to do this, plugs were removed from control mice (no added VEGF-A and bFGF) at the end of day 4, 7 and 11. The plugs that contained VEGF-A and bFGF were removed from mice at the end of days 2, 4, 7 and 11 after injecting Matrigel. Standardizing the optimal time for neovascularisation, a dose response study using 3-azidoWA was performed in which the compound was administered at 1, 10, 20, 30, and 50 mg/kg/d, i.p. for 3 days (8th, 9th, and 10th). At the end of duration, Matrigel plugs were removed for visualization and quantification. After identifying doses of 3-azido WA that inhibits vascularization, the number of doses to inhibit neovascularisation (preventive study) or disrupt established vasculature (treatment study) was investigated. For the preventive study mice were dosed with (30 mg/kg/d, i.p.) for 1–4 days after 24 h Matrigel injection. Seven days after injection of Matrigel plugs mice were sacrificed and the plugs removed for visualization and quantification. For the treatment study, neovascularisation was allowed to develop over a 7-day period. Groups of animals were dosed with compounds on day 8 (1-day dosing) or days 8–10 (3 days dosing) at 30 mg/kg/d, i.p. The effects on established vasculature were assessed 11 days after the injection of the Matrigel plugs. Statistical Analyses Data were expressed as mean 6 SEM. Comparisons used Student’s t test. P,0.05 values were assigned significance. Results 3-azidoWA is an Anti-proliferative Agent and Induces Apoptosis in PC-3 and HeLa Cells Withaferin A is a potent cytotoxic agent and showed growth- inhibitory properties in tumor cell culture experiments [19,26]. As good as parent molecule Withaferin A, the derivative of With- aferin, 3-azidoWA (Fig. 1, A) significantly exhibited anti-prolifer- ative effect in a panel of cell lines tested (having IC50 within a range of 800 nM -1.0 mM) [25] (Fig. 1, B). For further confirmation of apoptosis by 3-azidoWA, we performed Annexin V-FITC staining of the 3-azidoWA treated HeLa cells. As depicted in Fig. 1, C 59.1% early apoptotic cells compared to 72.5% with 25 nM staurosporine, appeared when HeLa cells were treated with 1.0 mM 3-azidoWA for 24 h. By DAPI staining we observed that 1.0 mM of 3-azidoWA, not 0.5 mM induced apoptosis in Hela and PC-3 cells within 24 h of incubation as compared to positive control staurosporine (Fig.1, D). A residual passaging related early apoptoic cells were observed in FACS data (at lower concentration of 0.25 mM of 3-azidoWA treatment), however, higher concentration (1.0 mM) of 3-azidoWA, lead the cells towards apoptosis with chromatin condensation around the nuclear periphery accompanied by nuclear size reduction (white arrowheads, Fig. 1, D). Active caspase-3, quantified by western blot analysis showed cleavage of caspase-3 at 1.0 mM of 3- azidoWA treatment but not at sub-toxic doses (0.5 mM) (Fig. 1, E). Collectively, these results demonstrate that 3-azidoWA is a prospective cytotoxic and apoptosis inducing natural product derivative. 3-azidoWA Inhibits Cell Motility, Invasion and Colony Formation As withanolides were known to inhibit invasion capability of cancer cells [27], we intended to study the effect of 3-azidoWA on the invasive potential of HeLa and PC-3 cells in vitro. Wound healing assays were used to determine whether sub-toxic concentration of 3-azidoWA could inhibit motility of Hela and PC-3 cells. After 48 h, cell monolayer’s were wounded, the vehicle 3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 DMSO treated cells had completely filled in the cleared area (Fig. 2, A), whereas treatment with 0.50 and 0.75 mM of 3- azidoWA significantly (p,0.05) inhibited motility of Hela and PC- 3 cells, as did treatment with staurosporine (Fig. 2, A and B) (Fig. S1, A and B). Colony formation ability of HeLa and PC-3 cells were attenuated by 3-azidoWA treatment in a dose dependent manner. As shown in Fig. 2, C sub-lethal doses of 3-azidoWA decreased colony formation ability of HeLa cells in statistically significant manner (P,0.05) (Fig. 2, D) (Fig. S1, C and D). A critical event in tumor invasion and metastasis is the ability of tumor cells to invade through the extracellular matrix, allowing tumor cells to move beyond the confines of primary tumor environment [28]. To examine the effect of 3-azidoWA on cell invasion, Boyden chamber invasion assay was carried out to determine the ability of HeLa and PC-3 cells to invade through biological matrices in vitro. As shown in Fig. 2, E and F, (Fig. S1, E and F, P,0.01) treatment with 3-azidoWA (0.50 and 0.75 mM) inhibited cell invasion (P,0.05).To overrule the possibility of alteration of cell invasion due to programmed cell death, we carefully choose the physiologically relevant concentrations of 3- azidoWA. All these results collectively indicate that sub-toxic doses of 3-azidoWA alter the growth kinetics of HeLa and PC-3 cells. This could be a positive indicator for testing its antineoplastic activity in cervical and prostate cancer cells. Inhibition of Matrix Metalloproteinase 2 Gelatinase Activity and Expression by 3-azidoWA Tumor cell invasion through matrix and tissue obstruction needs the combined effects of increased cell motility and controlled proteolytic degradation of matrix. High levels of MMP’s in tumor tissues have been correlated with cancer cell matrix degradation, invasion and metastasis [29]. As 3-azidoWA inhibited cell motility, we investigated whether 3-azidoWA exerts anti-gelatinase activity. As seen in Fig. 3, A and B (Fig. S2, A), upper row, increasing concentration of 3-azidoWA selectively abrogated gelatinase activity and expression of 72 KDa MMP-2 band as confirmed by gelatin zymography and Western blot analysis. The effect of MMP-2 inhibition by 3-azidoWA was more pronounced than parent molecule with- aferin A (Fig. 3, C) (Fig. S2, B).Further we examined whether 3- azidoWA could inhibit other gelatinase (MMP-9) and results revealed that 3-azidoWA specifically block the MMP-2 activity in HeLa and PC-3 cells, but not MMP-9 (Fig. 3, A middle row) (Fig. S2, A middle row). Together, these findings suggest that 3- azidoWA strongly and selectively abrogates MMP-2 expression and activity in a dose dependent manner. 3-azidoWA Induces Extracellular Par-4 Secretion by Classical Pathway As couples of apoptosis inducing agents facilitate the secretion of extracellular Par-4 [3], we sought to examine a panel of medicinal plants derived pure natural products that could promote apoptosis and/or enhance secretion of Par-4 (data not shown). Surprisingly we found that as low as 0.5 mM (much below cytotoxic dose) of 3- azidoWA, but not the parent molecule Withaferin A (data not shown) induced the extracellular Par-4 secretion in dose depen- dent manner (Fig. 3, B middle row), verified by Western blot. Moreover, we observed 3-azidoWA treatment augmented the secretion of extracellular Par-4 in the conditional media that essentially mimicked the effects of 300 ng TRAIL (TNF- related apoptosis inducing ligand) and 1.0 mM of Tunicamycin treatment (Fig. 3, D and E middle row). To assess whether this secretion of PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44039