Abstract A45: Hydroxylated polymethoxyflavones: a novel class of agents for colon cancer prevention

A45 Orange peels have been used as flavoring and traditional Chinese medicine for many centuries. Polymethoxyflavones (PMFs) are a unique class of flavonoids, and almost exclusively exist in the citrus genus, particularly in the peels of sweet oranges (Citrus senensis) and mandarin oranges (Citrus reticulata). Major PMFs are permethoxylated PMFs (Me-PMFs) such as nobiletin, 3,5,6,7,8,3’,4’-heptamethoxyflavone (HMF), and tangeretin, which have been shown to inhibit cancer cell growth in vitro and in vivo. Recently, we have isolated a class of novel PMFs, namely hydroxylated PMFs (OH-PMFs), from sweet orange peel. These OH-PMFs can be formed from their corresponding Me-PMFs counterparts by hydrolysis naturally. Herein, we studied the effects of three major OH-PMFs, namely 5-hydroxy-6,7,8,3’,4’-pentamethoxyflavone (5HPMF), 5-hydroxy-3,6,7,8,3’,4’-hexamethoxyflavone (5HHMF), and 5-hydroxy-6,7,8,4’-tetramethoxyflavone (5HTMF), on colon cancer cells, and compared their effects with their corresponding Me-PMFs counterparts, namely nobiletin, HMF, and tangeretin, respectively. Our results showed that OH-PMFs significantly inhibited colon cancer cell (HCT116 and HT29) growth in a dose dependent fashion, and these effects were much stronger than those produced by their corresponding Me-PMFs counterparts. Cell cycle analysis by flow cytometry demonstrated that at 24 h, 5HPMF caused cell cycle arrest at G2/M phase, while 5HHMF resulted in arrest at G0/G1 phase. In contrast, at much lower concentration, 5HTMF significantly increased the sub-G0/G1 cell population, indicating possible DNA degradation due to cell death. Annexin V/PI co-staining assay was used to detect possible apoptosis after treatments with OH-PMFs for 48 h. It was found that all three OH-PMFs increased the apoptotic cell population, but 5HTMF showed a much stronger pro-apoptotic effect at much lower concentration in comparison to 5HPMF and 5HHMF. The distinct effects of these three OH-PMFs on cell cycle and apoptosis of colon cancer cells suggest that each OH-PMFs may have different molecular targets in the cancer cells. Moreover, three OH-PMFs profoundly modulated proteins related to cell proliferation and apoptosis. OH-PMFs, namely 5HPMF and 5HHMF, produced profound tumor-suppressive changes on these proteins, while their corresponding Me-PMFs, namely nobiletin or HMF did not cause any noticeable change on the same proteins tested. On the other hand, tangeretin caused various tumor-suppressive changes on the proteins tested, however, its effects were either similar or to less extent than those produced by 5HTMF at much lower concentrations. Overall, at 48 h, three OH-PMFs decreased the level of K-RAS and phosphorylation of AKT, increased the level of p16, and activated caspase cascade, while 5HTMP also increased the level of p16 and decreased the level of Mcl-1. Our results demonstrated that OH-PMFs are promising novel agents for colon cancer prevention. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A45.