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The Prostate 34:130–136 (1998)
Quantification of Matrix Metalloproteinases and
Tissue Inhibitors of Metalloproteinase in
Prostatic Tissue: Analytical Aspects
Klaus Jung,1* Michael Lein,1 Norbert Ulbrich,2 Birgit Rudolph,3
Wolfgang Henke,1 Dietmar Schnorr,1 and Stefan A. Loening1
1
Department of Urology, University Hospital Charité, Humboldt University, Berlin, Germany
2
German Research Center for Rheumatology, Berlin, Germany
3
Department of Pathology, University Hospital Charité, Humboldt University,
Berlin, Germany
BACKGROUND. The balance between matrix metalloproteinases (MMP) and the tissue inhibitors of metalloproteinases (TIMP) has been seen as important during tumor invasion and
progression. The determination of these components needs a special strategy of tissue preparation. This analytical problem has not been considered for prostatic tissue.
METHODS. We adapted an extraction method consisting of two extraction steps with 0.25%
Triton X-100/CaCl2 solution and two heat extraction steps at 60°C for 4 min. This combination
allowed a complete extraction of MMP (measured as enzyme activity) and TIMP-1 (measured
with an ELISA test) from cancerous and normal prostatic tissue samples.
RESULTS. The median values for cancerous vs. normal MMPs (50.8 mU/g wet tissue and
1,580 mU/g protein vs. 88.8 and 2,497) and TIMP-1 (4.49 mg/g wet tissue and 96.7 mg/g
protein vs. 12.4 and 237.8) were significantly lower, whereas the respective ratios for MMP/
TIMP-1 (11.1 vs. 4.0 on wet weight and 15.5 vs. 5.3 on protein basis) were significantly higher.
CONCLUSIONS. An optimized extraction procedure was elaborated for determining MMPs
and TIMP-1 in prostatic tissue samples. The increased ratio of MMP/TIMP-1 can be interpreted as an indicator of the imbalance between MMP and TIMP, characteristic of prostate
carcinoma tissue. Prostate 34:130–136, 1998. © 1998 Wiley-Liss, Inc.
KEY WORDS:
prostate; prostate carcinoma; matrix metalloproteinase; tissue inhibitor
of metalloproteinase
INTRODUCTION
Matrix metalloproteinases (MMP) form a group of
enzymes with the common ability to degrade various
components of the extracellular matrix such as collagen, elastin, and gelatin [1]. These enzymes play an
important role in tumor invasion and metastasis [2].
Both in vitro and in vivo investigations have shown
that increased levels of MMPs are associated with the
invasive and metastatic potential in several human
malignant tumors, e.g., in breast, colon, gastric, and
lung cancers [3–6]. The catalytic activities of MMPs are
controlled in part by specific inhibitors, the so-called
tissue inhibitors of metalloproteinases (TIMPs). Low
TIMP expression correlates with enhanced invasive
© 1998 Wiley-Liss, Inc.
and metastatic properties of human tumors [7,8].
Thus, the balance between MMPs and TIMPs as both
positive and negative modulators of the invasive and
metastatic processes has been seen as decisive [2].
There are sparse data on these components in the
prostate [9–13]. To prove the biological significance of
metalloproteinases and their inhibitors, measurements
of these components as proteins, enzymatic activities,
and mRNA quantities are necessary [14]. As metallo*Correspondence to: Prof. Klaus Jung, M.D., Department of Urology, University Hospital Charité, Humboldt University Berlin,
Schumannstraße 20/21, D-10098 Berlin, Germany. E-mail:
jung@rz.charite.hu-berlin.de
Received 30 September 1996; Accepted 19 February 1997
Matrix Metalloproteinases in Prostatic Tissue
proteinases are bound to extracellular matrix, their reliable determination in tissue needs a special strategy
of tissue preparation [15]. This analytical problem has
attracted little attention so far. Numerous tissue extraction approaches have apparently been recommended because of special tissue characteristics
[5,9,11,12,16–22]. When we prepared prostatic tissue
for measuring MMPs and TIMPs, we found incomplete results and therefore proceeded to a systematic
study. This problem is of general interest for future
prostate research.
