3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 Figure 8. Schematic represents the extracellular Par-4 induction due to 3-azidoWA could abrogate invasion. doi:10.1371/journal.pone.0044039.g008 In addition to degrading ECM components, MMP’s were shown to confer apoptosis resistance by modulating Fas-FADD mediated death signaling [46] and cleavage of Fas by MMP-2 resulted in decreased sensitivity of HT-29 colon carcinoma cells to Fas mediated apoptosis [32]. Extracellular Par-4, on the other hand has been documented to exert extrinsic apoptotic effect by activating downstream caspase-3 [3]. Earlier studies have shown that downregulation of MMP-2 with adenovirus mediated delivery of MMP-2 (ad-MMP-2) siRNA reduced invasion, migration and angiogenesis in A549 cells in vitro and inhibited tumor growth and metastasis in vivo [47]. Further the authors also revealed that Ad- MMP-2 mediated tumor growth inhibition occurred through cleavage of caspases-8,-9 and -3, and activation of FAS-mediated signaling pathway [32]. Consequently, we try to demonstrate here 3-azidoWA as well as TRAIL mediated inhibition of MMP-2 in cervical (data not shown) and prostate cancer cell line through induction of extracellular Par-4 which has been shown to induce extrinsic apoptotic pathway by FADD-caspase-8-caspase-3 activa- tion [3]. Interestingly, we have observed that ectopically over expressed GFP-Par-4 which secreted spontaneously in the conditioned medium [3], significantly inhibited MMP-2 within 48 h of post transfection. Moreover, 3-azidoWA treatment caused activation of caspase-3 and inhibition of MMP-2 expression compared to vehicle treated cells and blocking of caspase-3 activation by caspase inhibitor could not restore the MMP-2 expression level. Hence, that inhibition of MMP-2 by 3-azidoWA was apoptosis independent. Interestingly, this azido derivative (3- azidoWA) did not confer any significant change in the cell proliferation data what we obtained and as reported earlier with parent Withaferin A [25], but it enhanced the biological activity of the said derivative tremendously. To address the rational discrimination of 3-azidoWA in controlling cell motility versus apoptosis we carefully choose a range of drug concentrations and observed that 0.5 mM 3-azidoWA did not contribute to DNA fragmentation (data not shown) but altered cell motility. There- fore, there may be a logical explanation that at a low dose of (0.5 mM) 3-azidoWA inhibited MMP-2, but had no effect on cleaved caspase-3 (data not shown) activation, but at the dose level of 1.0 mM, not only MMP-2 activity was strongly downregulated but also caspase-3 mediated apoptotic machinery might be unregulated via extracellular Par-4. Thus it might be possible that low dose of 3-azidoWA induced basal secretion of extracel- lular Par-4 that might be utilized to downregulate MMP-2 and additional increase of 3-azidoWA/prolong drug treatment might add excess pool of secretory Par-4 in the conditioned media that accelerated apoptosis by extrinsic pathway. But, how the intracellular Par-4 level was correlated with extracellular Par-4 secretion due to 3-zidoWA treatment is under current investiga- tion in our laboratory. The increased occurrence of spontaneous tumors in Par-4 mice lends credence to the identification of Par-4 as a tumor suppressor protein but secreted form of Par-4 in the culture medium of both normal and malignant prostate cells has been characterized as a new physiologically relevant therapeutic target in cancer [3]. TRAIL on the other hand is one of the most studied and clinically relevant inducers of apoptosis [48] and enhanced TRAIL sensitivity has been observed in Jurkat cells after Par-4 transfection [49]. Here we have noticed a dose dependent increase in extracellular Par-4 level when HeLa/PC-3 cells were treated with TRAIL and Tunicamycin (with fairly high concentration of 300 ng/ml TRAIL and 1.0 mM Tunicamycin), which also inhibited MMP-2 gelatinolytic activity due to extrinsic apoptotic induction by secretory Par-4. In view of finding the role of 3- azidoWA induced extracellular Par-4, that escalated gradually with increasing concentration of 3-azidoWA (below toxicity), we found the concomitant weaken of MMP-2 band in the zymogram (having equal loading control BSA), that resembled anti-invasive property of conditioned media containing secretory Par-4. As cancer cells are prone to ER stress, 3-azidoWA treatment might add more ER stress to escalate the level of secretory Par-4 and promote signaling pathways that control cellular motility (Fig. 8). When these extracellular Par-4 in the conditional media was depleted with Par-4 neutralizing antibody, HeLa cells invaded through the membrane of the Boyden chamber (Fig. 4, C) and PLOS ONE | www.plosone.org 12 September 2012 | Volume 7 | Issue 9 | e44039