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9 by Interleukin-1· in S100A4-Positive Liposarcoma Cell Line: Correlation with Cell Invasiveness

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9 by Interleukin-1· in S100A4-Positive Liposarcoma Cell Line: Correlation with Cell Invasiveness ANTICANCER RESEARCH 24: 967-972 (2004) Activation of Metalloproteinases-2 and –9 by Interleukin-1· in S100A4-positive Liposarcoma Cell Line: Correlation with Cell Invasiveness LAURA PAZZAGLIA, FRANCESCA PONTICELLI, GIOVANNA MAGAGNOLI, GIORGIA MAGAGNOLI, GABRIELLA GAMBERI, PAOLA RAGAZZINI, ALBA BALLADELLI, PIERO PICCI and MARIASERENA BENASSI Department of Muscoloskeletal Oncology, Rizzoli Orthopaedic Institute, 40100, Bologna, Italy Abstract. Background: The S100A4 gene may affect the Matrix metalloproteinases (MMPs) have been reported invasive properties of tumor cells through modulation of to be involved in the early phase of the invasive process metalloproteinases (MMPs) and their inhibitors (TIMPs). (2,3). MMPs represent a family of at least 24 proteolytic Materials and Methods: In the human liposarcoma cell line, enzymes. Among these, MMP2 (gelatinase A) and MMP9 SW872, we analyzed the expression of S100A4 protein by (gelatinase B) are secreted as latent pro-enzymes (72KDa immunohistochemistry and flow cytometry. The production of pro-MMP2 and 92KDa pro-MMP9) and transformed into MMP2, MMP9, TIMP1 and TIMP2 was assessed by gelatin active forms (62 KDa act-MMP2 and 82 KDa act-MMP9) zymography and enzyme-linked immunoabsorbent assay before after cleavage of a peptide of 10 KDa amino-terminal and after interleukin-1· (IL-1·) and interleukin-6 (IL-6) domains (4). stimulation; cell invasiveness was measured by Matrigel invasion MMPs are inhibited by endogen tissue inhibitors, TIMPs, assay. Results: S100A4-positive SW872 cells responded to IL-1· a family of four different members which form non-covalent with induction of immunoreactive MMP2 and TIMP1 and with complexes with MMPs. Imbalances between MMPs and activation of both MMPs, the latter significantly associated with TIMPs could direct tumor cells towards a metastatic an increase of cell invasiveness. Treatment with IL-6 induced phenotype. TIMP1, a glycoprotein with a molecular mass of less significant variations resulting in a more stable invasive about 28KDa, inhibits MMP9 function and its expression has behavior. Conclusion: These data show that S100A4-positive been associated with decreased tumor growth and SW872 respond to interleukins by influencing the behavior of invasiveness in many tumor cell lines (5,6). However, some factors involved in extracellular matrix degradation and reports have shown that TIMP1 may also act as a growth emphasize the predominant role of MMP activity status on the factor, by promoting the growth of a variety of cells (7,8). positive regulation of cell migration mechanisms. TIMP2 is a 21KDa protein capable of binding to both latent and activated forms of MMP2 (9). Contrasting studies have The invasion of malignant cells through basement reported growth inhibitory and promoting effects of TIMP2 membrane and their ability to metastasize is a multistep in different cell types (10). Previous findings demonstrated process that includes detachment of the cells from their that MMPs may play an important role in the proteolytic primary tumor mass, attachment to extracellular matrix process accompanying bone-turnover in osteosarcoma, (ECM) binding sites, degradation of ECM and migration to especially in the presence of cytokines (11). MG-63 the adjacent tissues (1). osteosarcoma cells responded to interleukin-1· (IL-1·) increasing MMP1 and MMP3 mRNA, as well as the mRNA from TIMP1 which was increased when compared with normal conditions. At the protein level, zymography detected Correspondence to: MariaSerena Benassi, Oncologic Research an increase in gelatinolytic activity corresponding to pro- Laboratory, Istituti Ortopedici Rizzoli, Via di Barbiano 1/10, 40136 MMP2 in the presence of IL-1·. On the other hand, IL-1· Bologna, Italy. Tel: +39-051-6366930, Fax: +39-051-6366761, e- mail: [email protected] and interleukin-6 (IL-6) stimulated the MMP2 level and activity in both MG-63 and Saos2 tumor cells (12). However, Key Words: S100A4 protein, metalloproteinases, liposarcoma cells, Andersen et al. (13) suggested that the effects of growth extracellular matrix. factors and cytokines on the matrix metalloproteinase and 0250-7005/2004 $2.00+.40 967 ANTICANCER RESEARCH 24: 967-972 (2004) their inhibitor synthesis depend on the presence of a small rabbit IgG (R&D System, Minneapolis, MN, USA) for 30 min at calcium binding modulator protein called S100A4 or CAPL. RT. After washing, 25 Ìg/ml swine anti-rabbit FITC conjugate The S100 protein family is involved in various cellular (DAKO) was added for 30 min at RT in the dark. After further washing, the cells were analyzed on a FACStar Plus cytometer processes including