3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 Figure 5. 3-azidowithaferin induce MMP-2 inhibition is apoptosis independent. (A) HeLa cells (0.56106 cells/well) were plated in cover slip in 6 well tissue culture plates and were treated with 1.0 mM 3-azidoWA along with vehicle and staurosporine (25 nM) in presence (right panel) and absence (left panel) of Pan Caspase inhibitor. After 24 h treatment cells were washed, fixed and immunostained with caspase-3 antibody (Texas red), counterstained with DAPI (blue) to score apoptotic nuclei and analyzed random fields with Zeiss LSM-510 metaconfocal microscope and images were captured at 63x magnification. (B) Conditional media collected from above experimental group were employed for MMP-2 activity by zymography. doi:10.1371/journal.pone.0044039.g005 was no increase in hemoglobin level of Matrigel plugs when retrieved from animals over a 7-11-days period. On the other hand plugs containing VEGF and bFGF revealed a marked degree of neovascularisation (n = 5, P,0.05) with maximum increase being achieved by 7-day period. To determine the effects of 3-azidoWA on VEGF/bFGF- induced angiogenesis in vivo, mice were dosed intraperitonally with 3-azido WA for 7 days, starting 24 h after Matrigel injection. Visual inspection of the plugs after removal revealed the marked increase in vascularization, seen as a deep red appearance that occurred in plugs of untreated animals (Fig. S3, B). Animals dosed with 3-azido WA 1,10, 20, 30 mg/kg/d, i,p dose but not with 50 mg/kg/d i,p (due to toxic effects and animals died) there was a marked reduction (n = 5, P,0.05) in the level of neovascularisation at 30 mg/kg/d as compared to untreated plugs (VEGF +bFGF). Having established that 30 mg/kg/d, i,p dose effectively abolished Matrigel plug vascularisation, this dose was used to investigate the number of doses required to prevent neovascularization (preventive study) or to disruptive established blood vessels (treatment study), as would be encountered in a therapeutic setting. For preventive study administration of 30 mg/kg/d of 3-azido WA daily for 1, 2, 3, or 4 days, 24 h after Matrigel injection, revealed that dosing for 4 days was sufficient to completely blocked plug neovascularisation as shown in Fig.7, A (n = 5, P,0.05). After letting neovascularisation become established over a 7-days period for treatment study, dosing for 3 days (i,e days 8–10) resulted in a marked significant reduction (n = 5, P,0.05) in plug vascularisation compared to control and one day (i,e day 8) dosing (Fig 7, B).Thus, these experiments clearly PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e44039