3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 Figure 6. Effect of 3-azidoWA on phosphorylation of Akt, ERK and expression of TIMP-1. (A) HeLa and (B) PC-3 cells were treated with increasing concentrations of 3-azidoWA as indicated, phosphorylated and total Akt, ERK expressions were determined by Western blotting. TIMP-1 expression was verified by same method. doi:10.1371/journal.pone.0044039.g006 exhibit that 3-azidoWA may capable of suppressing VEGF induced neovessel formation and abolishe in vivo angiogenesis. Discussion a-b-unsaturated carbonyl moiety exists in a plethora of natural products exhibiting cancer chemo-preventive and chemo-protec- tive activities. Their biological activities are mostly attributed to their ability to act Michael acceptors. Currently, irreversible binding at active sites has been considered to be one of the reasons to drug resistance in cancer treatment [36]. Thus, in order to include a-b-unsaturated carbonyl group in drug development strategies, the potentially adverse effects must be circumvented. This can be achieved by fine tuning the reactivity of the a-b- unsaturated carbonyl unit leading to a high degree of specificity. Different approaches for comparing similar natural products and synthetic molecules have been followed in order to predict the reactivity of a-b-unsaturated carbonyl compounds. The a-b- unsaturated carbonyl moiety permits the direct adjustment of electrophilicity and reduction potential through modification of the peripheral substituents [36]. A structural modification of natural products represents an elegant approach towards finding new biological activities. In the light of promising therapeutic prospective of Withaferin A as an anticancer molecule reported by our institute [37,38], we were keen to investigate modifications of the ring A with a-b- unsaturated carbonyl functionality. We envisaged that substitution of the a-b-unsaturated carbonyl system of Witheferin A at the b- position may be a practical approach to find new activities of the modified compounds as this will obviate irreversible covalent binding in active sites by biological nucleophiles, a reason behind drug resistance in cancer treatment [25]. This led to the discovery of pure compound 3-azidoWA (for compound purity supporting NMR, CMR, COSY data are provided in the supplementary section), a potent anticancer molecule than the parent withaferin A. In the present study, we have identified a highly selective MMP- 2 inhibitor, 3-azidoWA, a natural product withanolide derivative from traditional medicinal plant W. Somnifera. Though, with- anolides have been well documented for diverse biological functions, viz, antiproliferative, anti-invasive, radiosensitizing and antiarthritic, here, for the first time, we uncover the target specific MMP-2 inhibition by 3-azidoWA in moderately aggressive cervical and prostate cancer cells. We demonstrate here that 3- azidoWA is a potent inducer of extracellular Par-4. Although, the intracellular Par-4 induction by Withaferin A had been empow- ered the apoptotic induction of these class compounds in several cancers [39], interestingly, we have explored the role of extracellular systemic Par-4 which was robustly increased in the conditioned media due to 3-azidoWA treatment as compared to Withaferin A treatment. Our data reveal that inhibition of MMP-2 expression and gelatinase activity by 3-azidoWA was extracellular Par-4 dependent. PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e44039