131
heated at 60°C under agitation for 4 min. Then the
mixture was cooled on ice for 5 min and centrifuged as
described. The supernatant was removed; the heat extraction of the pellet and the centrifugation of the suspension were repeated several times to study the extraction effect of MMP and TIMP. All the supernatants
were collected separately and stored at −80°C not
longer than 7 days until analysis.
The recommended final extraction procedure consisted of two extractions with Triton solution followed
by two heat extractions, as described. The supernatants collected are combined.
MATERIALS AND METHODS
Tissue Samples
Prostate tissue samples were obtained from the cancerous and noncancerous parts of the same prostates
(n = 9; mean age of patients, 63 years) which had been
surgically removed by radical prostatectomy. Small
pieces of tissue were dissected immediately after removal of the prostate and stored in liquid nitrogen
until analysis. The cut edges within the prostate were
inked so that the dissected pieces could be easily assigned to the adjacent prostate tissue examined histopathologically [23]. Histological analysis of all tissue
pieces used was carefully performed by a clinical pathologist (B.R.) to ensure that the material used was
either malignant or nonmalignant tissue. The differentiation of tumors were classified according to the
conventional grading scale 1–3. Of the 9 tumor
samples investigated, 2 were classified as G1, 6 as G2,
and 1 as G3. The use of this human tissue for research
purposes was approved by the Ethical Committee of
the Charité Hospital (Berlin, Germany).
Preparation of Tissue Extracts
The extraction procedure was based on the general
recommendations of Woessner [15]. Thirty milligrams
of tissue were weighed (wet weight), minced, and homogenized with 0.1 ml of a solution containing 0.25%
Triton X-100 and 10 mM CaCl2 in a Wheaton glass
homogenizer (Wheaton, Millville, NJ) by 10 up-anddown strokes on ice. The homogenate was transferred
to a 1.5-ml tube (Eppendorf GmbH, Hamburg, Germany). The homogenizer was rinsed twice with 0.1 ml
of Triton solution. This mixture of 0.3 ml was centrifuged at 23,000g at 4°C for 15 min. The supernatant
was removed. To investigate the effect of the extraction procedure, the pellet was resuspended and the
extraction was repeated several times. The pellet obtained after Triton extraction was resuspended in 0.2
ml of a solution containing 50 mM Tris/HCl buffer,
pH 7.5, 150 mM NaCl, and 100 mM CaCl2, and was
Quantification of MMP, TIMP, and Protein
The previously described supernatants were removed from the freezer and thawed with agitation at
room temperature. MMP activity was determined
with the continuous fluorimetric assay (spectrofluorimeter LS 50B, Perkin-Elmer, Überlingen, Germany)
according to Knight et al. [24], using 25 mM (7methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu (3[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-Ala-ArgNH2 (Bachem GmbH, Heidelberg, Germany) as substrate in an assay buffer of 50 mM Tris/HCl, pH 7.5,
200 mM NaCl, and 5 mM CaCl2. Aminophenylmercuric acetate (1 mM) was added to activate latent MMPs.
Blanks were made with 1 mM 1,10-phenanthroline to
inhibit metalloproteinase activity. We measured metalloproteinase activities in crude extracts without pretreatment of reduction/alkylation to destroy possible
inhibitors, as we did not observe any effect of this
pretreatment. Enzyme activities were calculated from
the linear part of the reaction curve. One enzyme unit
was defined as that enzyme amount that cleaves 1
mmol substrate per min.
