3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 Figure 1. 3-azidoWA is apoptosis inducing anti-proliferative agent. (A) Synthesis of 3-azidoWA. (B) Effects of 3-azidoWA on the cell proliferation of A549 (lung cancer), PC-3, DU-145 (prostrate cancer) and HeLa (cervical cancer) cell lines were determined by MTT assay. (C) 3-azidoWA along with vehicle DMSO and positive control staurosporine treated cells were analyzed by FACS, (as indicated); graphical representation of the results show proportion of non-apoptotic cells (Q3), early apoptotic (Q4), necrotic (Q1) and late apoptotic (Q2) cells. (D) HeLa cells (56104) were cultured in chamber slides and treated with 3-azidoWA (0.25, 0.50 and 1.0 mM) along with vehicle DMSO and positive control staurosporine (25 nM) for 24 h. After fixation, cells were stained with nuclear stain DAPI and photographed under fluorescence microscope (100x magnifications) to identify the apoptotic nuclei (white arrowheads). (E) HeLa cells were treated with increasing concentrations of 3-azidoWA as indicated, procaspase-3 and cleaved caspase-3 expressions were determined by Western blotting along with loading control b-actin. doi:10.1371/journal.pone.0044039.g001 Par-4 occurred via the classical BFA-sensitive pathway involving the ER/golgi route, we terminated protein trafficking from the ER to golgi with Brefeldin A (BFA) and found secreted Par-4 level was decelerated in the conditioned media after 3-azidoWA and TRAIL treatment in presence of Brefeldin A (Fig. 4, A and B upper row). Here, we suggest that 3-azidoWA induces extracel- lular Par-4 secretion by classical BFA sensitive pathway. Depletion of Extracellular Par-4 Restores Cell Invasion Due to its immense physiological functions reported [30], Par-4 contributes major role in tumor regression and inhibition of NFkB activity [31]. Hence, we rationally examined the anti- metastatic role of tumor suppressor secretory Par-4. We pre- treated conditioned medium obtained from 3-azidoWA treated (48 h) HeLa cells with control PTEN antibody and Par-4 neutralizing antibody for 30 min and then added into the upper well of Boyden chamber containing Hela cell suspension. Interestingly these results showed cells pre-treated with Par-4 neutralizing antibody invaded through membrane in presence of 3-azidoWA compared to control PTEN antibody pre-treated cells (Fig. 4, C). The number of migrated cells received Par-4 antibody pre-treatment was highly invasive and as good as vehicle + PTEN antibody treated control cells (Fig. 4, D). PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44039