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Type IV collagenase (matrix
metalloproteinase-2 and -9)
in prostate cancer
L Zhang
1
, J Shi
1
, J Feng
1
, H Klocker
2
, C Lee
3
& J Zhang
1
*
1
The Key Laboratory of Bioactive Materials, Ministry of Education, Institute for
Molecular Biology, Nankai University, Tianjin, P.R. China;
2
Department of Urology,
University of Innsbruck, Innsbruck, Austria; and
3
Northwestern University Feinberg
School of Medicine, Chicago, USA
Background: The type IV collagenases/gelatinases matrix metalloproteinase-2
(MMP-2) and -9 (MMP-9) play an important role in cancer invasion and metastasis.
In the present study, we measured the expression of mRNAs and enzymatic
activities of MMP-9 and -2 in prostate tissues and serum samples from men with
or without prostate cancer.
Methods: A total of 44 tissue samples (three from healthy volunteers, 21 from
patients with benign prostate hyperplasia, 10 from patients with localized prostate
cancer and 10 from patients with metastatic disease) and 71 serum samples were
collected (20 from healthy volunteers, 26 from patients with benign prostatic
hyperplasia, 10 from patients with localized cancer, 15 from patients with
metastatic cancer). The level of mRNA for MMP-2 and -9 was determined
by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR).
The enzymatic activity of MMPs was determined by zymography.
Results: Expression of MMP-9 mRNA was significantly higher in malignant than
in nonmalignant prostate tissues (Po0.001), while no significant difference of
MMP-2 expression was detected in different prostate tissues. Results of
zymography showed that there was significant difference in the enzymatic activity
of MMP-9, but not MMP-2, among normal prostate, BPH, localized and metastatic
prostate cancer tissues, serum samples (Po0.05). The active form of MMP-2, with a
molecular mass of 62 kDa, was detected in normal prostate, BPH and prostate
cancer tissues, but not in the serum samples. Moreover, there was a significant
difference in the ratio of the active form (62 kDa) and proform (72 kDa) of MMP-2
among normal, BPH and prostate cancer tissues. This ratio was further increased
in metastatic prostate cancer tissues.
Conclusion: The activity of MMP-9 and the ratio of active form/proform of
MMP-2 are associated with the progression and metastasis of prostate cancer.
Prostate Cancer and Prostatic Diseases (2004) 7, 327–332. doi:10.1038/sj.pcan.4500750
Published online 7 September 2004
Keywords: metastasis; MMP-2; MMP-9; zymography; RT-PCR
Introduction
Tumor metastasis involves extensive interactions
between the invading cancer cells and the surrounding
stromal cells. Such interactions promote degradation of
the extracellular matrix (ECM) by specialized proteolytic
enzymes, which are produced by both the cancer cells
and the stromal cells, and are likely to affect both
primary and metastatic sites. Among these enzymes,
urokinase and a variety of matrix matalloprotienases
(MMPs) play important roles.
1–4
Among different MMPs, MMP-2 (or gelatinase A) and
MMP-9 (or gelatinase B) collectively referred to as type
IV collagenases or gelatinases, have been found to be
specifically associated with prostate cancer metastasis.
Elevated levels of MMP-2 and -9 in the plasma and urine
have been correlated with metastasis in prostate cancer
patients.
5,6
Secretion of MMP-2 and -9 induce tumor
Received 3 March 2004; revised 29 April 2004; accepted 14 June 2004;
published online 7 September 2004
*Correspondence: J Zhang, Institute for Molecular Biology, Nankai
University, Tianjin 300071, P.R. China.
E-mail: zhangju@nankai.edu.cn
Prostate Cancer and Prostatic Diseases (2004) 7, 327–332
&
2004 Nature Publishing Group All rights reserved 1365-7852/04
$30.00
www.nature.com/pcan
angiogenesis in prostate cancer cells.
7,8
Pettaway and
colleagues found that the expression of MMP-2 and -9
mRNA, in comparison with E-cadherin expression, at
biopsy could predict advanced prostate cancer at radical
prostatectomy.
