Figure 4. Feeding of pure flavonolignans did not affect angiogenesis and normal histology in non-target organs. (A– B) Lungs, liver and kidneys from each mouse were collected and analyzed for CD31 immunoreactivity as well as for histopathological analyses. Quantitative analyses were performed using Zeiss Axioscope 2 microscope (Carl Zeiss, Germany) and photographs were originally Prostate Cancer Angioprevention by Flavonolignans captured (at 400x) with a Carl Zeiss AxioCam MrC5 camera with Axiovision Rel 4.5 software. Abbreviations: Sil A: Silybin A; Sil B: Silybin B; Iso A: Isosilybin A, Iso B: Isosilybin B. doi:10.1371/journal.pone.0034630.g004 Immunoblotting HUVEC were treated with 30 mM dose of each flavonolignan with or without VEGF stimulation, lysates were prepared and analyzed by standard immunoblotting method as described earlier [33,34]. a-tubulin and b-actin were used to confirm equal protein loading. Three Dimensional Spheroid Formation Assay In 24 well plates, 100 mL of matrigel was added. Thereafter, 100 ml of RPMI-1640 and matrigel mixture (50:50) containing ,16103 DU145 cells was added. After 15 min, 1 mL RPMI-1640 medium with 10% FBS containing DMSO vehicle or 90 mM concentration of each flavonolignan was added in the well; treatments were replaced every 48 h for 2 weeks. At the end of the experiment, spheroid formation was counted under Zeiss inverted microscope at 100x. Statistical Analyses Statistical analyses were performed using Sigma Stat software version 2.03 (Jandel Scientific, San Rafael, CA). The statistical significance of differences between control and treated-groups was determined by Student’s t test and p , 0.05 value was considered significant. One way ANOVA followed by Tukey’s test was used for multiple comparisons. The autoradiograms/bands were scanned, and mean density of bands (where mentioned) was determined using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA). Results Effect of Flavonolignans on PCA DU145 Xenograft Growth In terms of anti-tumor efficacy, the oral administration of flavonolignans effectively inhibited the growth of DU145 xeno- grafts in nude mice, and this was discernible from the third week onwards (Fig. 1A). At the end of the experiment (10 wks), silybin A treatment inhibited the tumor volume by 44 and 50% with 50 and 100 mg/kg body weight doses, respectively (p, 0.05) (Fig. 1A); while silybin B inhibited the tumor volume by 38 and 47% at same doses, respectively (p, 0.05) (Fig. 1A). In isosilybin A treated mice, tumor volume was inhibited by 44 and 50% (p, 0.05) (Fig. 1A), while in mice treated with isosilybin B the tumor volume was inhibited by 46 and 67% with 50 and 100 mg/kg body weight doses, respectively (p, 0.05) (Fig. 1A). Overall, isosilybin B was most effective in inhibiting the DU145 xenograft growth, followed by silybin A or isosilybin A and silybin B. Although, these differences in the biological effect of each diastereoisomer did not achieve statistical significance; these results were consistent with previously reported in vitro studies [21,22]. At the time of necropsy, all animals were examined for gross pathology, and we did not observe any signs of abnormality in all the vital organs examined. Furthermore, the administration of these compounds through oral gavage did not cause any significant change in the diet consumption pattern or body weight gain of mice (data not shown). Also, we did not observe any adverse effect in terms of general behavior of animals, suggesting an overall safe nature of these compounds. PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34630