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Int. J. Cancer (Pred. Oncol.): 74, 220–223 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
MATRIX METALLOPROTEINASES 1 AND 3, TISSUE INHIBITOR
OF METALLOPROTEINASE-1 AND THE COMPLEX
OF METALLOPROTEINASE-1/TISSUE INHIBITOR
IN PLASMA OF PATIENTS WITH PROSTATE CANCER
Klaus JUNG1*, Lars NOWAK1, Michael LEIN1, Friedrich PRIEM2, Dietmar SCHNORR1 and Stefan A. LOENING1
of Urology, University Hospital Charité, Humboldt University Berlin, Berlin, Germany
2Department of Clinical Chemistry, University Hospital Charité, Humboldt University Berlin, Berlin, Germany
1Department
We analyzed blood plasma concentrations of matrix metalloproteinase-1 and -3 (MMP-1; MMP-3), the tissue inhibitor of
metalloproteinase-1 (TIMP-1) and the complex MMP-1/
TIMP-1, and looked for any correlation with prostate cancer
stage. These components were measured by ELISA tests
specific for these proteins in healthy male controls (n 5 35),
and in patients with benign prostatic hyperplasia (BPH;
n 5 29), with prostate cancer (PCa) without metastasis
(T2,3pN0M0; n 5 29) and with PCa with metastatic disease
(T2,3,4pN1,2M1; n 5 18). Mean values of MMP-1 and of the
complex MMP-1/TIMP-1 were not different among the 4
groups studied. The mean MMP-3 and especially TIMP-1
concentrations were significantly higher in PCa patients with
metastases compared with controls, BPH and PCa patients
without metastases. Ten of these 18 patients had TIMP-1
concentrations higher than the upper reference limit. TIMP-1
concentrations were correlated with staging but not with
grading. Our results point towards plasma TIMP-1 concentration as a potential marker of malignant progression of PCa.
Int. J. Cancer 74:220–223, 1997.
r 1997 Wiley-Liss, Inc.
Matrix metalloproteinases (MMP) are a family of enzymes with
the common ability to degrade various components of the extracellular matrix such as collagen, elastin and gelatin (Liotta and
Stetler-Stevenson, 1991). These enzymes are involved in all
physiological processes occurring during tissue remodelling and
repair and play a crucial role in pathological conditions such as
rheumatoid arthritis, tumor invasion and metastasis (Crawford and
Matrisian, 1995). Both in vitro and in vivo investigations have
shown that increased concentrations of MMPs are associated with
the invasive and metastatic potential in several human malignant
tumors, e.g., in breast, colon, gastric and lung cancers (Anderson et
al., 1995; Zucker et al., 1995; Murray et al., 1996). It is assumed
that these proteases destroy the integrity of the basement membrane and enable cancer cells to invade normal tissue and facilitate
tumor cell dissemination via blood vessels and lymphatics. The
catalytic activities of MMPs are controlled by various mechanisms
including enzyme synthesis, secretion as zymogens which undergo
extracellular activation and inhibition of the activated enzymes by
specific inhibitors, the tissue inhibitors of metalloproteinases
(TIMPs). Thus, the balance between MMPs and TIMPs as both
positive and negative modulators of the invasive and metastatic
processes appears to be decisive (Crawford and Matrisian, 1995).
Since these changes in cellular concentration may be reflected in
body fluids, determinations of MMPs, TIMPs and of their complexes in the blood have been recommended as simple, noninvasive
tools in cancer diagnostics and monitoring (Zucker et al., 1995).
Increased plasma concentrations of the complex gelatinase B:tissue
inhibitor have been observed in patients with gastrointestinal tract
cancer (Zucker et al., 1995). There has been only one report on
MMPs in the blood of prostate cancer (PCa) patients: Baker et al.
(1994) noted changes in the concentration of MMP-1, MMP-3,
TIMP-1 and TIMP-2 in the blood of such patients using in-house
tests. Although these results are of great interest, no confirmatory
data have been presented so far. Serum samples as used by these
authors not being suitable for reliable measurements of these
components (Jung et al., 1996), and since commercial assays for
determining MMP-1, MMP-3, TIMP-1 and the complex of MMP1/TIMP-1 have since been introduced, our objective was to
evaluate their potential usefulness in patients with PCa.
