Prostate Cancer Angioprevention by Flavonolignans Figure 1. Flavonolignans inhibit PCA DU145 xenograft growth through targeting proliferation and apoptosis. DU145 xenografts were initiated and mice were administered either vehicle (CMC) or 50 and 100 mg/kg body weight doses of each diastereoisomer. (A) Tumor volume was measured and plotted as a function of time (days). Each value in the curves is mean 6 SEM of 10–12 mice. (B–D) Xenograft tissues were analyzed for PCNA, TUNEL and cleaved caspase-3 (CC3) by IHC. The data shown in the bar diagrams is the mean6 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; #, p # 0.01; $, p # 0.05. doi:10.1371/journal.pone.0034630.g001 ment in patient’s survival, these treatment options are extremely expensive and have shown unacceptable levels of toxicity [18,19]. Therefore, we rationalized the need to identify non-toxic natural agents with broad spectrum anti-angiogenic efficacy; and in this regard, in the present study, we focused on the anti-angiogenic efficacy of four pure diastereoisomers from Milk Thistle (Silybum marianum) extract, namely silybin A, silybin B, isosilybin A and isosilybin B. These diastereoisomers are flavonolignans with an identical flavonoid moiety and differ only in their configurations about the lignan moiety, and their chemical and biological properties have been detailed earlier [20,21,22,23,24]. Here, for the first time we analyzed the angiopreventive efficacy of these flavonolignans in PCA xenograft model and various ex vivo, in vivo and in vitro angiogenesis assays. Methods Cell Line and Reagents Human PCA DU145 cells were from ATCC (Manassas, VA) and cultured as described earlier [25]. HUVEC were from Lonza (Walkersville, MD) and were grown in EBM2 media with EGM-2 SingleQots supplements. Matrigel and invasion chamber were from BD Biosciences (New Bedford, MA). TUNEL assay kit was from Promega (Madison, WI). Carboxymethylcellulose (CMC), Harris hematoxylin and b-actin antibody were from Sigma (St. Louis, MO). DAB kit was from Vector Laboratories (Burlingame, CA). Streptavidin and PCNA antibody were from Dako (Carpinteria, CA). Antibodies for CD31, VEGF and nestin were from Abcam (Cambridge, MA). Antibodies for VEGFR1, VEGFR2, and HIF-1a [used for immunohistochemistry (IHC) analysis] as well as antibodies for Cdk2, Cdk4, Cdc2, cyclin D1, cyclin D3, cyclin B1, p21, p27, Skp 2, Cdc25A and Cdc25C and normal goat serum were from Santa Cruz Biotechnology (Santa Cruz, CA). p27 antibody was from Neomarkers (Fremont, CA) and p21 antibody was from Upstate (Charlottesville, VA). Antibodies recognizing the phosphorylated and/or total protein levels of VEGFR1, VEGFR2, Src, ERK1/2, Akt, Bad, mTOR, p70S6K, cleaved caspase 3, cleaved caspase 9, and goat anti- rabbit HRP-conjugated secondary antibodies were from Cell Signaling (Beverly, MA). ECL detection system and anti-mouse PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34630