Prostate Cancer Angioprevention by Flavonolignans Figure 9. Differential effect of flavonolignans on human PCA DU145 cells and endothelial HUVEC in cell culture assays. (A) Effect of flavonolignans (at 90 mM dose) on the three dimensional growth of DU145 cells was studied as detailed in ‘Materials and Methods’. Representative spheroids photomicrographs are shown at 100x and 400x. (B) In Transwell invasion assay, either DU145 cells (plated in the lower chamber) or HUVEC (plated in the upper chamber) were treated with individual diastereoisomers (at 30 mM dose), and HUVEC invasiveness was measured. (C) DU145 cells were treated with individual diastereoisomers (at 30 mM dose) and conditioned media was collected. HUVEC were plated on matrigel along with 0.5% FBS or conditioned media from different treatment groups; and tube formation was analyzed. Representative photomicrographs of tubular network are shown at 100x. Abbreviations: Sil A: Silybin A; Sil B: Silybin B; Iso A: Isosilybin A, Iso B: Isosilybin B; CM: Conditioned media; *, p # 0.001; #, p # 0.01. doi:10.1371/journal.pone.0034630.g009 diagnosed pre-malignant tumors or for the therapeutic regression of the already advanced disease [16,45]. Milk thistle has been used for centuries to treat chronic liver disease, and to protect the liver against toxins [46]. In recent years, Milk thistle research use has grown significantly with close to two dozen clinical trials underway or already completed evaluating its efficacy against variety of diseases including chronic hepatitis, diabetes, asthma, mushroom poisoning and various cancers (these studies are listed at ClinicalTrials.gov). In the past, there have been numerous efforts in isolating and purifying the individual constituents in Milk thistle to better exploit its clinical usefulness. Members of our team were the first to purify and elucidate seven distinct flavonolignans from Milk Thistle extract, namely: silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, and isosilychristin, and one flavonoid, taxifolin [47]. Their biological effects as pure compounds were assessed on several anti- proliferative end points in human PCA cell lines, where isosilybin B ranked as the most potent flavonolignan for nearly all the end points, including the inhibition of cell growth, clonogenic potential, PSA and androgen receptor levels, and topo IIa promoter activity [20,21,22,24]. Despite these advances in the Milk Thistle research, the in vivo biological efficacy and related toxicity of these pure flavonolignans remained unknown because of insufficient compound quantities. In particular, isosilybin B was the most challenging of the major diastereoisomer to purify, due to its limited abundance in the natural extract [21] and its relatively PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e34630