5-demethylnobiletin and Colonic-Neoplasms

The intake of common polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), is strongly correlated to the initiation of colon cancer. BaP is a well-known pro-carcinogen that is metabolically activated by xenobiotic-metabolizing enzymes. Studies indicate that polymethoxyflavones, including 5-demethylnobiletin (5-DMNB), exhibit anti-inflammatory and anti-carcinogenic properties. However, the effects of 5-DMNB on xenobiotic-metabolizing enzymes and BaP-induced carcinogenesis remain unclear. The combination of BaP and a promoting agent-dextran sulfate sodium (DSS)-has been demonstrated to induce tumors in mouse models. Thus, this study aimed to determine the protective effect of 5-DMNB on carcinogen biotransformation and BaP/DSS-induced colon carcinogenesis. Our results showed that 5-DMNB had a substantial inhibitory effect on CYP1B1 induced by BaP and upregulated the detoxification enzymes UDP-glucuronosyltransferases (UGTs) and glutathione S-transferases (GSTs). Furthermore, subsequent analyses confirmed that the dietary administration of 5-DMNB markedly ameliorated tumor formation in BaP/DSS-treated mice. Exposure to BaP/DSS also significantly elevated TNF-α levels, and the administration of 5-DMNB reversed this increase. Taken together, we determined that 5-DMNB attenuates BaP/DSS-induced colon cancer through the regulation of inflammation and xenobiotic-metabolizing enzymes. These results indicate that 5-DMNB has significant potential as a novel chemopreventive agent for preventing carcinogen activation and inflammation-associated carcinogenesis.

Topics: Animals; Anti-Inflammatory Agents; Anticarcinogenic Agents; Benzo(a)pyrene; Carcinogenesis; Colon; Colonic Neoplasms; Cytochrome P-450 CYP1B1; Dextran Sulfate; Flavones; Glucuronosyltransferase; Glutathione Transferase; Hep G2 Cells; Humans; Male; Mice, Inbred BALB C; Mice, Inbred ICR

Biotransformation of dietary components is important for their in vivo biological activities after oral ingestion. Herein, we investigated biotransformation of 5-demethylnobiletin (a polymethoxyflavone found in citrus fruits) in mice, and its implication in the inhibition of human colon cancer cells.. Urine samples were collected from mice fed with 5-demethylnobiletin (1) and analyzed by LC-ESI-MS and HPLC coupled with an electrochemical detector. Three major metabolites were identified as 5,3'-didemethylnobiletin (2), 5,4'-didemethylnobiletin (3), and 5,3',4'-tridemethylnobiletin (4) by comparing their ESI-MS and HPLC profiles with those of authentic standards synthesized by a multistep route. Cell viability assay in human colon cancer cells demonstrated that all three metabolites of 5-demethylnobiletin, especially 5,3'-didemethylnobiletin (2), showed much stronger inhibitory effects on cancer cell growth than 5-demethylnobiletin. For example, metabolites (2-4) showed IC50 of 0.12, 5.5, and 4.2 μM in SW620 cells, respectively, while 5-demethylnobiletin at 10 μM only caused 37% inhibition after 72 h of treatment.. Three novel metabolites were identified in mice after oral administration of 5-demethylniobiletin. These metabolites exhibited strong inhibitory effects against human colon cancer cells. Our results provide a first report on these bioactive metabolites and warrant further investigation on their molecular mechanism of actions.

Topics: Administration, Oral; Animals; Biotransformation; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; Chromatography, Liquid; Citrus; Colonic Neoplasms; Flavones; Humans; Male; Mass Spectrometry; Mice; Plant Extracts

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