and tumor invasion/metastasis [7,8]. During the developmental phase, many of the MMP inhibitors failed in the early phase clinical trials because of extensive homology between catalytic domains of MMP’s. Moreover, most of the synthetic/semi- synthetic inhibitors of MMP’s were withdrawn during clinical trials due to unanticipated long term drug intolerance reduced drug compliance [9]. On the other hand, recently, natural products or natural product derivatives have been considered extremely potential to abrogate MMP-2 and -9 mediated invasion/metastasis either in vitro or in vivo set up. These include aqueous cinnamon extract [10], green tea extract [11], curcumin [12], and steroidal saponin from fenugreek [4], chitooligosachar- ides (COS) from marine natural products [13]. Withaferin A (WFA) is a prototype of the withanolide class of natural products that exhibit diverse pharmacological activities, including antitumor, antiangiogenic, cardioprotective, anti-inflam- matory, and immunomodulatory effects [14,15]. The bioactive properties of Withaferin A includes cytoskeletal remodeling by binding to Annexin II [16], antiangiogenic [17,18] and antitumor activity [19,20] by inhibition of proteasomal chymotrypsin [21] and apoptotic induction by inhibition of protein kinase C [22]. Recently, Oh et al have demonstrated the caspase-3 activation through Withaferin A [23]. Apart from its anti-cancerous activity, Withaferin A has also been documented for its anti-inflammatory property by suppressing alpha-2-macroglobulin [24]. With our recent success towards the development of a library of Withaferin A semisynthetic analogues, the rational screening strategy lead to the generation of 3-azidoWA, the potent anticancer candidate [25]. Although the importance of a-b-unsaturated functionality of ring A of Withaferin A and the anticancer potential of 3-azidoWA became obvious, still its mode of action was not clear. In this study we evaluated the mechanistic role of 3-azidoWA (3-azido WA), an azido Withaferin derivative on motility and invasion of cancer cells. We also wanted to co-relate this study with the signaling pathways viz, ERK, Akt, which are activated in different cancers and potentiates invasion and metastasis of diverse cancer cells. The results of these studies provide us with important information on the mechanistic role of 3-azidoWA on abrogation of invasion of cervical and prostate cancer cells that might be en- routed through extracellular Par-4. Materials and Methods Cell Culture and Antibodies All cell lines were purchased from European Collection of Cell Culture (ECACC), Fetal Bovine Serum (FBS), RPMI-1640, Minimum Essential Medium (MEM), Penicillin G, Streptomycin, Trypsin-EDTA were obtained from Invitrogen Corp. 5-diphenyl- tetrazolium bromide (MTT), Paraformaldehyde, Crystal violet, Staurosporine, Phenylmethylsulfonyl fluoride (PMSF), Dithiothre- tol (DTT), NP-40, Protease Inhibitor Cocktail, Gelatin, Brij-35, Comassie Brilliant Blue (R-250), Brefeldin A (BFA), Tunicamycin, TRAIL, Annexin V-FITC apoptosis detection assay kit, Dimethyl- sulfoxide (DMSO), Bradford reagent was obtained from Sigma Chemicals Co. (St. Louis, MO) Propedium Iodide and Ultracruz DAPI mounting medium was obtained from Santa Cruz Biotechnology Inc.(Santa Cruz, CA), Z-VAD Pan caspase inhibitor, recombinant human VEGF-165 and recombinant human FGF (basic 146 aa) were obtained from R&D Systems (Minneapolis, MN). Cells were cultured in RPMI 1640/MEM containing 10% fetal bovine serum in presence of 70 mg/L penicillin and 0.1 g/L streptomycin and were incubated at 37uC with 95% air and 5.0% carbon dioxide. All cells were used in experiments during the linear phase of growth. Antibodies were 3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 obtained from following commercial sources: anti-Par-4, anti- MMP-2, anti-Akt, anti-ERK, and anti-TIMP-1 from Santa Cruz Biotechnology (Santa Cruz, CA), anti-p-Akt from and anti- caspase-3 (Cell Signaling) and anti-b-actin, anti-p-MAPK (Sigma Chemical, St. Louis, MO). Synthesis of 3-azido WA Triethylamine was added to a solution of TMSN3 (1.2 equiv.) in dry methanol (3.0 ml) at room temperature (RT) to maintain the pH of 8.5. Withaferin A (1.0 equiv., 470 mg, and 1.0 mmol) was separately dissolved in dry methanol (2.0 ml) and the solution was added to the methanolic solution of TMSN3 and kept for the 3.5 h. The progress of the reaction was monitored through TLC. After the completion, the reaction mixture was dried completely, dissolved in water (5.0 ml) and extracted with CHCl3 (10 ml) three times to obtain the product 3-azidoWA. For 3-azidoWA treatment, 3-azidoWA was dissolved in 100% DMSO and diluted with culture medium so that the working concentration of DMSO was less than 0.2%. Clonogenic Assay The assay was performed according to the previously described method [10]. Briefly, HeLa cells were plated at a seeding density of (16103 cells/well) in 6 well tissue culture grade plates. After 24 h the culture medium was changed and new medium was added and cells were exposed to various concentrations of 3-azidoWA/ vehicle DMSO for 5 days at 37uC incubator in 5% CO2. Later on, the obtained colonies were fixed with 4% paraformaldehyde and were stained with 0.5% crystal violet solution. The colonies from the plates were counted and averaged from the observed fields randomly (n = 3) and photographed with Olympus c-7070 wide 700 M inverted microscope camera. Cell Proliferation Assay The cell viability was determined by standard MTT dye uptake method [10]. Briefly, HeLa, PC-3, A549 and DU-145 cells (36103 cells/well) were plated into a 96 well tissue culture plate and treated with different concentration of 3-azidoWA in triplicate so that the final concentration of DMSO solvent was 0.2%. After 48 h incubation, MTT solution was added and cells were cultured for another 4 h at 37uC in 5.0% CO2 incubator. The amount of colored formazan derivative was determined by measuring optical density (OD) using TECAN microplate reader (Infinite M200 PRO) at 570 nm. The percentage viability was determined according to the protocol described [10]. Scratch Motility (Wound Healing) Assay The assay was performed as described previously [4]. Briefly, HeLa and PC-3 cells were plated in a 6 well plate at a concentration of (5.56105 cells/well) and allowed to form a confluent monolayer for 24 h, it was then serum starved for 24 h. After that the monolayer was scratched with a sterile pipette tip (200 mL), washed with serum free medium to remove floated and detached cells and photographed (time 0 h). Cells were succes- sively treated in medium containing low serum (1.0%) in presence of different concentrations of 3-azidoWA (0.25, 0.50, and 0.75 mM) along with vehicle DMSO for 24 h. Wounded areas were progressively photographed with Olympus c-7070 with 700M camera (100x magnification).The percentage of wound closure was estimated by the following equation: wound closure % = [1-(wound area at t1/wound area at t0) x 100% ], where t1 is the time after wounding and t0 is the time immediately after wounding. PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44039