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 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 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 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 , 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 for 3 min Invasion assay. The cells were seeded into 100-mm culture plates between each step. Dilution of the antibodies was performed (5x105 cells/plate) and maintained for 24 hours in culture medium with PBS supplemented with 2% FCS and 0.1% NaAz. To supplemented with 10% fetal bovine serum. After recovery of the prevent non-specific binding, the cells were incubated with adherent cells with trypsinization, they were collected, washed in 2mg/ml of human immunoglobulin (Sclavo, Siena, Italy) for 30 PBS and suspended in serum-free medium. Three hundred Ìl of min at room temperature (RT) and, after washings, the cells cell suspension containing 5x105 cells/ml in serum-free media and were incubated with 10 Ìg/ml rabbit polyclonal antibody against increasing concentrations of IL-1· and of IL-6 (25ng/ml and human S100A4 (DAKO, Glostrupp, Denmark) for 30 min at RT. 50ng/ml) were added to each insert of an invasion chamber The control specimen consisted of cells incubated with 10 Ìg/ml (Chemicon International, INC., CA, USA) (18). The invasion

968 Pazzaglia et al: Metalloproteinase Proteolytic Pathway Modulation

Figure 1. Moderate to strong cytoplasmatic immunoreactivity of S100A4 in the majority (>50%) of SW872 cells (IHC, 20X).

Table I. ELISA: levels of MMP and their inhibitior expression following interleukin stimulation.

C IL-1a IL-6 25ng 50ng 25ng 50ng

MMP2 52.5±4.6 92.5±5.6 110.0±6.8 71.0±5.2 100.5±7.5 ng/ml *p < 0.001 0.001 0.05 0.05 MMP9 10.3±1.5 10.5±2.0 9.8±1.7 9.5±1.0 10.0±2.3 ng/ml *p < ns ns ns ns TIMP2 195.0±6.2 195.0±9.3 191.0±10.1 201.0±5.4 200.5±5.0 ng/ml *p < ns ns ns ns TIMP1 662.5±7.5 2021.0±8.5 2665.7±8.1 700.3±6.7 830.0±9.0 ng/ml *p < 0.0001 0.0001 0.05 0.01 Figure 2. Flow cytometric analysis of S100A4 in SW872 under basal conditions. Open histogram represents the histotype control (rabbit IgG). *p = statistical significance Shaded histogram represents the sample (S100A4 specific antibody). ¢ MCFI represents the increment of fluorescence intensity of the sample versus control.

chamber was incubated for 18 to 48 h at 37ÆC in 5% CO2. Then, cells on the lower surface of the membrane. Then, the cells were non invading cells were removed as well as ECMatrix gel from dissolved in 10% acetic acid and measured on an Automated the interior of the insert, using a cotton-tipped swab. The inserts Microplate Reader EL311sx (Bio-Tek Instruments, Winooski, were dipped into staining solution for 20 min to stain invasive VT, USA) at 550 nm.