9,10
In the present study, we investigated the expression of
MMP-2 and -9 in prostate tissue samples using a
semiquantitative RT-PCR. Zymographic techniques are
used for the detection of enzymatic activities.
11
We found
that MMP-9 was associated with prostate cancer in tissue
and serum specimens. We also detected a change in the
ratio of the active form and proform of MMP-2 in
prostate cancer.
Materials and methods
Tissue and serum samples
BPH and prostate cancer tissue samples were obtained
during open prostatectomy surgery, and immediately
frozen in liquid nitrogen and stored at 801C. Prostatic
tissues were obtained from automobile accident victims
(age 22–28 y, median 24), patients with BPH (age 54–67 y,
median 63), patients with organ-confined (age 57–72 y,
median 69), and patients with metastasis disease (age
58–74 y, median 71). For gelatin zymography, three nor-
mal prostate, 21 BPH, 10 organ-confined prostate cancer
and 10 metastatic tissues were sampled.
Serum samples were collected from 10 patients with
organ-confined prostate cancer (age 57–76 y, median 71),
15 patients with metastatic prostate cancer (age 58–74 y,
median 71), 26 patients with BPH (age 51–69 y, median
66) and 20 healthy volunteers (10 male and 10 female;
age 23–45 y, medium 27).
Procurement of the above tissue and serum specimens
has been approved by the Institutional Review Board
of Nankai University. Clinical specimens received the
exemption status, while specimens from automobile
accident victims received consent for research from
family members.
RNA extraction
Total RNA was prepared from tissue samples using
Trizol (Gibco) according to the manufacturer’s instruc-
tions. RNA was suspended in DEPC-H
2
O and stored at
801C until use. The purity of the RNA was established
by reading the optical density of each sample at 260 and
280 nm, using Ultrospec 1100 pro Spectophotometer
(American Pharmacia).
Reverse transcriptase-polymerase chain reaction
(RT-PCR)
An aliquot of 1.0 mg of RNA was added to RNase-free
water to a final volume of 10 ml, denatured for 5 min at
721C and cooled immediately on ice, followed by the
addition of RT mixture (10 ml), which contained first-
strand buffer, 200 U of Moloney murine leukemia virus,
20 U of RNasin, 10 mM DTT, 4.75 mM random hexamers
and 500 mM deoxynucleotides (Promega, Madison, WI,
USA). The reaction was carried out at 371C for 2 h,
followed by an enzyme inactivation step for 5 min at
951C. The resulting cDNA was stored at 201C until use.
The PCR reaction was carried out in 25 ml of final
volume containing 1.0 ml of cDNA, 0.5 mM of each primer,
1 PCR buffer, 0.2 mM each dNTP, 1.5 mM MgCl
2
, 0.75 U
AmpliTaq Gold DNA polymerase. The following primers
were used:
MMP-2 forward primer, 5-ACC TGG ATG CCG TCG
TGG AC-3;
MMP-2 reverse primer, 5-TGT GGC AGC ACC AGG
GCA GC-3 (for amplification of a 447-bp product for
human MMP-2, accession number J03210);
MMP-9 forward primer, 5-GGT CCC CCC ACT GCT
GGC CCT TCT ACG GCC-3;
MMP-9 reverse primer, 5-GTC CTC AGG GCA CTG
CAG GAT GTC ATA GGT-3 (for amplification of a
640-bp product for human MMP-9, accession number
NM-004994);
b-microglobulin forward primer, 5-ATG CCT GCC
GTG TGA ACC ATG T-3;
b-microglobulin reverse primer, 5-AGA GCT ACC
TGT GGA GCA ACC T-3 (for amplification of a 285-bp
product for human b-microglobulin, accession number
NM-004048).
The PCR reaction was conducted with the following
steps: after an initial denaturation step of 5 min at 941C,
34 cycles of denaturation at 951C for 25 s, primer
annealing at 581C for 1 min and extension at 721C for
1 min were performed (21 cycles for b-microglobulin).