MATERIAL AND METHODS
Patients and samples
A total of 111 men was studied, including a control group of 35
healthy individuals (median age 48, range 30–68 years), 29 patients
with benign prostate hyperplasia (BPH) (median age 64, range
51–82 years) and 47 with PCa (median age 65, range 46–79 years).
The diagnosis of BPH was based on histological analysis of tissue
obtained by transurethral resection of the prostate. The diagnosis of
PCa was established histopathologically by microscopic examination of prostatic specimens after biopsy or prostatectomy. Clinical
staging was made according to the TNM system by digital rectal
examination, transrectal ultrasonography, determination of prostatespecific antigen (PSA) and radionuclide bone scan including
surgical assessment of lymph node status. Computed tomography
and/or magnetic resonance imaging was performed in some
individuals. Of the 47 patients with PCa, 29 (median age 65, range
46–79 years) had PCa without metastasis (T2,3pN0M0) and 18
(median age 67, range 52–77 years) had PCa with metastasis
(T2,3,4pN1,2M1).
Blood was collected before examination in plastic tubes for
preparation of serum samples (for PSA and other conventional
clinical chemistry parameters) and in lithium-heparin-coated plastic tubes for preparation of plasma samples (for MMP and TIMP)
(Monovette Systems 03.1528, 03.1589; Sarstedt, Nümbrecht, Germany). The tubes were centrifuged within 30 min after venipuncture at 1,600g for 15 min at 4°C. The supernatants were carefully
removed, divided into aliquots and stored at 280°C until analysis.
ELISA tests for MMP and TIMP
We used BIOTRAK ELISA assays for MMP-1, MMP-3, TIMP-1
and the MMP-1/TIMP-1 complex (RPN 2610–2612; Amersham,
Little Chalfont, UK). The assays are based on the 2-side sandwich
principle. Standards, controls and samples are incubated in microtiter wells precoated with respective antibodies. The components
MMP-1, TIMP-1 and MMP-1/TIMP-1 complex, respectively, bind
to the wells, and other components of the samples are removed by 4
washing steps. A second antibody specific to the respective
component is added to the wells and incubated. After washing, the
second antibody bound to MMP-1, MMP-3, TIMP-1 or complex is
detected using horseradish-labelled antibodies and tetramethyl-
Contract grant sponsor: Fund of the German Chemical Industry, contract
grant number 400770; Contract grant sponsor: Family-Klee-Foundation.
*Correspondence to: Department of Urology, University Hospital Charité,
Humboldt University Berlin, Schumannstrasse 20/21, D-10098 Berlin,
Germany. Fax: (1149-30)2802-1402. E-mail: jung@rz.charite.hu-berlin.de
Received 20 November 1996; revised 24 December 1996
BLOOD METALLOPROTEINASES IN PROSTATE CANCER
benzidine as substrate. The reaction is terminated by addition of
sulphuric acid and the absorbance is measured on a microplate
(HTIII, Anthos Labtec, Salzburg, Austria) at 450 nm using the
cubic-spline method for calculation of concentrations (EIA/KINStar software, version 7.0, WEPAH-MED, Berlin, Germany).
According to the data provided by the manufacturer, the respective
assays specifically recognize the corresponding proteins and there
is no crossreaction with other metalloproteinases. All tests were
performed in duplicate. The limits of detection for MMP-1,
MMP-3, TIMP-1 and the MMP-1/TIMP-1 complex, respectively,
defined as the corresponding concentrations located 3 standard
deviations above the measured average blanks (n 5 10), were 0.13
µg/l, 5.0 µg/l, 1.1 µg/l and 4.8 µg/l, respectively. The precision of
the data determined with pooled sera were between 3.0–10.7%.
PSA and other routine laboratory parameters
PSA was measured by the AxSym test (Abbott, Abbott Park, IL).
