RESEARCH ARTICLE Inhibitory effects of 5-hydroxy polymethoxyflavones on colon cancer cells Peiju Qiu 1,2 , Ping Dong 2 , Huashi Guan 1 , Shiming Li 3 , Chi-Tang Ho 4 , Min-Hsiung Pan 5 , David Julian McClements 2 and Hang Xiao 2 1 Marine Drug and Food Institute, Ocean University of China, Qingdao Shandong, P. R. China 2 Department of Food Science, University of Massachusetts, Amherst, MA, USA 3 WellGen, Inc., North Brunswick, NJ, USA 4 Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA 5 Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan Received: December 18, 2009 Revised: January 29, 2010 Accepted: February 2, 2010 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 0 ,4 0 -pentamethoxyflavone, 5-hydroxy-3,6,7,8,3 0 ,4 0 -hexamethoxyflavone, and 5-hydroxy-6,7,8,4 0 -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 0 ,4 0 -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 0 ,4 0 - pentamethoxyflavone caused cell cycle arrest at G2/M phase in HT29 cells, while 5-hydroxy- 3,6,7,8,3 0 ,4 0 -hexamethoxyflavone led to significant G0/G1 phase arrest. In contrast, 5-hydroxy- 6,7,8,4 0 -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). Keywords: 5-hydroxy polymethoxyflavones / Apoptosis / Cell cycle arrest / Cell growth inhibition / Colon cancer 1 Introduction Polymethoxyflavones (PMFs) refer to flavonoid compounds that bear two or more methoxy groups on their basic benzo- g-pyrone skeleton with a carbonyl group at the C4 position (Fig. 1). Naturally, PMFs were mainly found in citrus plants, particularly in the peels of sweet oranges (Citrus sinensis) and mandarin oranges (Citrus reticulata) [1]. Currently, more than 20 PMFs have been isolated and identified from different parts of citrus plants. They exhibited a broad spectrum of biological activity, such as anti-inflammatory, anti-carcinogenic, anti-atherogenic, anti-viral, and anti- oxidative ones [1–3]. The most studied PMFs are perme- thoxylated PMFs, such as nobiletin, 3,5,6,7,8,3 0 ,4 0 -hepta- methoxylflavone (HMF) and tangeretin (Fig. 1). These PMFs are also most abundant ones found in orange peel extract from cold-pressed sweet orange peel oil [1]. Recently, a class of hydroxylated PMFs have been isolated from aged orange peel extracts, and they could be formed by auto-hydrolysis of their permethoxylated counterparts Abbreviation: 5HHMF, 5-hydroxy-3,6,7,8,3 0 ,4 0 -hexamethoxy- flavone; 5HPMF, 5-hydroxy-6,7,8,3 0 ,4 0 -pentamethoxyflavone; 5HTMF, 5-hydroxy-6,7,8,4 0 -tetramethoxyflavone; HMF, 3,5,6,7,8,3 0 ,4 0 -heptamethoxyflavone; MTT, 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly ADP ribose polymerase; PI, propidium iodide; PMF, polymethoxyflavone Correspondence: Dr. Hang Xiao, Department of Food Science, University of Massachusetts, 100 Holdsworth Way, Amherst, MA 01003, USA E-mail: hangxiao@foodsci.umass.edu Fax: 11-413-545-1262 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com S244 Mol. Nutr. Food Res. 2010, 54, S244–S252 DOI 10.1002/mnfr.200900605