A final extension step was performed at 721C for 5 min
in order to complete the PCR reaction. Possible DNA
contamination was monitored by performing PCR in the
same conditions without the addition of cDNA. PCR for
the housekeeping gene (b-microglobulin) and MMP
genes were performed at the same annealing tempera-
ture in the same cycle run for all samples. This procedure
was followed so that comparison of gene expression in
different samples was performed under the same
conditions of amplification.
Semiquantitative analysis of PCR products
A detailed procedure for the semiquantitative analysis of
PCR products for MMP-2 and -9 was reported earlier.
12
In the present study, we used b-microglobulin as the
internal standard. The PCR products for MMPs and
b-microglobulin were analyzed by 1.5% agarose gel
electrophoresis. PCR products were visualized by ethi-
dium bromide staining. The density of each band was
measured by a computer-assisted image analysis system
(Syngene). Integrated density intensity of the band for
b-microglobulin, the housekeeping gene, of each sample
was arbitrarily set as 1 and the density of the band of
individual genes was adjusted to this value.
Zymography
The zymography was conducted according to
established reports
11,13
with minor modifications. Briefly,
fresh prostatic issues were cut into small pieces and
mixed with ice-cold extraction buffer (50 mM Tris-HCl
pH7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA). The
mixture was homogenized at 41C and centrifuged at
10 000 g for 10 min. The supernatant fraction from each
Type IV collagenase in prostate cancer
L Zhang et al
328
Prostate Cancer and Prostatic Diseases
preparation was transferred into a new tube, and the
protein concentration was estimated, using the Bio-Rad
protein assay reagent (Bio-Rad). An aliquot of 40 mgof
protein was mixed with the sample buffer in nonredu-
cing conditions (in the absence of mercaptoethanol or
DTT) and loaded onto a 10% polyacrylamide gel, which
has been incorporated with 1.0 mg/ml gelatin (Sigma)
for electrophoresis. The serum samples were treated with
the same protocol. Conditions for electrophoresis were
100 V for 1.5 h alongside with a broad-range molecular
weight marker (Bio-Rad). At the conclusion of electrophor-
esis, SDS was removed by washing the gel twice for 30 min
with 2.5% Triton X-100 in 50 mM Tris-HCl (pH 7.5) and
once for 20 min with 50 mM Tris-HCl (pH 7.5). The gels
were incubated overnight at 371Cwith50mMofTris-HCl
(pH 7.5), 0.15 M of NaCl, 10 mM of CaCl
2
,0.1%ofTritonX-
100. Staining was carried out for 1 h at room temperature
with 0.5% Coomassie brilliant R-250 in 45% methanol and
10% acetate, followed by destaining with 45% methanol
and 10% acetate until clear bands over a blue background
were observed. Purified human MMP-2 and -9 (Chemicon)
were loaded at 10 ng per lane as controls. The relative
intensity of each gel was normalized against the respective
controls and was expressed as the fraction of the control.
The intensity of bands corresponding to MMP-2 and -9
was measured using a computer-assisted image analysis
system(Syngene).Theratioofthebandsoftheactiveform
(62 kDa) over the proform (72 kDa) of MMP-2 (active
form/proform) was also calculated.
Statistics analysis
The normalized expression of MMP-2 or -9 was esti-
mated by their median values and ranges. Comparison
of the means of different groups was performed using
t-tests. A P-value less than 0.05 was considered as sta-
tistically significant. The Statistical Package for Social
Science (SPSS) was used for the present study.
Results
RT-PCR analysis of MMP-2 and -9 expression
in prostate tissue samples
Figure 1 shows the result of semiquantitative RT-PCR
analysis of MMP-2 and -9 expression in human prostate
tissues. A similar procedure was described earlier.
12
The
density of the band of individual genes was normalized
with that of the housekeeping gene (b-microglobulin).
