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 . These enzymes play an important role in tumor invasion and metastasis . 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 . 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 . 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: email@example.com 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 . 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 . 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 . 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. , 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 . 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  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 . 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. , 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 . However, the above-mentioned MMPs cleave that substrate differently . 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 . 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 . 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 . 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 . Metalloprotease activities of different human tumors grown in nude mice correlated with their invasive characteristics . 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 . 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 . 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 . 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. 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