Prostate Cancer Angioprevention by Flavonolignans Figure 7. Effect of flavonolignans on viability, cell cycle distribution and apoptosis in HUVEC. (A–B) HUVEC were treated with DMSO or individual flavonolignan and analyzed for total cell number and cell cycle distribution. (C) HUVEC were treated with flavonolignans, and 24 h later, total cell lysates were prepared and analyzed for cell cycle regulators. The densitometry values presented below the bands are ‘fold change’ compared to control after loading control (a-tubulin) normalization. (D) HUVEC were treated with flavonolignans (at 30 mM dose) for 36 h and analyzed for morphology (representative photomicrographs are shown at 100x), levels of cPARP, cleaved caspase 3 and 9, and percentage apoptotic cells. 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.g007 microvessels (Fig. 2). VEGF is a potent pro-angiogenic factor [17,35], and IHC analyses of tumor sections clearly showed that treatment with these diastereoisomers decreases both VEGF and VEGFR2 expression in tumor tissues (Fig. 2). Notably, except silybin A, VEGFR1 expression was also significantly decreased by these agents (Fig. 3). Overall, isosilybin B was relatively more potent in its efficacy on VEGF, VEGFR2 and VEGFR1 expression. HIF-1a is the master transcriptional factor that is stabilized under hypoxic conditions in growing tumors and controls tumor metabolism as well as expression of pro-angiogenic factors such as VEGF [36,37,38]. Although HIF-1a stabilization occurs under low oxygen conditions, its expression is also controlled by the serine/threonine kinase Akt [39]. Akt also plays an important role in multiple cellular processes, such as cell survival, apoptosis, migration and metabolism [40,41]. IHC analyses of tumors showed that all four of the flavonolignans strongly decrease HIF-1a and pAktser473 levels, with marginal effect on total Akt expression only by isosilybin A and isosilybin B (Fig. 3). Effect of Flavonolignans on Angiogenesis in Non-target Organs To confirm that the anti-angiogenic effects of these flavono- lignans are specific to tumor tissues, we analyzed the non-target organs (liver, lung, and kidney) for CD31 expression to determine the microvessels density. There was no difference in the microvessel density in liver, lung and kidney between control and mice fed with pure flavonolignans (Fig. 4A, CD31 quantification data not shown). H & E analyses also showed that these diastereoisomers have no adverse effect on the histology of normal organs (Fig. 4B). Effect of Flavonolignans on Angiogenesis in Ex Vivo and In Vitro Assays We further examined the anti-angiogenic activity of the flavonolignans in ex vivo capillary formation assay using mouse dorsal aortas. After six days of culture on matrigel under angiogenic conditions, we observed a significant number of vessels sprouting from the mouse aortas (marked by arrows), which was inhibited in a dose-dependent manner by flavonolignans treatment (Fig. 5A). One of the important steps during neo-angiogenesis is the formation and merging of tubes produced by endothelial cells forming a complex network of vessels and capillaries [42,43]. To understand the effect of flavonolignans on this biological event, we performed tube formation assay. As shown in Fig. 5B, bottom panel, all four compounds significantly inhibited the VEGF- induced tube length in HUVEC. As shown in the pictures (Fig. 5B, top panel), VEGF treatment induced the formation of tubular networks by HUVEC, which was disrupted by flavonolignan treatments. Effect of Flavonolignans on VEGF-induced Proliferation and Chemotactic Motility VEGF plays an important role during neo-angiogenesis through its mitogenic and motogenic effect on endothelial cells [17,35]. In our studies, VEGF treatment induced the HUVEC growth that was strongly inhibited by flavonolignans treatment (Fig. 6A). Such treatments also inhibited the chemotactic motility of HUVEC towards VEGF in the Transwell invasion assay (Fig. 6B). Importantly, isosilybin B was the least effective compared to the other diastereoisomers in terms of inhibitory effect on the chemotactic motility of HUVEC. Effect of Flavonolignans on Viability, Cell Cycle Progression and Apoptosis in HUVEC To further elucidate the biological effect of these flavonolignans on endothelial cells, we analyzed viability, cell cycle and apoptosis in HUVEC. As shown in Fig. 7A, the four flavonolignans (5– 30 mM) inhibited HUVEC viability in a dose- and time-dependent manner. Cell cycle analyses revealed that all the diastereoisomers induced G1 arrest after 24 h of treatment, but only silybin A, silybin B and isosilybin A also caused G2/M arrest (Fig. 7B). After 48 h of treatment, the noticeable effect of silybin A, silybin B and isosilybin A in HUVEC was the induction of G2/M arrest, which was missing with isosilybin B treatment (Fig. 7B). Isosilybin A treatment (at 30 mM) significantly induced S-phase arrest after 48 h of treatment, which could be linked to strong apoptotic death induced by isosilybin A (Fig. 7B). We next examined the effect of these flavonolignans on various cell cycle regulatory molecules, namely cyclins, Cdks and Cdk inhibitors as well as their regulators Skp2 and phosphatases Cdc25. As shown in Fig. 7C, the four flavonolignans decreased the levels of Cdk2 and Cdk4 with marginal effect on Cdc2. These compounds also decreased the levels of cyclin D1, cyclin D3, cyclin A, and cyclin B1. Silybin A and isosilybin A moderately increased the p27 expression, but it was decreased by isosilybin B (Fig. 7C). On the contrary, p21 expression was decreased by silybin A, silybin B and isosilybin A but not by isosilybin B (Fig. 7C). However, all four diastereoisomers strongly decreased the expression of Skp2 and Cdc25C with no or moderate inhibitory effect on Cdc25A level (Fig. 7C). These results suggested that these four diastereoisomers have few similar (but differing in the extent) and few contrasting effects on the expression of cell cycle regulatory molecules. We then examined the effect of the pure flavonolignans (at 30 mM) on apoptosis after 36 h of treatment in HUVEC. As shown in Fig. 7D, isosilybin B was the least efficacious in terms of inducing apoptosis related morphological features (detachment and rounding), signaling molecules involved in apoptosis (cPARP, cleaved caspase 3 and 9) and percentage of apoptotic cell population in HUVEC. Conversely, silybin A, silybin B and isosilybin A strongly induced apoptotic death in HUVEC (Fig. 7D). Effect of Flavonolignans on VEGF-induced Signaling in HUVEC Next, we examined flavonolignans effect on the VEGF-induced signaling cascade that controls proliferation, motility and tube formation in endothelial cells [35]. VEGF treatment strongly increased the VEGFR2 phosphorylation at Ser1175 site, a reliable marker for its activity, which was inhibited by pre-treatment with flavonolignans (Fig. 8). Similarly, VEGF increased the phosphor- PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e34630