Figure 8. Effect of flavonolignans on VEGF-induced signaling cascade in HUVEC. HUVEC were serum starved for 22 h, treated with diastereoisomers for 2 h and stimulated with VEGF (10 ng/ml) for 10 minutes. Total cell lysates were prepared and analyzed for mentioned signaling molecules. The densitometry values presented below the bands are ‘fold change’ compared to control after loading control (b-actin) normalization. doi:10.1371/journal.pone.0034630.g008 Prostate Cancer Angioprevention by Flavonolignans ylation of VEGFR1, Src, ERK1/2, Akt, BAD, mTOR, and p70S6K. As shown in Fig. 8, except ERK1/2 and mTOR, phosphorylation at other sites was inhibited by these compounds albeit to different extent. Compared to its other diastereoisomers, isosilybin B was the least effective in terms of effect on the VEGF- induced signaling molecules in HUVEC. Differential Sensitivity of Flavonolignans Towards PCA and Endothelial Cells Earlier findings [20,21,22,24] and the present study suggested that among the four pure flavonolignans, isosilybin B is the most effective in terms of efficacy against PCA cells but, surprisingly, it has the least efficacy in terms of its effect on HUVEC (inhibition of chemotactic motility or apoptosis induction) (Figs. 6 and 7). Therefore, we performed a series of experiments to elucidate the relative efficacy of these four diastereoisomers against PCA cells vis-a`-vis endothelial cells. First, we analyzed their effect on the three dimensional growth of DU145 cells in matrigel. Treatment with these compounds strongly inhibited the number and size of spheroid formed by DU145 cells with isosilybin B being most effective (Fig. 9A). Next, we performed co-culture studies using Transwell chambers. We plated HUVEC in the upper chamber, while DU145 cells were cultured in the lower chamber. In two separate experiments, HUVEC or DU145 cells were treated with pure flavonolignans, and thereafter, HUVEC invasion through matrigel was studied in each case. As shown in Fig. 9B, isosilybin B was the most effective in decreasing the HUVEC migration when DU145 cells were treated but was least effective when HUVEC were treated. To follow this, we treated DU145 cells with these diastereoiso- mers (30 mM) and collected the conditioned media. The pro- angiogenic potential of the conditioned media was analyzed in a tube formation assay using HUVEC. As shown in Fig. 9C, conditioned media from DU145 cells increased the tube length as well as tubular network formed by HUVEC. Conditioned media from the flavonolignan treated DU145 cells significantly decreased the tube length as well as tubular network (Fig. 9C). In this assay too, isosilybin B was the most effective diastereoisomer (Fig. 9C). On the contrary, silybin A was more effective when HUVEC were treated directly with these diastereoisomers (Fig. 5B). Together these studies further confirmed the differential sensitivity of these diastereoisomers towards PCA and endothelial cells. Discussion Neo-angiogenesis is the critical step in the development and progression of most of the human cancers. Beyond the critical size of 1–2 mm, oxygen and nutrients have difficulty in diffusing to the core cells of the tumor, causing a state of cellular hypoxia. Under hypoxic conditions, cancer cells secrete several pro-angiogenic factors such as VEGF and bFGF, which cause recruitment of endothelial cells from the neighboring blood vessels [6,44]. But the continuous and excessive presence of pro-angiogenic stimuli in the tumor microenvironment interferes with the normal maturation of vessel network, and as a result, vessels in the tumor area show abnormal morphology and physiology. Hence, it is possible to specifically target abnormal tumor angiogenesis by inhibiting endothelial cell recruitment as well as their proliferation in the tumor microenvironment. Notably, the results from the present study clearly suggest the strong efficacy of four pure flavonolignans from Milk Thistle extract, on these aspects, which signifies their angiopreventive efficacy against PCA. These findings are aslo transnationally noteworthy, as currently, anti-angiogenic strategies are extensively pursued towards preventing the progression of PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34630