5-demethylnobiletin and 5-hydroxy-3-6-7-8-3--4--hexamethoxyflavone

Previously, we reported that 5-hydroxy polymethoxyflavones (5OH-PMFs) isolated from orange, namely 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone, 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5HHMF) and 5-hydroxy-6,7,8,4'-tetramethoxyflavone (5HTMF), potently induced apoptosis and cell-cycle arrest in multiple human colon cancer cells. Herein, using isogenic variants of HCT116 human colon cancer cells, we investigated the effects of p53, Bax and p21 on the apoptosis and cell-cycle arrest induced by different 5OH-PMFs.. Annexin V/PI co-staining assay demonstrated that 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (p53(+/+) ) cells but not in HCT116 (p53(-/-) ) cells. Furthermore, 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (Bax(+/-) ) cells, whereas their pro-apoptotic effects on HCT116 (Bax(-/-) ) cells were marginal. All three 5OH-PMFs increased G0/G1 cell population of HCT116 (p53(+/+) ) cells, and these effects were abolished in HCT116 (p53(-/-) ) and HCT116 (p21(-/-) ) cells. Immunoblotting analysis showed that 5HHMF and 5HTMF increased the levels of cleaved caspase-3, cleaved PARP in both HCT116 (p53(+/+) ) and HCT116 (Bax(+/-) ) cells and these effects were much weaker in HCT116 (p53(-/-) ) and HCT116 (Bax(-/-) ) cells.. Our results demonstrated that 5OH-PMFs, especially 5HHMF and 5HTMF, induce apoptosis and cell-cycle arrest by p53-, Bax- and p21-dependent mechanism.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Flavones; HCT116 Cells; Humans; Osmolar Concentration; Poly(ADP-ribose) Polymerases; Tumor Suppressor Protein p53

Hydroxylated polymethoxyflavones (PMFs) are a class of novel flavonoid compounds mainly found in citrus plants. We studied the effects of three major 5-hydroxy PMFs, namely: 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone, 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone, and 5-hydroxy-6,7,8,4'-tetramethoxyflavone, on human colon cancer HCT116 and HT29 cells. Their effects were compared with those produced by their permethoxylated counterparts, namely: nobiletin, 3,5,6,7,8,3',4'-heptamethoxylflavone, and tangeretin. 5-Hydroxy PMFs showed much stronger inhibitory effects on the growth of the colon cancer cells in comparison with their permethoxylated counterparts, suggesting the pivotal role of hydroxyl group at 5-position in the enhanced inhibitory activity by 5-hydroxy PMFs. Flow cytometry analysis demonstrated that three 5-hydroxy PMFs produced different effects on the cell cycle and apoptosis, which may suggest that three 5-hydroxy PMFs act through different mechanisms. For example, 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone caused cell cycle arrest at G2/M phase in HT29 cells, while 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone led to significant G0/G1 phase arrest. In contrast, 5-hydroxy-6,7,8,4'-tetramethoxyflavone increased sub-G0/G1 cell population, which has been confirmed to be due to enhanced apoptosis. Our results further demonstrated that the inhibitory effects of 5-hydroxy PMFs were associated with their ability in modulating key signaling proteins related to cell proliferation and apoptosis, such as p21(Cip1/Waf1), CDK-2, CDK-4, phosphor-Rb, Mcl-1, caspases 3 and 8, and poly ADP ribose polymerase (PARP).

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Proliferation; Cell Survival; Colonic Neoplasms; Drug Screening Assays, Antitumor; Flavones; HCT116 Cells; HT29 Cells; Humans; Inhibitory Concentration 50; Necrosis; Protein Biosynthesis; Structure-Activity Relationship