Routine clinical chemistry parameters, including alanine aminotransferase (normal upper limit: 41 U/l), g-glutamyltransferase (49 U/l)
and C-reactive protein (5 mg/l), were measured by standard assays
on the Hitachi 717 analyzer (Boehringer Mannheim, Germany).
Statistical analyses
Statistical calculations were performed by the statistical package
Statgraphics, version 5.01 (Statistical Graphics, Rockville, MD).
The Mann-Whitney U test with independent samples, the correlation coefficient according to Spearman, analysis of variance
(ANOVA) and a distribution fitting procedure (KolmogorovSmirnov test) were used. The central 95% reference intervals in the
control group were calculated according to the procedure recommended by the IFCC (Solberg, 1987); p , 0.05 was considered
statistically significant.
RESULTS
The control group consisted of 35 healthy men. In the control
group, plasma concentrations of MMP-1, MMP-3, TIMP-1 and the
complex MMP-1/TIMP-1 did not correlate with each other, and did
not correlate with alanine aminotransferase and the C-reactive
protein. The preliminary upper reference limits were calculated as
means 1 1.96 SD (Solberg, 1987). The corresponding limits were
19.7 µg/l for MMP-1, 19.8 µg/l for MMP-3, 1,740 µg/l for TIMP-1
and 110 µg/l for the complex MMP-1/TIMP-1.
Figure 1 shows the values of the MMP-1, MMP-3, TIMP-1 and
MMP-1/TIMP-1 complex in plasma of controls and the patients
with BPH and PCa. Patients with PCa were subdivided in 2 groups,
i.e., patients without metastasis and patients with distant metastasis
and/or metastasis in regional lymph nodes as having locally
advanced and/or metastatic disease. The mean age of BPH and PCa
patients did not differ but it was somewhat higher than in healthy
controls (64 and 65 vs. 48 years). However, there was no
correlation between the 4 analytes and ages (range, 30–82 years) in
the noncarcinoma group of healthy controls and BPH patients
(rs 5 20.19, 20.18, 0.09, 0.22; p . 0.05). Thus, no age-effect on
the concentrations of these analytes has to be taken into account
when comparing concentrations measured in the groups studied.
The study groups revealed PSA values typical for each kind of
patient. Thus, PSA values in BPH patients, and in PCa patients
without metastasis and with metastatic disease were clearly different (median values, 4.2 vs. 6.5 and 23.7 µg/l). However, the mean
MMP-1 and MMP-1/TIMP-1 complex concentrations were not
increased in the 3 groups of patients compared with those in the
control group (Fig. 1a,d; p . 0.05). There was a tendency for
rather lower values, and only a few patients were outside the
reference limits. In contrast, the mean MMP-3 and especially
TIMP-1 concentrations in PCa patients with metastases were
significantly higher than those in the control group, BPH patients
and PCa patients without metastasis (Fig. 1b,c; p , 0.05). These
changes were distinctly more pronounced in the case of TIMP than
in that of MMP-3. Ten of the 18 PCa patients with metastases had
221
values above the upper TIMP-1 reference limit, but only 3 were
above the MMP-3 limit. Although the mean TIMP-1 value in PCa
patients without metastasis was not increased compared with the
controls and BPH patients, 7 of these 29 patients showed values
higher than the upper reference limit. Ten patients of that group had
PSA values ,4 µg/l, generally considered as being the normal
upper PSA cutoff limit. It was interesting that 4 patients of that
subgroup showed increased TIMP values. There were no correlations of TIMP-1 and MMP-3 values in these patients ( p . 0.05)
with those of alanine aminotransferase (rs 5 20.10 and 20.02),
g-glutamyltransferase (rs 5 0.28 and 20.13) and C-reactive protein (rs 5 0.07 and 0.07).
The correlation matrix between staging, grading, PSA and the
metalloproteinase parameters is shown in Table I. MMP-1, MMP-3
and the MMP-1/TIMP-1 complex did not correlate with staging or
with grading, whereas TIMP-1 concentrations showed a correlation
with staging (N/M state) similar to that of PSA (rs 5 0.383;
p , 0.02), but not with grading. Thus, the correlation of TIMP
corresponds to the TIMP concentrations found in the 2 PCa groups
with and without metastasis.
