Prostate Cancer Angioprevention by Flavonolignans Figure 2. Flavonolignans inhibit angiogenesis in vivo. DU145 xenograft tissues were analyzed for CD31, nestin, VEGF and VEGFR2 by IHC. Quantitative analyses were performed using Zeiss Axioscope 2 microscope (Carl Zeiss, Germany) and photographs were originally captured (at 400x) with a Carl Zeiss AxioCam MrC5 camera with Axiovision Rel 4.5 software. The data shown in the bar diagrams is the mean 6 SEM of 4–5 samples. 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.g002 HRP-conjugated secondary antibody were from GE Healthcare (Buckinghamshire, UK). VEGF was from R & D (Minneapolis, MN). Silybin A, silybin B, isosilybin A and isosilybin B were isolated (purity .97%) from powdered extract of the fruits of Silybum marianum (L.) Gaertn. [obtained from Euromed, S.A. (Barcelona, Spain)]. The hybrid chromatographic/precipitative techniques and procedures for the gram scale purification of these flavonolignans are already reported previously [26]. In Vivo Tumor Xenograft Study Athymic (nu/nu) male nude mice were from the NCI (Frederick, MD). The treatment protocol was approved by the Institutional Animal Care and Use Committee of the University of Colorado Denver. About 3 6 106 DU145 cells were suspended in 50 mL of serum free medium, mixed with 50 mL of matrigel, and injected s.c. in the right flank of each mouse. The day following xenograft implantation, mice were randomly divided into nine groups, and all treatments were done by oral gavage as: Group I mice (vehicle control group) with 200 mL of 0.5% CMC (w/v) in sterile water; Groups II and III mice with 50 and 100 mg/kg body weight dose of silybin A; Groups IV and V mice with 50 and 100 mg/kg body weight dose of silybin B; Groups VI and VII mice with 50 and 100 mg/kg body weight dose of isosilybin A; and Groups VIII and IX mice with 50 and 100 mg/kg body weight dose of isosilybin B, respectively. All these treatments (5 days/week) were given in 200 mL of 0.5% CMC. As all four compounds have same molecular weight (482.1), each dose-level was equimolar across these agents. Once tumor xenograft growth commenced, tumor sizes were measured twice weekly using digital caliper and tumor volume was calculated by the formula: 0.5236 L1(L2)2, where L1 is long diameter, and L2 is short diameter. IHC Analyses Tumor samples were processed and immuno-stained following published methods [27,28,29]. Percentage of PCNA, TUNEL, cleaved caspase 3 and HIF-1a positive cells was calculated by counting the number of positive stained cells (brown stained) and the total number of cells at five arbitrarily selected fields from each tumor at 400x magnification. Microvessels stained with CD31 and nestin were quantified in 5 random microscopic (400x magnifica- tion) fields per tumor. VEGF, VEGFR1, VEGFR2, pAktser473, and Akt immunoreactivity was analyzed in 5 random areas for each tumor tissue and was scored as 0+ (no staining), 1+ (weak staining), 2+ (moderate staining), 3+ (strong staining), 4+ (very strong staining). PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34630