mediated control of tumor growth is in fact important in the
growth of S2–020 cells in nude mice. Under the NK-depleted condition
induced by the antibody, tumor growth did not show any obvious
differences between PI-C-secreting clone and nonsecreting clone
(Figure 7A)
. This finding suggests that NK activity is involved, at
least partly, in the effects of PI-C on the tumor growth in
vivo, and PI-C may modulate the host NK-cell activity in favor
of the tumor cells.
|
PI-C Synthesis on NK Cytotoxicity in
Vitro
Finally, NK sensitivities of the clones were compared in
vitro. As shown in Figure 7B
, all PI-C-secreting clones showed
decreased sensitivity to NK cytotoxicity of nonactivated nude mouse
spleen cells. It should be emphasized that the level of NK sensitivity
is inversely correlated with the in vivo aggressiveness of
the cells shown in Table 2
. Although ATIM 1 secreted similar or a
little less PI-C compared with ATIM 8 (Figure 3C)
, ATIM 1 was more
resistant to NK cytotoxicity than ATIM 8. The reason for this finding
is uncertain at present, and there may exist an additional deviation of
the phenotype in ATIM 1
Synthesis of PI by Adenocarcinoma Cell Lines in
Vitro
To determine whether human tumor cells other than SUIT-2
synthesize PI, we have analyzed PI antigen levels in
culture-conditioned media of various human tumor cell lines by enzyme
immunoassay. As shown in Table 3
, PI
was frequently secreted by adenocarcinoma cell lines. A sarcoma cell
line (HT1080), a glioblastoma cell line (MGM-1), and a primary culture
of chordoma also secreted PI. By contrast, normal intestinal
epithelial (FHs74int) and a fibroblast (Flow 2000) cell line did not
synthesize detectable amounts of PI.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PI-C, a
C-terminal fragment of PI generated by MMPs, can enhance the growth
and aggressiveness of the invasive behavior and lymph node metastasis
of S2–020 human pancreas adenocarcinoma cells in vivo.
Although the precise mechanism by which PI-C increases tumor growth
in vivo remains to be clarified, the results described here
suggest that the phenomenon is possibly due to its modulatory effects
on NK-cell activity against the tumor cells. S2–020 is a highly
invasive subline in the in vitro matrigel invasion assay but
exhibits less invasiveness and fewer metastases in nude mice as
compared with S2–007. Therefore, although S2–020 has capability of
invading ECM in vitro, additional phenotypes to sufficiently
express its aggressive potential are lacking in this subline of nude
mice. As S2–020 was NK sensitive and the ectopic expression of PI-C
in S2–020 resulted in decreased sensitivity of the cells to NK
cytotoxicity as well as the increased in vivo growth and
invasiveness, one of the lacking phenotypes required for in
vivo aggressive behavior of this subline may be the resistance to
NK cytotoxicity. In fact, the metastatic subline S2–007 was more
resistant to NK cytotoxicity than S2–020. However, the ectopic
expression of PI-C in S2–020 did not significantly improve its
capability of the spontaneous lung metastasis, and it is possible that
the differences of lung metastases observed between the clones may
simply be derived from the differences in tumor sizes. Thus, one or
more other phenotypes to establish a sufficient number of metastatic
colonies in the lung may still be lacking in this subline. Secretion of
a MMP, such as matrilysin, may be a candidate for the lacking
phenotype, as S2–020 secreted much less matrilysin than S2–007.
Importantly, the findings described in this report could be in
accordance with the previous findings of a CRISPP peptide that is
highly homologous or identical to PI-C and is a suppressor of NK and
LAK cytotoxicity in vitro.16,17
Therefore, this
report confirms the effects of the CRISPP peptide in vivo
for the first time. The CRISPP peptides were reported to be frequently
found in blood plasma of cancer patients but not of healthy
individuals.16
The origin of the CRISPP peptide is
uncertain at present.25
However, theoretically,
MMP-dependent cleavage of PI can also generate a CRISPP-like
peptide, PI-C, in the tumor-cell microenvironment and thus may
contribute to tumor progression. Although we do not have any direct
evidence that a sufficient amount of PI-C is generated by MMPs in
human tumor tissues in vivo, a tumor-cell-derived PI and
its cleaved fragment were concomitantly present in SFCM of some human
adenocarcinoma cell lines7
and the metastatic subline
(S2–007) of SUIT-2. Furthermore, in S2–007, the experimental evidence
suggested that MMPs were involved in the generation of the cleaved
PI. In addition to the tumor-cell-derived PI, interstitial fluid
also contains PI that can be bound to ECM.26
Such an
ECM-bound PI, as well as a soluble form, can also be a substrate for
MMPs.26
These lines of evidences suggest that local
production of PI-Cs would be expected in vivo in invading
tumor tissues with high levels of MMPs, such as matrilysin,
stromelysin-1 and 3, neutrophil collagenase, and interstitial
collagenase. Therefore, together with the fact that the CRISPP peptide
(PI-C homologue) is found in the blood plasma of most cancer
patients,16
the in vivo relevance of the
experimental system described in this report to human cancer seems to
be reasonably supported.
