Role of Matrix Metalloproteinase (MMP) 2 and MMP-9 in Soft Tissue Sarcoma

Immunohistochemical staining

All procedures for the immunohistochemical staining were performed with the MicroProbe staining system based on capillary action.18) Immunohistochemical staining was performed with the Cap-Plus Detection Kit (Zymed Laboratories, South San Francisco, CA, USA). Paraffin wax sections, 4-µm-thick, mounted on probe slides, were immunostained with anti-mouse monoclonal antibodies for MMP-2, MMP-9, TIMP-1, and TIMP-2 antigens (Oncogene, La Jolla, CA, USA) by the avidin-biotin peroxidase complex method. Sections were deparaffinized and immersed in 60% methanol with 0.3% hydrogen peroxide for 7 minutes to block endogenous peroxidase activity, then incubated with blocking solution (Zymed Laboratories) for 3 minutes at 45℃. The sections were incubated with the anti-mouse monoclonal antibodies to MMP-2, TIMP-1, and TIMP-2 (1:50) for 2 hours at room temperature in a humidified chamber. For the primary mouse monoclonal antibodies to MMP-9, sections were treated with citrate buffer, pH 6.0, at 85℃ for 15 minutes and incubated with MMP-9 antibodies (1:25) overnight at 4℃. Biotin-labeled secondary antibody (Zymed Laboratories) was added for 7 minutes at 45℃. The streptavidin-horseradish peroxidase (Zymed Laboratories) was then applied to the capillary channels, and incubated for 7 minutes at 45℃. After drainage, the tissue sections were ready for chromogen reaction with 3-amino-9-ethylcarbazole (AEC, Zymed Laboratories). The sections were counterstained with hematoxylin and mounted in Universal Mount (Research Genetics, Huntsville, AL, USA).

Immunohistochemical staining was evaluated by scoring the pattern and staining intensity of the cells under a ×100 light microscope. The staining level was classified into four classes according to the distribution of stained cells under the light microscope as 0%, < 25%, 25%-50%, and > 50% of total tumor cells, classified as none, mild, moderate, and diffuse, respectively. Two specialists assessed the staining level of each case twice to determine interobserver and intraobserver reliability. Observer agreement was then assessed with the unweighted kappa coefficient (κ).

RNA isolation and RT-PCR

Malignant fibrous histiocytoma tissue fragments were acquired at surgery. Tissue was flash-frozen in liquid nitrogen. Total RNA from the tissue was extracted using the methodology for TRIzol reagent (Invitrogen, San Diego, CA, USA). RNA (1 µg) was reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen).

Primers for MMP-2, MMP-9, TIMP-1, TIMP-2, and actin, a housekeeping gene that is expressed constitutively, were designed. All primers were checked against the GenBank database to ensure no similarities with other known human DNA sequences. The primer sequences are shown in Table 2. The polymerase chain reaction (PCR) reaction mixture contained 2 µL of each cDNA sample, 10 pM each of sense and antisense primers, and other PCR reagents in a final volume of 20 µL. PCR reagents, dNTP, Taq DNA polymerase, and 10× reaction buffer (40 mM KCl, 10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl₂, stabilizer, and tracking dye) obtained from Accupower PCR PreMix (Bioneer, Daejeon, Korea). PCR reactions were conducted using a Perkin-Elmer GeneAmp PCR system 2700 (Perkin-Elmer, Norwalk, CT, USA) as follows. PCR cycles were 94℃ for 5 minutes, then 35 cycles of denaturation at 94℃ for 1 minute, annealing at 60℃ (65℃ for MMP-9) for 1 minute, and polymerization at 72℃ for 2 minutes, followed by 72℃ for 7 minutes.

Table 2
Primer Sequences and Size of Products of Target Genes

• Click for larger image

The RT-PCR products were visualized on 1% agarose gels electrophoresed in 0.5× TBE buffer containing 0.5 µg/mL ethidium bromide. The fidelity of the RT-PCR product was verified by comparing the size of the amplified products to the expected cDNA bands and by sequencing the PCR products. With a housekeeping gene, β-actin, as an internal control, expression levels of MMP-2 and MMP-9 genes were analyzed by semi-quantitative RT-PCR using Image-Pro Plus (Media Cybernetics, Bethesda, MD, USA).

Western blot analysis

To investigate whether TIMP-1 and TIMP-2 could inhibit the expression of MMP-2 and MMP-9 in human MFH cells, protein levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were determined by Western blotting. Samples were homogenized in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% NaDOC, 50 mM Tris, pH 8.0, 1 mM PMSF, and 1 µg/mL aprotinin and leupeptin) and the supernatant was collected following centrifugation. Protein concentrations were determined by the bicinchoninic acid method (BCA kit, Pierce, Rockford, IL, USA). Equal amounts of proteins of MFH cell were electrophoresed in 10% sodium dodecyl sulfate (SDS) polyacrylamide gel using a MiniProtean (Bio-Rad, Hercules, CA, USA) gel apparatus, and the electrophoresed gel slabs were electrotransferred to nitrocellulose membranes (Amersham Life Science, Buckinghamshire, UK) overnight at 4℃ in transfer buffer (30 mM Tris, 240 mM glycine, 25% methanol) at a constant voltage of 20 V. The membrane was blocked in 5% defatted milk powder in TBS-T, pH 7.6 (100 mM Tris buffer with 150 mM NaCl and 0.1% Tween 20), and the blot was incubated with specific primary antibodies. To detect MMP-2, MMP-9, TIMP-1, TIMP-2, and β-actin, we used monoclonal antibodies (Oncogene) at a final concentration in blocking buffer of 1 µg/mL (1:1,000 dilution). These antibodies recognize both the latent (92 kDa) and active forms (82 kDa) of MMP-9, and the latent (72 kDa) and active forms (62 kDa) of MMP-2. Horseradish peroxidase goat anti-mouse immunoglobulin (BD Bioscience, San Jose, CA, USA) was used as the secondary antibody (1:2,000) for 1 hour at room temperature, and then the membrane was washed four times for 15 minutes each in TBS-T. Bands were developed using the Enhanced Chemiluminescence (ECL Plus) system (Amersham Bioscience, Buckinghamshire, UK). The MMP-2 and MMP-9 were semi-quantified using an Image-Pro Plus (Media Cybernetics).

Gelatin zymography

This is an in vitro assay using gelatin-substrate gel electrophoresis to measure the level of MMP activity in MFH samples. Frozen MFH tissues were pulverized in liquid nitrogen and homogenized in buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl₂, 200 mM NaCl) and a homogenizer. Protein concentrations were determined by the BCA method (BCA kit, Pierce). Samples were mixed with an equal volume of 4× sample buffer (200 mM Tris-HCl, 8% SDS, 0.4% bromophenol blue, 40% glycerol). Samples were electrophoresed on 8% SDS polyacrylamide gels containing 2 mg/mL gelatin (type A, Sigma, St. Louis, MO, USA). Following electrophoresis, the gel was washed three times for 30 minutes in 2.5% Triton X-100 at room temperature, and incubated for 18 hours at 37℃ in incubation buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl₂, 200 mM NaCl). The gel was stained for 1 hour with Coomassie Brilliant Blue R-250 (0.2% Coomassie Brilliant Blue R-250, 20% methanol, 10% acetic acid in H₂O) and destained in washing solution (30% methanol, 10% acetic acid). White bands on the blue background indicated zones of digestion corresponding to the presence of different pro-MMPs and activated MMPs on the basis of their molecular weight. The MMP-2 and MMP-9 were semi-quantified using Image-Pro Plus (Media Cybernetics).