There was no significant difference in the normalized
expression of MMP-2 in normal, BPH and prostate cancer
tissues. However, for MMP-9, the median normalized
expression was significantly different between nonmalig-
nant and malignant prostate tissues (Po0.001), and there
was a further significant increase in the metastatic prostate
cancer compared to that of the localized prostate cancer.
Zymography analysis of MMP-2 and -9 expression
in prostate tissue samples
Enzymatic activities for MMP-2 and -9 were determined
by the gelatinolytic activity in SDS-PAGE zymograms.
Figure 2 shows an example of zymography for MMP-2
and -9 in prostate tissue samples. MMP-2 showed two
distinct bands. The proform has a higher molecular
weight and the active form has a lower molecular
weight. As indicated in Figure 3a, there was a significant
difference in the activity of MMP-9 between normal
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a
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Figure 1 Normalized expression of MMP-2 and -9 in normal, BPH,
localized and metastatic prostate cancer tissues in comparison to the
housekeeping gene b-microglobulin. Vertical bars denote standard devia-
tion. *Denotes that the value is significantly different from that of the
normal (Po0.05). **Denotes that the value is significantly different from
that of the BPH (Po0.05).
Figure 2 Analysis of enzymatic activity of MMP-9 and -2 in prostate
tissue samples by zymography.
Type IV collagenase in prostate cancer
L Zhang et al
329
Prostate Cancer and Prostatic Diseases
tissues and BPH tissues (Po0.05), as well as between
BPH and metastatic prostate cancer (Po0.05). The
enzymatic activity for MMP-9 in metastatic prostate
cancer was about three-fold higher than that in BPH and
about nine-fold higher than that in normal tissues.
Figure 3b shows that, although the enzymatic activity
for MMP-2 in the normal prostate was significantly
lower than that BPH and prostate cancer tissues
(Po0.001), the MMP-2 activity was not significantly
different between BPH and prostate cancer (P40.05).
Interestingly, there was a significant difference (Po0.01)
in the ratio of the active form of MMP-2 (62 kDa) over the
proform (72 kDa) between the normal prostate and BPH,
as well as between BPH, and metastatic prostate cancer
tissues (Figure 3c).
Zymography analysis of MMP-2 and -9 expression
in prostate serum samples
Figure 4 shows an example of the zymography for
MMP-2 and -9 in serum samples. Figure 5a shows that,
similar to the values observed in prostate tissues, the
enzymatic activity for MMP-9 was significantly different
between the normal prostate and BPH tissues (Po0.05),
as well as between BPH and metastatic prostate cancer
(Po0.05). Also similar to the findings in the tissues, there
was no significant difference in the enzymatic activity for
MMP-2 among the normal prostate, BPH and prostate
cancer tissues. The active form of MMP-2 was not
detected in any of the serum samples.
Discussion
Results of the present study demonstrated that the
expression of MMP-9 mRNA as well as the enzymatic
activity was significantly higher in malignant than in
nonmalignant prostate tissues. The present study also
indicated that, although the expression of MMP-2 mRNA
was not significantly different. However, the ratio of the
active form of MMP-2 (62 kDa) over its proform (72 kDa)
was significantly different between the normal prostate
and BPH, as well as between BPH and metastatic prostate
cancer tissues. Findings from the serum specimens also
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Normalized Units
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Normalized Units
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b
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Figure 3 Relative activity of MMP-2 and -9 in prostate tissue samples. Vertical bars denote standard deviation. *Denotes that the value is significantly
different from that of the normal (Po0.05). **Denotes that the value is significantly different from that of the BPH (Po0.05).
Figure 4 Zymography analysis of activity of MMP-9 and -2 in serum
samples.
Type IV collagenase in prostate cancer
L Zhang et al
330
Prostate Cancer and Prostatic Diseases
demonstrated a significant difference in MMP-9, but not
in MMP-2, among the normal subjects, BPH and prostate
cancer patients. Our study also demonstrated that the
active form of MMP-2 was not found in any of the serum
specimens. These findings offer a possibility that MMP-9
in tissues and in sera and the ratio of active/proform for
MMP-2 in prostate tissues could be potential prognostic
markers for prostate cancer patients.