The mechanism underlying the suppression of NK cytotoxicity by PI-C
is uncertain at present. It has been reported that the CRISPP peptide
induces structural changes in mitochondria (SCM) of
lymphocytes16,27,28
and that the amino acid sequence of the
CRISPP peptide corresponding to Phe366-Lys387
of PI is responsible for the immunosuppresive effects in
vitro.17
This amino acid sequence of PI-C also
contains a hydrophobic pentapeptide domain,
Phe370-Met374, corresponding to the SEC
(serpin-enzyme complex) receptor recognition sequence,29,30
suggesting that the sequence may somehow modulate a certain cellular
function. However, the existence of SEC receptors on lymphocytes is
undefined at present. A number of different cell types, including
hepatocytes, mononuclear phagocytes, neutrophils, neuronal cells, and
intestinal epithelial cell line Caco-2 cells express this receptor on
their surface.31
The SEC receptor recognition sequence, as
well as PI-C, is chemotactic for neutrophils and can induce an
increase in the intracellular free Ca2+
concentration.32-34
It should be noted that this sequence
is exposed when PI is complexed with its physiological target
elastase or with other serine proteinases,29,30
and these
PI-proteinase complexes can be generated in sites of inflammation or
in tumor tissues. In this respect, it would be very interesting to know
whether the PI-elastase complex also exhibits NK-suppressive
effects. Clearly, further efforts would be necessary to explore the
biological activity of PI-C and the precise mechanisms underlying
the activity. In addition to PI-C, an immunosuppressive effect of a
noncleaved form of PI has been reported,35-37
and the
effect seems to be independent of its proteinase inhibitory
activity.37
Previous immunohistochemical studies revealed that patients with
PI-positive adenocarcinomas of colon and lung had a worse prognosis
than PI-negative ones, particularly in the early
stage.8,9
Similar, if not identical, results were also
reported in gastric adenocarcinoma.10,38
Production of
PI by the tumor cells themselves was previously observed and, in
this report, in a variety of adenocarcinomas in
vitro.7,21,39
Matrilysin is a very potent proteinase
in generating PI-C,4,5
and an enhanced expression of
matrilysin in tumor cells, including adenocarcinoma cells, has been
reported by a number of groups.1,22,40-42
Recently it
appeared that the expression of matrilysin is associated with
tumorigenicity,12,13
suggesting that this enzyme may have
an undefined important function in the early stage tumor progression.
These lines of evidence, together with the results described in this
report, may support a hypothesis that generation of PI-C from
tumor-cell-derived or host-derived PI by matrilysin in a tumor-cell
microenvironment may contribute, at least partly, to the early-stage
tumor progression via modulation of tumor-host immune
interactions. In addition, our results may gain insight into the
biological role of stromelysin-3 in vivo, which has been
largely undefined. Human stromelysin-3, in its active form, is
specifically secreted by fibroblasts located in the vicinity of cancer
cells.1,42,43
It is associated with poor prognosis of
cancer patients44,45
and somehow contributes to the
survival of tumor cells in nude mice, particularly in the early stage
of the tumor development.11
At present, PI is the only
known physiological substrate for the mature forms of human
stromelysin-3.6
Although a possibility of the presence of
undefined specific stromelysin-3 substrates, which are critical to
early-stage tumor progression, does remain, our results suggest that
stromelysin-3 may have a role in cancer cell-host interaction in favor
of the cancer cells, not only by inactivation of PI but also by
generating PI-C in the pericellular microenvironment.
In conclusion, this study provides experimental evidence for an
in vivo role of the carboxyl-terminal fragment of PI,
which could be generated by MMPs. Future investigations to explore a
pathophysiological role of this proteolytic fragment are required. The
results will add constructively to the existing body of knowledge
relating to the role of MMPs in tumors and support the recently
emerging concept that MMPs have a much more complex role in tumor
progression than previously believed.14
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by a Grant-in-Aid for Encouragement of Young Scientists 06857018 (to H. Kataoka) from the Ministry of Education, Science, and Culture and a grant from the Special Coordination Funds of the Science and Technology Agency (to M. Koono), Japan.
Accepted for publication October 26, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 antitrypsin by human stromelysin. FEBS Lett 1991, 279:91-94[Medline]
1-proteinase inhibitor by matrilysin and stromelysin. Biochim Biophys Acta 1994, 1199:224-228[Medline]
1-antitrypsin. Biochem Biophys Res Commun 1994, 204:613-620[Medline]
1-antitrypsin secreted by human adenocarcinoma cell lines. FEBS Lett 1993, 328:291-295[Medline]
-1-antitrypsin in early stage of colorectal carcinomas. Int J Cancer 1990, 45:244-250[Medline]
-1-antitrypsin expression in adenocarcinomas of the lung: an immunohistochemical analysis. Br J Cancer 1992, 65:300-302[Medline]
-1-antitrypsin-protease complexes. J Biol Chem 1991, 266:11281-11288
1-antitrypsin deficiency and Alzheimer's disease. Pediatr Res 1994, 36:271-277[Medline]
1-Proteinase inhibitor is a neutrophil chemoattractant after proteolytic inactivation by macrophage elastase. J Biol Chem 1988, 263:4481-4484-1-antitrypsin-elastase complexes and amyloid-ß peptide. J Clin Invest 1992, 90:1150-1154
1-Antitrypsin is an effector of immunological stasis. Nature 1978, 274:589-590[Medline]
1-antitrypsin inhibit natural killing and antibody-dependent cell-mediated cytotoxicity. Scand J Immunol 1982, 15:109-113[Medline]
1-antichymotrypsin and 1-proteinase inhibitor on NK cytotoxicity. Only 1-proteinase inhibitor inhibits natural killing. Clin Chim Acta 1990, 190:163-174[Medline]
1-antichymotrypsin and 2-macroglobulin in human gastric carcinomas: a retrospective immunohistochemical study. Hum Pathol 1984, 15:957-964[Medline]
1-proteinase inhibitor. Biol Chem Hoppe-Seyler 1996, 377:301-311[Medline]
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