TIMP-1 was measured by the BIOTRAK™ ELISA
kit (RPN 26112, Amersham International, Little Chalfont, UK). The assay is based on the two-sided sandwich principle. The supernatants were diluted with
1% bovine albumin in 50 mM phosphate-buffered saline, pH 7.5. All tests were performed with at least two
dilutions for each supernatant. Standards, controls,
and samples were incubated in microtiter wells precoated with anti-TIMP-1 antibodies. TIMP-1 was
bound to the wells, while other components of the
samples were removed by four washing steps. A second antibody specific to TIMP-1 was added to the
wells and incubated. After washing, the second antibody-bound TIMP-1 was detected using horseradishlabelled antibodies and tetramethyl-benzidine as substrate. The reaction was terminated by addition of sulphuric acid, and absorbance was measured on a
microplate reader (HTIII, Anthos Labtec Instruments,
Salzburg, Austria) at 450 nm using the cubic-spline
132
Jung et al.
method for calculation of concentrations (EIA/KINStar software, version 7.0, WEPAH-MED, Berlin, Germany). According to the manufacturer, the ELISA specifically recognizes TIMP-1 and there is no crossreaction with other TIMPs and metalloproteinases. The
limit of detection for TIMP-1, defined as the corresponding concentrations located 3 standard deviations above the measured average blanks (n = 10), was
4.8 mg/l. The interassay-precision data determined
with pooled sera were 5.8%.
Protein concentrations were measured with Coomassie brilliant blue assay reagent, using bovine serum albumin as standard [25].
Statistical Analysis
Statistical calculations (Student’s t-test with paired
data and Wilcoxon signed ranks test) were carried out
with the statistical package Statgraphics, version 5.1
(Statistical Graphics Corp., Rockville, MD). Differences of P < 0.05 were considered statistically significant.
(±17%) and 75% (±6%) for the malignant tissue and
88% (±12%) and 70% (±12%) for the corresponding
normal counterparts, respectively (t-test of paired
data; P > 0.05).
It can be concluded from all these results that two
extraction steps with the Triton X-100 solution and
two heat extraction steps result in an appropriate final
extraction procedure both for normal and cancerous
prostatic tissue samples, because about 95% of these
components are extracted under these conditions.
Table I presents our provisional data of MMP,
TIMP-1, and the ratio of MMP/TIMP-1 related to tissue wet weight and to tissue protein. Median MMP
and TMP-1 values were significantly lower, but the
ratio of MMP/TIMP-1 was significantly higher in cancerous tissue samples than in their normal counterparts. A correlation of MMP or TIMP values with the
state of tumor differentiation was not established.
However, it should be considered that there was a
small number of cases in the classes G1 (n = 2) and G3
(n = 1).
DISCUSSION
RESULTS
Figure 1A,B summarizes our experimental results
of the extraction of MMP and TIMP-1 from prostatic
tissue. We also included the data of protein extraction
(Fig. 1C), because it is common to use tissue protein as
reference basis for the analytes under study. Because
we did not find significant activation of MMP activity
by aminophenylmercuric acetate, we measured MMP
without that activator. Three extraction steps using the
detergent Triton X-100 solution did not guarantee a
complete extraction of the respective analytes. Using
the Triton X-100 solution, the percentage extraction
rate of TIMP-1 was additionally significantly lower
than that of MMP (P < 0.05). About 30% of the total
TIMP-1 and about 15% of MMP remained unextracted. Thus, the additional heat extraction was necessary to complete the extraction of these components.
To test the usefulness of the heat extraction we made
additional recovery experiments. Known amounts of
MMP and TIMP-1 were subjected to the heat extraction procedure, and the recovery rate was measured (n
= 5). About 85% of the added amounts was recovered.
This showed that the heat extraction procedure was
able to extract the remainder of MMP and TIMP-1
after Triton X-100 extraction.