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slightly higher levels with IL-1· than IL-6 (Table I). Neither induced secretion of immunoreactive MMP9. On the other hand, zymography analysis with APMA incubation in non- stimulated cells (Figure 3) showed gelatinolytic activity corresponding to both latent and active forms of MMP2 and MMP9, with a predominant production of latent pro-enzyme MMP2. Incubation with IL-1· and IL-6 determined an increased signal of all detected bands, as evidenced by the respective molecular weights (Figure 3 A, B). However, when the active/inactive enzyme ratios were considered, IL-1· incubation resulted in a gradual activation of MMP2 and MMP9 (0.24±0.03; 0.31±0.05; 0.53±0.04 and 0.78±0.01; 1.05±0.03; 1.2±0.01, respectively), while IL-6 induced a more relevant gelatinolytic activity of latent forms (0.20±0.05; 0.04±0.01; 0.01±0.001 and 0.80±0.02; 0.78±0.04; 0.50±0.02, respectively) (Figure 4). Concerning MMP inhibitors, higher levels of TIMP1 than TIMP2 were seen in non-stimulated SW872. Stimulation with IL-1· and, in a less evident manner, with IL-6, led to a further gradual significant increase of immunoreactive TIMP1, while the level of TIMP2 was not affected by increasing concentrations of IL- 1· and IL-6 (Table I). Figure 3. Zymography amount of MMP2 and MMP9 as pro-enzymes (pro-MMP2, pro-MMP9) and active enzymes (act-MMP2, act-MMP9) in SW872 invasiveness. In the Matrigel invasion assay, SW872 culture medium of SW872 cell line by stimulation with APMA. (A) were moderately, but significantly, affected by IL-1·. Gelatin zymogarphy is performed without and with 25ng and 50ng of IL- 1· and (B) without and with 25ng and 50ng of IL-6. A high range Matrigel invasion increased up to 20% with regard to non- prestained SDS-PAGE standard was used. stimulated cells (p<0.05) (Figure 5). A strong association between cell invasiveness was seen with activation of MMP2 (r2=0.93) and MMP9 (r2=0.90). Not significant variation of cell invasiveness was found in IL-6-stimulated SW872 (Figure 5). Results Conclusion S100A4 expression. In SW872 without IL-1· or IL-6 stimulation, IHC and flow cytometry showed positive Proteolytic activity produced by tumor cells is fundamental expression of S100A4 protein, which critically affects cancer to invasion and metastastatic phenotype (19). In a large cell motility and progression throughout the cell cycle. The series of soft tissue sarcomas, we previously demonstrated majority of liposarcoma cells (>50%) had a moderate to that a deregulated expression of MMPs and TIMPs is strong positive immunostaining, revealing high levels of associated with poor prognosis in terms of metastasis expression and uniform distribution (Figure 1). Flow incidence (20,21). cytometry recorded an increment (¢) of mean channel of In vitro studies on osteosarcoma cells showed that the fluorescence of the sample (anti-S100A4) versus control expression of MMPs and their inhibitors was modulated by (rabbit IgG) of 85 MCFI (Figure 2). Neither IL-1· nor IL- S100A4 protein, thus affecting their invasive properties (17). 6 altered S100A4 immunoreactivity, nor did the MCFI of The down-regulation of S100A4 reduced cell motility and sample versus control change after cytokine stimulation caused an up-regulation of TIMP2, associated to small compared to non-stimulated cells. changes in MMP2. On the other hand, TIMP1 was up- regulated at both mRNA and protein levels, emphasizing MMP and TIMP expression. In non-stimulated SW872 cell the multifunctional role of MMP inhibitors (22,23). medium, ELISA showed a high production of In our study SW872, a liposarcoma cell line with up- immunoreactive MMP2, while MMP9 detection was regulation of S100A4 protein, secreted high amounts of minimal. After cytokine treatment (25ng and 50ng), MMP2 MMP2, predominantly as the inactive pro-enzyme pro-MMP2, secretion gradually and significantly increased, reaching as was evidenced by APMA incubation. Furthermore, when

970 Pazzaglia et al: Metalloproteinase Proteolytic Pathway Modulation

Figure 4. Variations of active/inactive enzyme ratios in IL-1· and IL-6-stimulated SW872 compared to non-stimulated cells.

without changing S100A4 expression. Different results were obtained by Andersen et al. (13) concerning the modulation of IL-1· induction of MMPs and their inhibitors by S100A4 protein in osteosarcoma cell culture. They demonstrated that high S100A4 protein expression inhibited MMP induction by IL-1·, but acted in synergy with IL-1· to reduce TIMP1 synthesis. Although, in our study, ELISA detected only small amounts of immunoreactive MMP9, the analysis of molecular weights, in incubation with APMA after IL-1· stimulation, evidenced an increase of the activated forms of both MMP2 and MMP9, strongly associated with cell invasiveness. These data agree with previous findings Figure 5. IL-1· more significantly increased Matrigel invasion of SW872 correlating in vitro invasion with MMP and S100A4 compared to IL-6 stimulation. expression (24) and emphasize the critical role of MMP activity status on cell migration mechanisms. On the other hand, the high levels of TIMP1 associated comparing the immunoreactivity of TIMP2 and TIMP1, which with S100A4 , in vitro, suggest the need to are respectively inhibitors of MMP2 and MMP9, higher levels better define the dual function of TIMP1, which can either of TIMP1 were found. After IL-1· stimulation, SW872 further promote or inhibit cell growth and spread depending on increased secretion of immunoreactive MMP2 and TIMP1, upstream events.

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Exp Cell Res 224: associated gene S100A4 in human neuroblastoma and primitive 110-115, 1996. neuroectodermal tumor cells J Pediatric Surg 36: 1040-1044, 2001. 11 deBart AC, Quax PH, Lowik CW and Verheijen JH: Regulation of plasminogen activation, matrix metalloproteinases and urokinase-type of plasminogen activator-mediated extracellular matrix degradation in human osteosarcoma cell line MG63 by interleukin-1 alpha. J Bone Miner Res 10: 1374-1384, 1995. 12 Damiens C, Fortun Y, Charrier C, Heymann D and Padrines M: Modulation by soluble factors of gelatinase activities released by osteoblastic cells. Cytokine 12: 1727-31, 2000. 13 Andersen K, Maelandsmo GM, Hovig E, Fodstad O, Loennechen T and Winberg JO: Interleukin-1 alpha and basic fibroblast growth factor induction of matrix metalloproteinases and their Received November 17, 2003 inhibitors in osteosarcoma cells is modulated by the metastasis- Revised January 20, 2004 associated protein CAPL. Anticancer Res 18: 3299-303, 1998. Accepted February 25, 2004

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