3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 Figure 3. 3-azidowithaferin induces extracellular Par-4 secretion and inhibits MMP-2. (A) HeLa cells were left untreated or treated with 0.50 mM and 0.75 mM of 3-azidoWA for 48 h, conditioned media was analyzed for MMP-2 & -9 gelatinase activity. (B) HeLa cells were left untreated or treated with 0.25, 0.50, 0.75 and 1.0 mM 3-azidoWA for 48 h, conditioned media obtained was employed for western blot analysis followed by Coomassie blue staining to reveal the 68 KDa BSA band for loading control. (C) HeLa cells were treated with various concentration of parent withaferin A for 48 h and the activity of MMP-2 was determined by gelatin zymography. (D) & (E) HeLa cells were treated with TRAIL for 180 minutes and with Tumicamycin for 60 minutes (as indicated). Conditioned media were subjected to gelatin zymography analysis for MMP-2 activity and Western blot analysis for extracellular Par-4, followed by coomassie blue staining for loading control. doi:10.1371/journal.pone.0044039.g003 azidoWA blocks MMP-2 expression in a dose dependent manner and that may mediate through extracellular Par-4. Inhibition of MMP-2 Activity by 3-azidoWA is Independent of Apoptosis We further envisaged into the direct or indirect correlation of 3- azidoWA induced MMP-2 inhibition and concurrence of apop- tosis. Previous studies have demonstrated the extrinsic pathway of apoptosis was induced by extracellular Par-4 [3] and siRNA mediated knock down of MMP-2 sensitized lung cancer cell apoptosis [32]. Therefore, we treated HeLa cells with vehicle, 1.0 mM of 3-azidoWA and 25 nM staurosporine in presence or absence of caspase inhibitor and conditioned medium was employed for analyzing MMP-2 activity. As shown in Fig. 5, A treatment of HeLa cells with 1.0 mM of 3-azidoWA and positive control staurosporine (25 nM) induced apoptosis, which was quantified by active caspase-3 staining and when apoptosis was blocked by Pan caspase inhibitor, MMP-2 activity level remained same as 1.0 mM 3-azidoWA alone treatment lane (Fig. 5, B). Our results suggest that inhibition of gelatinase activity of MMP-2 by 3- azidoWA is independent of apoptosis. 3-azidoWA Abrogates Akt-ERK Phosphorylation and Upregulate TIMP-1 Expression The importance of PI3K/Akt, ERK signaling pathways have been extensively studied in cancer cells invasion [33,34], we seek to determine the effect of 3-azidoWA on these signaling pathways. Our results revealed that Akt and ERK phosphorylations were reduced when HeLa and PC-3 cells were treated with increasing concentration of 3-azidoWA respectively (Fig. 6, A and B). On the other hand 3-azidoWA treatment elevated the expression of TIMP-1 which blocked the proteolytic potential of MMP-2 expression in both cell lines, in dose dependent manner (Fig 6, A and B). Collectively these results demonstrate that 3-azidoWA inhibited the Akt and ERK phosphorylation in cervical and prostate cancer cells and augmented TIMP-1 expression. PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44039