DISCUSSION
The MMP family can be divided into 4 subclasses according to
substrate specificity and structural similarity (Liotta and StetlerStevenson, 1991). These are the gelatinases (MMP-2 and MMP-9),
the collagenases (MMP-1 and MMP-8), the stromelysins (MMP-3
and MMP-10) and a subgroup including matrilysin (MMP-7),
stromelysin 3 (MMP-11) and metalloelastase (MMP-12). Furthermore, 3 specific TIMPs are recognized (TIMP-1, TIMP-2 and
TIMP-3). It is assumed that these proteins, when increased in
cancer tissue, may be elevated in the plasma of patients with more
invasive or metastatic cancers (Zucker et al., 1995). Measurements
have so far been performed by determining activities using
nonstandardized substrate degradations assays and immunological
assays using in-house ELISA approaches (Zucker et al., 1995).
Commercial tests for MMP-1, MMP-3, TIMP-1 and the MMP-1/
TIMP-1 complex have now been introduced, providing the opportunity for evaluating their potential as tumor markers (Zucker et al.,
1995). Our results show that the analytical sensitivity and reliability of these assays are indeed suited to determine these components
in human plasma.
On human prostate tissue sections, various investigations including immunohistochemical studies and in situ hybridization have
demonstrated, similar to findings in other carcinomas, several
changes in matrix metalloproteinases in malignant tissue, as
compared with adjacent noncarcinoma tissue parts (Pajouh et al.,
1991; Boag and Young, 1993; Stearns and Wang, 1993; Hamdy et
al., 1994; Knox et al., 1996; Stearns and Stearns, 1996). For
example, direct correlations were observed between the intensity of
MMP-2 expression and the Gleason score (Stearns and Wang,
1993; Stearns and Stearns, 1996). Changes in the expression of
MMP-7 and MMP-9 were also observed in benign prostatic
hyperplasia and PCa (Pajouh et al., 1991; Hamdy et al., 1994).
Therefore, it appears justified to also look for changes in the
concentration of metalloproteinases in the blood of patients with
PCa. We found only one report on metalloproteinase in the blood of
PCa patients (Baker et al., 1994). Baker et al. (1994), investigating
MMP-1, MMP-3, TIMP-1 and TIMP-2 in the serum of PCa
patients, found increased TIMP-1 and MMP-1, unchanged MMP-3
and decreased TIMP-2 concentrations.
Our measurements of MMP-1, MMP-3, TIMP-1 and the MMP1/TIMP-1 complex show that only TIMP-1 and MMP-3 are
elevated in PCa patients with metastases compared with controls,
BPH patients and PCa patients without metastases. The increases
of MMP-3 were markedly lower than those of TIMP-1. These data
are partly in contrast with the results of Baker et al. (1994), who
used serum samples. Our previous experiments have shown that the
sampling process is a decisive preanalytical condition for reliable
JUNG ET AL.
222
FIGURE 1 – Plasma concentrations of MMP-1 (a), MMP-3 (b), TIMP-1 (c) and MMP-1/TIMP-1 complex (d) in controls, and in patients with
benign prostate hyperplasia (BPH) and with PCa without (PCa, m2) and with metastasis (PCa, m1). Values are expressed as individual values
and as arithmetic means 6 SD. Horizontal lines indicate upper reference limits calculated from controls (see Results). Significance (at least
p , 0.05) in comparison with: (a) controls; (b) BPH patients; (c) PCa without metastasis; (d) PCa with metastasis.
TABLE I – CORRELATIONS OF MMP-1, MMP-3, TIMP-1 AND MMP-1/TIMP-1
COMPLEX WITH TUMOR STAGE, GRADE AND PSA1
Parameter
rs values
MMP-1
MMP-3
TIMP-1
MMP-1/TIMP-1 complex
0.04
20.22
20.08
20.10
0.363
0.26
0.25
0.343
0.28
0.523
0.12
0.363
0.333
0.09
20.05
0.24
Staging2
T
N/M
Grading
PSA
1R values shown are Spearman rank correlation coefficients and
s
were calculated by combining the 47 patients with PCa.–2Patients were
classified according to the TNM system and a grading scale of 1–3.
