Anti‐angiogenic Effects of Citrus Polymethoxyflavones and Their Major Metabolites

Abstract

Previous studies have demonstrated inhibitory effects of citrus polymethoxyflavones, nobiletin (NBT) and 5‐hydroxy nobiletin (5HN) on several types of cancer. We have identified major metabolites of NBT (i.e. M1: 3′‐hydroxy NBT; M2: 4‐hydroxy NBT; and M3: 3′,4′‐dihydroxy NBT) and 5HN (i.e. M4: 3′,5‐dihydroxy NBT; M5: 4′,5‐dihydroxy NBT; and M6: 3′,4′,5‐ trihydroxy NBT) in rodents fed with NBT and 5HN. In this study, we investigated the anti‐angiogenic activities of NBT, 5HN and their major metabolites using wound healing and tube formation assay with Human microvascular endothelial cells. Among NBT metabolites, M2 showed strongest inhibitory effects, while among 5HN metabolites, M4 had the most potent effects. In wound healing assay, M2, M1, NBT and M3 at 50 μM inhibits 50%, 40%, 30% and 10% of cell migration, respectively. M6, M5 and 5HN at 10 μM inhibited 50%, 40% and 30% of cell migration, respectively. Moreover, M4 at only 3μM inhibited more than 90% of cell migration. In tube formation assay, M3 and NBT at 50 μM inhibited 75% and 30% of capillary tubes formed, respectively, while M1 did not inhibit tube formation. M6, M5 and 5HN at 10 μM inhibited 90%, 77%, 54% of tube formation, respectively. Most interestingly, M2 at 50 μM and M4 at 3μM abolished all tube formation. Our results suggested that the number and position of hydroxyl groups in NBT, 5HN and their metabolites dictate their inhibitory effects on angiogenesis.Grant Funding Source : NIH: CA139174, AICR: 10A044