The percentage extraction rates of TIMP-1 and
MMP obtained with the Triton X-100 extraction and
the heat extraction were not different between cancerous tissue samples and their normal counterparts (n =
9). Using the Triton X-100 procedure, the mean (±SD)
extraction rates of MMP and TIMP-1 amounted to 83%
Several procedures have been described for the
preparation of tissue extracts to measure metalloproteinases and their inhibitors because these components are bound to extracellular matrix and need special tissue preparation [15,26]. For that purpose, both
simple buffer or salt solutions [9,16,22] and solutions
containing various detergents (e.g., Nonidet, Triton X100, Tween 80, Tween 20, SDS) of different concentrations were used for the extraction process [5,11,12,18–
21]. However, in general no data were given as to
whether the extraction was quantitative for metalloproteinases and their tissue inhibitors. Woessner [15]
reviewed the issue of sample preparation for metalloproteinase measurements in tissue extracts. He
pointed out that this problem has not been sufficiently
considered as a source of errors so far. Recommendations have been given for systematic studies of tissue
under investigation [15,26]. Our results confirm this
view and show that the use of an optimized extraction
procedure is a precondition for reliable measurements
of the components of the metalloproteinase system in
prostatic tissue.
The MMP family can be divided into four subclasses, according to substrate specificity and structural similarity [2]. 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, three specific TIMPs are known (TIMP-1, TIMP2, and TIMP-3). We measured MMP according to
Matrix Metalloproteinases in Prostatic Tissue
133
Fig. 1. Extraction of metalloproteinase (A), tissue inhibitor of
metalloproteinase-1 (B), and protein (C) from prostatic tissue.
Results are the means ± SD of 5 separate extraction experiments and are calculated as percentages of the sum of the total
amount of the respective analyte extracted. Extraction steps
1–3: 3 repeats of extraction using a solution containing 0.25%
Triton X-100, 10 mM CaCl2; extraction steps 4–6: 3 repeats of
extraction using a solution containing 50 mM Tris/HCl buffer, pH
7.5, 150 mM NaCl, 100 mM CaCl2 at 60°C for 4 min. MMP,
metalloproteinase; TIMP-1, tissue inhibitor of metalloproteinase1. For further details, see Materials and Methods.
Knight et al. [24], using 25 mM (7-methoxycoumarin4-yl)acetyl-Pro-Leu-Gly-Leu (3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 as substrate.
This is considered the most sensitive substrate for the
continuous measurement of total matrix metalloproteinases in crude tissue preparations [24]. However,
the above-mentioned MMPs cleave that substrate differently [24]. For example, gelatinase reacts approximately twice as sensitively as matrilysin with that substrate. Despite these limitations of different analytical
sensitivities to the respective enzymes, we think that
our general conclusion, of MMP extraction as incom-
plete using Triton solution alone and the requirement
of additional heat extraction, is justified.
A similar conclusion of incomplete extraction with
the Triton solution alone can be drawn for TIMP-1.
There have been no published data on this problem
regarding individual TIMP-1 so far, since immunoassays for that analyte have become available only recently. We measured TIMP-1 representative of the
three known TIMP-1, TIMP-2, and TIMP-3. Measurements by selective chemical destructions of TIMP are
cumbersome and cannot distinguish between the different TIMPs [27]. However, using the inhibitory effect
134
Jung et al.
TABLE I. Metalloproteinase Activity and Tissue Inhibitor of
Metalloproteinase-1 Values in Human Prostate*
MMP
mU/g wet tissue
mU/g protein
TIMP-1
mg/g wet tissue
mg/g protein
MMP/TIMP-1
Wet tissue basis
Protein basis
Normal tissue
Tumor tissue
88.8 (30.7–173)
2,497.0 (984–5,524)
50.8 (41.4–86.1)a
1,580.0 (1,127–2,447)a
12.4 (5.05–57.8)
237.8 (104–1,248)
4.0 (1.4–30.6)
5.3 (1.9–40.9)
4.49 (1.23–17.1)b
96.7 (25.7–411.8)b
11.1 (2.5–47.9)b
15.5 (3.4–60.9)b
*Values are medians (and ranges) of cancerous and corresponding
noncancerous tissue parts of the same prostates (n = 9) which had been
surgically removed by radical prostatectomy. MMP, metalloproteinase; TIMP-1, tissue inhibitor of metalloproteinase-1.
