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-1α) 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.
Highlights
Prostate cancer (PCA) is the most frequently diagnosed noncutaneous malignancy among men in the United States, and is the second leading cause of cancer-related deaths [1]
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)
In terms of anti-tumor efficacy, the oral administration of flavonolignans effectively inhibited the growth of DU145 xenografts in nude mice, and this was discernible from the third week onwards (Fig. 1A)
Summary
Prostate cancer (PCA) is the most frequently diagnosed noncutaneous 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]. Angiogenesis level in PCA has been correlated directly with Gleason score, tumor stage, progression, metastasis and survival [6,7,8]. 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
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