Angiopreventive Efficacy of Pure Flavonolignans from Milk Thistle Extract against Prostate Cancer: Targeting VEGF-VEGFR Signaling Gagan Deep1,2, Subhash Chander Gangar1, Subapriya Rajamanickam1, Komal Raina1, Mallikarjuna Gu 1 , Chapla Agarwal1,2, Nicholas H. Oberlies3, Rajesh Agarwal1,2* 1 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, Aurora, Colorado, United States of America, 2 University of Colorado Cancer Center, University of Colorado Denver, Aurora, Colorado, United States of America, 3 Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina, United States of America Abstract The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1a) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention. Citation: Deep G, Gangar SC, Rajamanickam S, Raina K, Gu M, et al. (2012) Angiopreventive Efficacy of Pure Flavonolignans from Milk Thistle Extract against Prostate Cancer: Targeting VEGF-VEGFR Signaling. PLoS ONE 7(4): e34630. doi:10.1371/journal.pone.0034630 Editor: Surinder K. Batra, University of Nebraska Medical Center, United States of America Received December 23, 2011; Accepted March 2, 2012; Published April 13, 2012 Copyright: 2012 Deep et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NCI R01 grants CA102514 and CA104286. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Rajesh.Agarwal@ucdenver.edu Introduction Prostate cancer (PCA) is the most frequently diagnosed non- cutaneous malignancy among men in the United States, and is the second leading cause of cancer-related deaths [1]. Clinical and experimental evidence have suggested that human tumors could persist for years as microscopic lesions in a state of dormancy and their further growth is critically dependent upon attaining an ‘angiogenic phenotype’ [2,3,4,5]. ‘Angiogenic switches’ involving the high VEGF and VEGF receptor (VEGFR) levels have been identified and considered responsible for PCA progression from low grade PIN (prostatic intraepithelial neoplasia) stage to high grade PIN and further to more aggressive, poorly differentiated, and androgen-independent malignant stages [6]. Furthermore, angiogenesis level in PCA has been correlated directly with Gleason score, tumor stage, progression, metastasis and survival [6,7,8]. Therefore, targeting angiogenesis has been the subject of several clinical investigations to improve the quality of life of cancer patients [9,10,11]. Furthermore, preventing the onset of angiogenesis in indolent tumors (referred as ‘angioprevention’) has been suggested as a novel and rationale approach to control PCA growth, malignant progression and metastasis to secondary sites. About four decades ago, Judah Folkman first predicted the potential role for anti-angiogenic inhibitors against solid cancers, and to date, several angiogenesis inhibitors have been tested against many malignancies [12,13,14,15]. Many of these inhibitors have already been FDA approved for their use either alone or in combination with cancer chemotherapeutic drugs [13,15,16]. For example, humanized VEGF antibody was approved against colorectal, brain, lung, and renal cancers [16]. Similarly, the tyrosine kinase inhibitors sorafenib and sunitinib, which target VEGFR activity, have been approved for use against advanced renal cell carcinoma [16]. Although inhibiting angiogenesis in cancer, in principle, is a sound preventive/therapeutic strategy, the current approach of targeting a single molecule such as VEGF or VEGFR is flawed, as cancer cells develop resistance through circumventing these molecules and continue to spread vascular networks [17]. Beside their limited efficacy in terms of improve- PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34630