Many studies have demonstrated that MMPs correlate
with cancer metastases.
5–10
Although MMP-2 and -9
were detected in tissues and cells of the prostate,
9
MMP-2 was also expressed in the stromal cells.
12
MMP-
2 and -9 were thought to be the key matrix matallopro-
teinases involved in cancer cell invasion and metastases,
since their overexpression could be induced by many
factors such as cytokines, growth factors and onco-
genes.
13–17
In the present study, we further demonstrated
that MMP-9 levels in prostate tissues could also be an
important MMP in cancer progression and metastasis. The
activity of MMP-9 in metastatic prostate cancer tissues
was about three-fold higher than that in BPH and about
nine-fold higher than that in normal tissues. Our future
studies will be directed toward factors in prostate cancer
that regulate expression of MMPs.
Although there was no significant difference between
BPH and prostate cancer samples in enzymatic activity of
MMP-2 and in the level of MMP-2 mRNA. The activated
form of MMP-2 was only detected in tissue samples, not
in any serum samples. The ratio of activated MMP-2/
proform in metastatic prostate cancer samples was about
two-fold higher than that in BPH samples. These results
suggest that the activity of MMP-2 alone could not
differentiate BPH from prostate cancer. These observa-
tions seem to underscore the potential role of MMP-2 in
prostate cancer metastasis. However, the significant
difference in the ratio of activated MMP-2/proform
between BPH samples and prostate cancer samples
suggest that BPH and prostate cancer may have such a
functional difference.
Brown et al
18,19
showed for the first time by the
approach of gelatin zymography that the ratio of active-/
proform of MMP-2 correlated with lymph node meta-
stasis in the human breast and lung cancers. Subsequent
studies demonstrated that this finding could apply to
many human cancers including thyroid cancer, oral
squamous cell carcinoma, stomach carcinomas, breast
carcinomas and non-small cell carcinoma of the lung.
20
Results of the present study also confirmed the above
conclusion for prostate cancer.
Serum levels of MMPs were found to be correlated
with invasion and metastasis of many malignancies,
including human lung cancers as well as breast
21
and
gastrointestinal cancers.
22
Results of the present study
verified that MMP-9 levels in the serum specimens
correlated with the presence of malignancy, as well as
with the metastatic status of prostate cancer. In serum
samples, the activity of MMP-9 in patients with meta-
static prostate cancer was about two-fold higher than
that in patients with BPH.
In the present study, we were unable to detect
any correlation in changes in serum MMP-2 levels
between BPH patients and patients with prostate
cancer. However, Gohji et al
6
detected MMP-2 in serum
specimens using a monoclonal antibody and found
that the density of MMP-2 in serum was associated
with the development and extension of prostate
cancer and that the serum MMP-2 level indicated the
degree of prostate cancer extension. The discrepancy
between this study and the study by Gohji’s group is
unclear. It is likely that a difference in detection
methodology used in two studies could account for such
a difference.
In conclusion, our results indicated that the expression
of MMP-9 in prostate tissues and serum and the ratio of
activated MMP-2/proform in tissues were associated
with metastatic prostate carcinoma. These findings were
supported by a most recent independent report.
23
These para-
meters may be useful as a prognostic marker for human
prostate cancer.
Acknowledgements
This research was funded by the following grants: 3 3
Canada China Biotechnology Seed Grant, the National
Natural Science Foundation of China (Grant No.
30271297), and the United States Department of Health
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MMP-2
Figure 5 Relative activity of MMP-2 and -9 in serum samples. Vertical
bars denote standard deviation. *Denotes that the value is significantly
different from that of the normal (Po0.05). **Denotes that the value is
significantly different from that of the BPH (Po0.05).
Type IV collagenase in prostate cancer
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and Human Services, National Institutes of Health (NCI)
Prostate Cancer SPORE (Grant No. P50 CA90386-01).
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