a,b
Significant differences (Wilcoxon signed rank test; aP < 0.05,
b
P < 0.01) between cancerous and noncancerous tissue samples.
of extracts on enzyme activity as measured by the
Azocoll technique, comparable data of about 80% and
20% of total TIMP were found in the Triton extract and
the heat extract, respectively [27]. We found that the
percentage Triton extraction of TIMP-1 was significantly lower than that of MMP. Thus, that difference
would cause a systematic error if the ratio of MMP/
TIMP was calculated from data obtained with the Triton extract.
A few studies on MMPs and TIMPs have been performed in human prostate tissue samples, prostate
cells, and prostate tumors grown in animals [9–13,28–
33]. The role of proteases in prostatic malignancy was
recently reviewed [34].
Immunohistochemical studies and in situ hybridizations demonstrated changes of various matrix metalloproteinases in carcinoma tissue compared with
adjacent noncarcinoma tissue parts. For example, direct correlations were observed between the intensity
of MMP-2 expression and Gleason score [9,12,13]. Increased MMP-2 and MMP-9 but reduced TIMP concentrations were found in conditioned media of epithelial cultures from neoplastic prostate [35]. Metalloprotease activities of different human tumors grown
in nude mice correlated with their invasive characteristics [32]. Changed expressions of MMP-7 and
MMP-9 were also observed in benign prostatic hyperplasia and prostate cancer [10,11]. With the help of our
optimized extraction procedure we simultaneously
determined decreased MMP as well as TIMP-1 levels
in malignant prostatic tissue compared with normal
counterparts. However, the reduction of TIMP-1 was
more evident, so that the ratio of MMP/TIMP-1 was
higher in cancerous tissue compared with normal tis-
sue. This decreased ratio indicates the imbalance between MMP and TIMP in favor of MMP as one phenomenon characteristic of carcinoma tissue [1].
It is important that TIMP-1 has two apparently opposite effects related to tumor progression. On the one
hand, TIMP-1 is a potent antagonist of metalloproteinases and inhibits important steps of tumor progression, especially proteolytic degradation of extracellular matrix and invasion. An imbalance between MMP
and TIMP can be an essential factor in tumor progression [1,35]. On the other hand, TIMP-1 stimulates the
growth of normal and malignant cells independent of
its inhibitory capacity [36]. Growth-promoting activities were found with TIMP concentrations between
10–100 ng/ml, whereas the inhibition of proteolytic
degradation of extracellular matrix needs concentrations higher than 1 mg/ml [37]. Thus, both increased
and decreased TIMP levels may be possible characteristics of cancer tissue. These characteristics are obviously tumor-specific. For example, elevated mRNA
TIMP-1 values found in colon tumors were strongly
correlated with lymph node and distant metastases,
whereas decreased levels were observed in pancreatic
cancer [38,39]. Thus, we consider the ratio of MMP/
TIMP as more important than the levels of the respective individual components. The measurement of
MMP expression by immunostaining and by nucleic
acid hybridization techniques should be completed by
activity determinations, because the essential determinant of invasive behavior is MMP activation.
In conclusion, the MMP and TIMP extraction procedure described in this study was demonstrated to be
a quantitative method. The present results of MMP/
Matrix Metalloproteinases in Prostatic Tissue
TIMP imbalance may bring into focus future experiments for correlating MMP and TIMP measurements
with tumor stage and histological grade.
ACKNOWLEDGMENTS
This work was partly supported by grants from the
Fund of the German Chemical Industry (400770, to
K.J.) and the Family-Klee-Foundation (to M.L.). We
thank Sabine Becker and Silke Klotzek for their valuable technical assistance.
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