MMP-2 Promotes avb3 Mediated Tumor Cell Migration Figure 3. The effect of GM6001 on the adhesion of human melanoma cells. (A) Human melanoma cells, A375, M21 and M21-L, were treated with different concentrations of GM6001, then fluorescently stained and seeded on 48-well plates coated with 10 mg/ml human fibronectin (FN). GM6001 (a broad spectrum hydroxamate MMPs inhibitor) was applied at different concentrations in order to address the role of MMP individuals on the tumor cell adhesion as described in MATERIALS AND METHODS. (B) A375 cells were seeded on human fibronectin (FN) or 0.5% bovine serum albumin (BSA)-coated 48-well plates for 30 min. The cells were treated with or without RGD peptides or 0.5 nM GM6001; (C) M21 and M21-L cells were seeded on human fibronectin (FN) or 0.5% BSA-coated 48-well plates for 30 min. The cells were treated with or without RGD peptides or 0.5 nM GM6001. The fluorescence intensity was measured using a fluorescence spectrophotometer. Statistical difference were determined by comparing the treated group with the normal control by t-test (***, P,0.05). doi:10.1371/journal.pone.0041591.g003 approximately 57% lower than that of the control group, and RGD peptides also reduced the cell migration nearly by 88% (Figure 5F). The results indicated that MMP-2 activity played a critical role in A375 cell migration. MMP-2 Enhances the avb3 Integrin-Mediated Adhesion of Human A375 Melanoma Cells by Cleaving Fibronectin MMP-2 cleaves various ECM proteins to implement different regulatory functions [17,47]. We investigated whether MMP-2 cleaved fibronectin to enhance the adhesion of melanoma cells mediated by avb3 integrin. Recombinant human MMP-2 was activated using APMA and incubated with full-length fibronectin (Figure 6A). The mixture was resolved by SDS-PAGE. Silver staining of the gel revealed several fibronectin fragments. To test whether the cleavage of fibronectin was functionally significant, cell adhesion assays were conducted on the cleaved fibronectin fragments at different time points (Figure 6B). As expected, the difference was not significant between MMP-2-cleaved fibronectin and control groups at 5 min. Thirty minutes later, approximately more than 36% A375 cells adhered to the culture wells coated with MMP-2–cleaved fibronectin compared with those coated with full-length fibronectin alone. The ratio was about 23% at 5 min, 36% at 15 min, 36% at 30 min, 22% at 45 min and 11% at 60 min respectively. It suggested that MMP-2 enhance the adhesion of A375 melanoma cells by cleaving fibronectin obviously at 30 min. This result was further confirmed by a nitrocellulose filter attachment assay (Figure 6C), in which full- length or MMP-2-cleaved fibronectin was resolved on a native Tris-HCl gel and transferred to nitrocellulose, and the cells that were bound to the filter were detected by amino black cell staining. PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e41591