Three situations of lymph node and distant metastasis were considered
for the calculation of correlation: N0M0; N1,2M0; N1,2M1 or
N3M1.–3Significant correlations: at least p , 0.05.
measurements of these components in the blood (Jung et al., 1996).
For example, MMP-1 and TIMP-1 measured in the serum were
about 5–7 times higher than in lithium heparin plasma because
these components are probably released during the clotting process.
Therefore, the results described by Baker et al. (1994) must be
interpreted with caution.
Our most important finding is that of an increase in TIMP-1
values in patients with metastases. Our results showed a stagedependence of TIMP-1 (Table I) and a correlation with PSA, a
classical PCa parameter. However, there were 4 cases with
increased TIMP values in the group of PCa patients without
metastases (n 5 10) and normal PSA values (,4 µg/l). This result
suggests the potential usefulness of assaying TIMP-1 in combination with PSA, but not instead of PSA. Follow-up studies will be
necessary to validate this assumption.
Increased TIMP-1 values have been found in plasma of patients
with liver diseases, systemic sclerosis, rheumatoid arthritis and
diabetes mellitus (Li et al., 1994; Manicourt et al., 1995). An
interesting aspect raised by our results concerns the cellular origin
of TIMP. Since there was no correlation of TIMP-1 with C-reactive
protein and liver enzymes in the PCa patients with metastases,
inflammatory processes or liver dysfunction may probably be
excluded. The increased plasma levels may be either a result of a
general reaction related to the tumor burden or, more probably,
BLOOD METALLOPROTEINASES IN PROSTATE CANCER
originate from the cancer tissue and/or metastatic tissue directly.
Increased amounts of TIMP-1 have been demonstrated in colon
cancer and hepatocellular carcinoma tissue (Zeng et al., 1995;
Nakatsukasa et al., 1996). Tissue TIMP concentrations were indeed
higher in advanced carcinomas cases.
Cytokines, such as transforming growth factor-b (TGF-b),
tumor necrosis factor-a and interleukins, are potent inducers of
TIMP-1 (Chua and Chua, 1990). There is a close correlation
between cytokines and TIMP-1 in tumor tissues. For example,
TGF-b1 has been found to be higher in cancerous than in normal
tissue of the prostate and was positively correlated with metastasis
(Kakehi et al., 1996). However, there is no information on tissue
TIMP values in PCa.
TIMP-1 exerts 2 apparently opposite effects on tumor progression. It is a potent antagonist of metalloproteinases and inhibits
important steps of tumor progression, especially proteolytic degradation of extracellular matrix and invasion. However, TIMP-1
stimulates the growth of normal and malignant cells independently
of its inhibitory capacity (Hayakawa et al., 1992). Growthpromoting activities have been observed with TIMP concentrations
composed between 10–100 ng/ml, whereas the inhibition of
proteolytic degradation of the extracellular matrix needs concentrations above 1 µg/ml. Thus, both increased and decreased TIMP
223
concentrations may be characteristic of cancer tissues. These are
tumor-specific (Zeng et al., 1995; Bramhall et al., 1996). For
example, elevated mRNA TIMP-1 values observed in colon tumors
are strongly correlated with lymph node and distant metastases
(Zeng et al., 1995), whereas decreased levels have been noted in
pancreatic cancer (Bramhall et al., 1996). We assume that the
increased plasma TIMP-1 amounts observed in PCa patients reflect
the situation in cancer tissues.
In conclusion, we found significantly increased plasma MMP-3
and TIMP-1 concentrations in patients with advanced PCa compared with controls, in PCa patients without metastasis and in
patients with benign prostatic hyperplasia, whereas MMP-1 and
MMP-1/TIMP-1 were not elevated. Our results point towards
plasma TIMP-1 as a potential marker of malignant progression in
PCa, and these results warrant further studies.
ACKNOWLEDGEMENTS
This work was supported by grants from the Fund of the German
Chemical Industry (to K.J.; 400770) and the Family-KleeFoundation (to M.L.). This study includes parts of the doctoral
thesis of L.N.
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