cell cycle progression, cell differentiation (Becton-Dickinson, Mountain View, CA, USA). The and motility via interaction with target proteins, such as fluorescence histogram and statistics were calculated using 2+ annexins, Ca release channels, myosin and protein kinases FACStar Plus software (Becton-Dickinson). To analyze the flow (14). Recently, S100 proteins have been suspected to play a cytometric data, the measurements of the distribution of cell significant role in malignancy progression of some types of fluorescence (the events) were considered. Once plotted in a cancer (15,16). In particular, the high expression of the histogram (x= fluorescence intensity expressed as channel, y= S1004A gene is related to a highly metastatic behavior number of events), the instrument calculates the values of central tendency (mode, mean and median). To identify the signal through stimulation of tumor cell motility and the modulation referring to the specific immunofluorescence of S100A4, we of MMP and TIMP synthesis (17). In order to identify calculated the difference (¢) between the mean channel of molecular interaction mechanisms in cell invasion, we fluorescence intensity (MCFI) of the signal given by the S100A4 analyzed the expression of MMPs, of their endogenous antibody and the MCFI of the signal given by the normal rabbit inhibitors TIMPs and of S100A4 protein in the human serum, used as control. liposarcoma cell line, SW872. Furthermore, we investigated the effects of inflammatory cytokines such as IL-1· and IL-6 Immunohistochemistry (IHC). SW872 cells (approximately 50,000) were smeared on microscope slides using a cyto-centrifuge (Cyto- on regulation of the metalloproteinase pathway in relation to Tek) and fixed with acetone.The slides were incubated at room cell invasive behavior. temperature for 2 h with primary antibody S100A4 (DAKO; dilution 1:40). Secondary biotinylated antibody (DAKO; dilution Materials and Methods 1:360) and streptoavidin-biotinylated alkaline phosphatase complex were applied. Then, the slides were developed in NBT/BCIP Cell lines and reagents. The human liposarcoma cell line, SW872, (Roche Ltd, Basel, Switzerland) and mounted for microscopic was obtained from the American Type Culture Collection examination. The control was performed using rabbit IgG instead (Rockville, MD, USA) and maintained in Leibovitz's L-15 Medium of primary antibody. (Sigma-Aldrich, Saint Louis, Missouri, USA) supplemented with 10% fetal bovine serum (Roche, Diagnostic Milan, Italy), L- ELISA. The amount of MMP2, MMP9, TIMP1 and TIMP2 in the glutamine (200mM) and antibiotics (100Uml penicillin, 100 Ìg/ml cell culture medium was measured with a two site ELISA streptomycin) at 37ÆC in a 5% CO2 humidified incubator. The cells ‘sandwich' format (Amersham Pharmacia Biotech, Piscataway, NJ, were seeded into 100-mm culture plates (5x105 cell/plate) and USA) using specific anti-human antibodies which recognize both maintained for 24 h in culture medium supplemented with 10% MMP inactive and active forms. The absorbance was measured at fetal bovine serum. 450 nm and the concentration of MMPs and TIMPs was calculated After washing with phosphate-buffered saline (PBS), using the known concentrations of the corresponding proteins as subconfluent cells were incubated for 24 h in serum-free medium standard. (5ml/plate) with increasing concentrations (25ng/ml, 50ng/ml) of IL-1· and IL-6 (Sigma-Aldrich). Aliquots of the resulting Gelatin zymography. SW872 cell culture medium was collected to conditioned medium were collected and analyzed by zymography determine the amount of latent and active MMPs. Activation of and enzyme-linked immunoabsorbent assay (ELISA). latent pro-enzymes was achieved by incubating the samples after The cells were utilized for flow cytometric analysis. concentration with 1mM 4-aminophenylmercuric acetate (APMA) (Sigma-Aldrich) for 18 h at 37ÆC. Gelatin zymography was Flow cytometric analysis. After recovery of adherent cells with performed using 10% SDS polyacrylamide gels containing 0.1% trypsinization, the cell count and viability were assessed. Then, the gelatin. The gels were stained with 0.125% Coomassie Blue. Each cells were fixed with 4% paraformaldehyde in PBS for 2 h at room protease was detected by zymography as a clear band against the temperature (RT), washed twice with PBS and kept at 4ÆC in blue-stained gelatin background, after destaining with 50% DEPC-treated PBS at a density of 4x106/ml until used. methanol and 10% acetic acid, and quantified by using a GS-670 An aliquot of cells (1x105/100 ml) was washed with PBS and imaging densitometer (Bio-Rad, Hercules, CA, USA). A high prewarmed at 80ÆC for 10 min to unmask intracellular antigen range prestained SDS-PAGE standard was used (Bio-Rad). sites. For the labeling experiment, the cells were processed in a 0.5 ml tube, washed and centrifuged at 3200 x g
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