Integrins directly bind components of the ECM and provide the traction necessary for cell motility and invasion. ECM remodeling is also controlled by integrins, which regulate the localization and activity of proteases. Proteases involved in these processes include serine proteases (the plasminogen activators uPA and tPA), matrix metalloproteinases (MMPs), and cysteine proteases (cathepsins B, D, L and H) [12]. GBM cells secrete MMPs and their mRNA and protein levels are elevated in patient biopsy tissues [13–15]. Significant correlation between MMP-9 levels and the histological grade of malignancy has been reported [16]. Furthermore, interactions between integrins expressed by glioma cells and the ECM and the activity of MMPs form the basis for glioma cell migration and invasion [17]. Expression of urokinase-type plasminogen activator receptor (uPAR) is much more robust in high-grade than in low- grade human gliomas [18]. Localization of uPAR mRNA in astrocytoma cells and the endothelial cells within brain tumor tissue has been reported and the expression of uPAR in the invading astrocytoma cells appears to have a critical role in the invasive behavior of glioblastoma [18]. Despite the controversy surrounding whether uPAR and integrins interact directly, many studies show that uPAR signaling requires integrin co-receptors. Furthermore, some non-integrin co-receptors of uPAR cooperate with integrins in signaling or influence uPAR-integrin interactions [19,20]. Additionally, uPA-uPAR binding results in the expression of cathepsin B [21], another important protease involved in ECM Silencing Proteases in Glioma degradation, and significantly higher levels of cathepsin B has been found in high-grade glioblastomas [22,23]. Taken together, the proteases MMP-9, uPA and Cathepsin B play critical roles in glioma pathology and have the combined ability to break down the ECM components. Moreover, the expression of one protease has a direct or indirect influence on the expression of other proteases. As such, the net proteolytic, and therefore invasive, potential of a given tumor cell might depend on the interplay between many proteolytic enzymes. In this scenario, targeting only one protease will definitely not result in the expected therapeutic outcome. Hence, in the present study, we investigated the effect of downregulating MMP-9, uPAR and ctahepsin B using both single as well as MMP-9/uPAR and MMP-9/cathepsin B bicistronic shRNA plasmid constructs on the expression of integrins and on the migrating and invasive potential of glioma xenograft cells. Results Effect of shRNA constructs on MMP-9, uPAR, and cathepsin B Gelatin zymography revealed an inhibition of MMP-9 activity with all the treatments (Fig. 1A). The activity/expression of one protease has a direct or indirect influence on the activity/ expression of other proteases. Our results were also in agreement with this fact and hence we noticed a prominent reduction in Figure 1. Efficiency of shRNA plasmid constructs and their effect on adhesion of xenografts to various ECM proteins. (A) Activity, protein and mRNA expressions of MMP-9, uPAR and cathepsin B in 4910 xenograft cells. MMP-9 and uPA activity was determined by gelatin and fibrin zymography after treatments with scrambled vector (SV-sh), MMP-9 (M-sh), uPAR (U-sh), cathepsin B (C-sh), MMP-9/uPAR (MU-sh) and MMP-9/ cathepsin B (MC-sh) plasmid shRNAs. Western blot analysis showing MMP-9, uPAR and cathepsin B protein expression levels were reduced after M-sh, U-sh, C-sh, MU-sh and MC-sh treatments. n = 3. RT-PCR of 4910 cells transfected with SV-sh, M-sh, U-sh, C-sh, MU-sh and MC-sh was performed as per standard protocols. Further, quantification of the Western blots (B) revealed significant reductions in protein expressions after M-sh, U-sh, C-sh, MU-sh and MC-sh treatments. n = 3. Values shown are the mean (6SEM). *p,0.05 vs. control (C) Adhesion assay was performed to evaluate the effect of SV- sh, M-sh, U-sh, C-sh, MU-sh and MC-sh treatments in 4910 and 5310 glioma xenograft cells on their adhesive potential to collagen (type I), vitronectin, fibronectin and laminin coated plates. Percent adhesion was calculated from the mean obtained from 3 independent experiments and values shown are the mean (6SEM). *p,0.05 vs. control. doi:10.1371/journal.pone.0011583.g001 PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11583