Prostate Cancer Angioprevention by Flavonolignans Figure 5. Flavonolignans inhibit angiogenesis in ex vivo and in vitro models. (A) Flavonolignans inhibit angiogenesis ex vivo. Mouse aortas were plated on matrigel and treated with flavonolignans. The arrows in the picture mark the emerging vessels from the aortas. (B) Flavonolignans inhibit VEGF-induced tube formation in HUVEC. HUVEC were plated on matrigel and effect of diastereoisomers treatment on VEGF-induced tube formation was analyzed. Representative tubular network photomicrographs are shown at 100x (top panel). Tube length was quantified as detailed in ‘Methods’ (bottom panel). Abbreviations: Sil A: Silybin A; Sil B: Silybin B; Iso A: Isosilybin A, Iso B: Isosilybin B; *, p # 0.001. doi:10.1371/journal.pone.0034630.g005 Effect of Flavonolignans on Proliferation and Apoptosis in DU145 Xenografts The IHC analysis of DU145 tumor samples showed that flavonolignans treatment significantly inhibits the immunostaining for PCNA (a biomarker for cell proliferation) (Fig. 1B), but increases the TUNEL and cleaved-caspase 3 positive cells (biomarkers for apoptosis) (Fig. 1C and 1D). Though statistically significant, the effect of flavonolignans on proliferation and apoptosis related biomarker was modest, and could not completely explain the observed slower xenograft growth and close to 50% growth inhibition with the flavonolignan treatment (Fig. 1A). Therefore, next we analyzed the tumor tissues for flavonolignans effect on angiogenesis, which is absolutely necessary for the tumors to grow beyond 1–2 mm size [3]. Effect of Flavonolignans on Angiogenesis in DU145 Xenografts To investigate whether these individual flavonolignans inhibited the xenograft growth by suppressing tumor angiogenesis, we stained the tumor section with CD31 (a biomarker for matured microvessels) and nestin (a biomarker for immature and newly formed microvessels). All four compounds inhibited the microves- sel density, both mature and newly forming, but isosilybin B was relatively more effective in its inhibitory effect on nestin-positive Figure 6. Flavonolignans inhibit VEGF-induced proliferation and invasion in HUVEC. (A) HUVEC were induced with VEGF and treated with each flavonolignan in 0.5% serum media, and total cell number was analyzed after 24 h. (B) HUVEC were plated in the upper chamber with DMSO or individual diastereoisomer, while VEGF was added in the lower chamber and HUVEC invasion was studied. Abbreviations: Sil A: Silybin A; Sil B: Silybin B; Iso A: Isosilybin A, Iso B: Isosilybin B; *, p # 0.001; #, p # 0.01; $, p # 0.05. doi:10.1371/journal.pone.0034630.g006 PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e34630