MMP-2 level was restored. Additionally, for further verification we diminished extracellular Par-4 secretion with BFA and found that depletion of extracellular Par-4 due to Brefeldin A treatment restored MMP-2 level compared to untreated vehicle. The inhibition of MMP-2 secretion by Brefeldin A might overcome by some mechanism need to be elucidated in future. Angiogenesis is a crucial step in tumor progression and invasion regulated by VEGF [35]. MMP-2 plays a pivotal role in this process. Interestingly, our in vivo results reveal 3-azidoWA is capable of suppressing VEGF-bFGF induced neovessel formation in vivo. Note, the treatment with increasing concentration of 3- azidoWA almost abolished the new blood vessel formation in the plugs containing VEGF and bFGF (Fig 7, A). Due to the importance of TIMP-1 in cancer cells invasion [28], and angiogenesis, we have demonstrated that 3-azidoWA treatment augmented the expression of TIMP-1 which is a modulator of MMP-2 expression in both cell lines, in dose dependent manner. Moreover, MAPK and PI3K/Akt pathways also contribute pivotal role in tumor development, maintenance and angiogenesis [50,51]. Therefore, we investigated the effect of 3-azidoWA on the activities of MAPK and PI3K/Akt signaling pathways. The results of our studies suggest that treatment with 3-azidoWA inhibited internal ERK and Akt phosphorylation in both HeLa and PC-3 (Supplementary Fig. 2, D) cells. In conclusion, we exhibit a novel pathway of 3-azidoWA induced MMP-2 inhibition, mediated through pro-apoptotic secretory protein Par-4, secreted by a classical BFA sensitive pathway. We have verified the possibility of apoptotic induction and abrogation of invasion could be controlled independently/ collaboratively by 3-azidoWA. Interestingly we disclose that 3- azidoWA mediated MMP-2 inhibition is apoptosis sovereign. How extracellular Par-4 modulates MMP-2 activity will be interesting topic to be explored in future. Taken together this report unveils a therapeutic potential of 3-azidoWA, derivative of dietary com- pound withaferin A to inhibit motility ability of cervical and prostate cancer cells through matrix metalloproteinase-2 by induction of extracellular Par-4, which has broader aspects of controlling tumorigenesis. Supporting Information Figure S1 (A) PC-3 cells (0.56105 cells/well) were grown to confluence in 6 well plate were scratched with sterile tip (200 mL) to create a wound, 3-azidoWA was added to cultures as indicated. Scratched areas were photographed (magnification 100x) at zero hour and then subsequently again at 24 h to assess the degree of wound healing. (B) The scratched areas were quantified in three random fields in each treatment, and the data were calculated from three independent experiments. (C) PC-3 (16103) cells/well were cultured and treated with various concentration of compound 3-azidoWA for 5 days at 37uC and then stained with crystal violet (for details see materials and methods), numbers of stained colonies were counted, photo- graphed (100x) and (D) data were calculated from three independent experiments. (E) Cell migration was determined via the modified Boyden chamber assay as described in Materials and Methods. PC-3 cells (26105) were seeded in top chamber in the presence or absence of absence of sub-toxic doses of 3-azidoWA. Cells were allowed to migrate for 24 h, at which point migratory cells on the bottom half of the insert membrane were stained with PLOS ONE | www.plosone.org 13 September 2012 | Volume 7 | Issue 9 | e44039 0.1% crystal violet and counted under 100x magnification. (F) Invasive cells were counted using image software as the number of invasive cells per high-power field (HPF). Five fields were counted in triplicate from each insert. Cell images were obtained using microscope Nikon Eclipse E200 inbuilt with camera. Columns, means; bars SD of three independent experiments. *P,0.05, **P,0.01 compared with untreated control. (TIF) Figure S2 (A) PC-3 cells were left untreated or treated with 0.50 mM and 0.75 mM of 3-azidoWA for 48 h, conditional media was analyzed for MMP-2 and -9 gelatinase activity. (B) PC-3 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 analyses followed by coomassie blue staining to reveal the 68 KDa BSA band for loading control. (C) PC-3 cells were treated with various concentration of parent molecule Withaferin A for 48 h and the activity of MMP-2 was determined by gelatin zymography. (TIF) Figure S3 (A). Time course for neovascularisation in Matrigel plugs. C57BL/J6 mice were injected subcutaneously with 0.5 ml Matrigel with or without VEGF+ bFGF. At the end of study plug were removed on days 2–11 from mice for visualisation and quantification of angiogenesis. (B). Effect of 3-azido WA on Matrigel plug neovascularisation. 3-azido WA was administered intraperitonially at the doses indicated for seven days starting 24 h after Matrigel injection. On day 8 animals were sacrificed and retrieve plugs for visualisation and quantification of angiogenesis within the Matrigel plugs achieved by haemoglobin estimation shows (n = 5,P,0.05) compared with the level of vascularisation in Matrigel plugs supplemented with VEGF +bFGF in animals. Representive of photographs of plugs from groups of five animals are shown. (TIF) Figure S4 1H NMR and 13C of 3-azido,2,3-dihydrowithaferin A. (TIF) Figure S5 1H-1H COSY of 3-azido,2,3-dihydrowithaferin A. (TIF) HSQC of 3-azido,2,3-dihydrowithaferin A. HMBC of 3-azido,2,3-dihydrowithaferin A. 3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 Figure S6 (TIF) Figure S7 (TIF) Acknowledgments aWe thank our Director Dr. R.A. Vishwakarma for encouraging us to complete this work. We would also like to thank P.R. Sharma, Zabeer Ahmed and Surjeet Singh for technical assistance. We are grateful to Dr. V.M. Rangnekar (University of Kentucky) for critically reading of the manuscript. Author Contributions Conceived and designed the experiments: AG DM. Performed the experiments: BR HA KY SK. Analyzed the data: GJ. Contributed reagents/materials/analysis tools: AG DM. Wrote the paper: AG.