3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 Figure 2. 3-azidoWA inhibits motility, invasion and colony formation ability of HeLa cells. (A) HeLa cells (0.56105 cells/well) were grown to confluence in six well tissue culture plate and scratched with sterile tip; 3-azidoWA was added to cultures as indicated. Scratched areas were photographed (magnification 100x) at zero hour and then subsequently again 24 h later to assess the degree of wound healing. (B) The scratched areas were quantified in three random fields in each treatment, and data were calculated from three independent experiments. (C) HeLa (16103) cells/well were cultured and treated with various concentration of compound 3-azidoWA for 5 days at 37uC and then stained with crystal violet (for details see materials and methods), numbers of stained colonies were counted, photographed (100x) and (D) data were calculated from three independent experiments. Columns mean; bars SD of three independent experiments. P,0.05, **P,0.01 compared with untreated control. (E) Cell migration was determined via the modified Boyden chamber assay as described in Materials and Methods. HeLa cells (26105) were seeded in top chamber in the presence or absence of 0.25, 0.50, and 0.75 mM of 3-azidoWA. Cells were allowed to migrate for 24 h, at which point migratory cells on the bottom half of the insert membrane were stained with 0.1% crystal violet and counted under 200x magnification. (F) Invasive cells were counted using image software as the number of migrated cells per high-power field (HPF). Five fields were counted in triplicate (n = 3) from each insert. Cell images were obtained using microscope Nikon Eclipse E200 inbuilt with camera. Columns mean; bars SD of three independent experiments. P,0.05, **P,0.01 compared with untreated control. doi:10.1371/journal.pone.0044039.g002 Inhibition of MMP-2 by 3-azidoWA is Mediated through Extracellular Par-4 To investigate the role of extracellular Par-4 which is known to promote apoptosis [3], we examined whether there is any inter- relation between two physiologically important secretory proteins Par-4 and MMP-2. Thus, we depleted the secretory Par-4 in the conditioned medium (3-azidoWA treated well) by using Par-4 neutralizing antibody. The neutralization of extracellular Par-4 restored MMP-2 level compared to the control antibody (PTEN) treatment (Fig. 4, E). Further we verified the ectopically expressed Par-4 could inhibit MMP-2, as over expressed Par-4 was secreted spontaneously in the conditioned media [3]. We demonstrate that secreted GFP Par-4 not GFP inhibited MMP-2 gelatinase activity (Fig. 4, F) in PC-3 cells. Collectively these results indicate that 3- PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44039