Published OnlineFirst July 28, 2009; DOI: 10.1158/1940-6207.CAPR-09-0081 Published Online First on July 28, 2009 as 10.1158/1940-6207.CAPR-09-0081 Cancer Prevention Research Flavones as Colorectal Cancer Chemopreventive Agents—Phenol-O- Methylation Enhances Efficacy Hong Cai,1 Stewart Sale,1 Ralf Schmid,2 Robert G. Britton,1 Karen Brown,1 William P. Steward1 and Andreas J. Gescher1 Abstract Flavonoids occur ubiquitously in plants, and some possess preclinical cancer chemopre- ventive activity. Little is known about molecular features that mediate chemopreventive ef- ficacy of flavonoids. Here, three related flavones, apigenin (4′,5,7-trihydroxyflavone), tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone), and 3′,4′,5′,5,7-pentamethoxyflavone (PMF), were compared in terms of their effects on (a) adenoma development in ApcMin mice, a model of human gastrointestinal malignancies; (b) growth of APC10.1 mouse adenoma cells in vitro; and (c) prostaglandin E-2 generation in HCA-7 human-derived colorectal cancer cells in vitro. Life-long consumption of PMF with the diet at 0.2% reduced ApcMin mouse adenoma number and burden by 43% and 61%, respectively, whereas apigenin was inactive. Tricin has previ- ously shown activity in this model. IC50 values for murine adenoma cell growth inhibition by PMF, tricin, and apigenin were 6, 13, and 18 μmol/L, respectively. In ApcMin mice that received flavones (0.2%) for 4 weeks, adenoma cell proliferation as reflected by Ki-67 staining was re- duced by PMF and tricin, but not by apigenin. On incubation with HCA-7 cells for 6 hours, PMF reduced prostaglandin E-2 generation with an IC50 of 0.8 μmol/L, a fraction of the respective values reported for tricin or apigenin. In silico PMF docked into the cyclooxygenase active site with greater affinity than tricin or apigenin. The results suggest that the rank order of cancer chemopreventive efficacy in ApcMin mice is PMF > tricin > apigenin, supporting the notion that the presence of O-methyl in the flavone molecular scaffold promotes gastrointestinal cancer chemopreventive efficacy. Flavonoids are polyphenolic phytochemicals that occur ubiq- ity on flavonoids would allow prioritization of agents for uitously in the leaves, seeds, bark, and flowers of plants, many preclinical development, but such information is extremely of which are consumed with the diet. Certain flavonoids ex- scarce. The flavone tricin (4′,5,7-trihydroxy-3′,5′-dimethoxy- emplified by the flavonol epigallocatechin-3-gallate contained flavone; Fig. 1) contained in rice bran and other grass species in green tea, the isoflavone genistein from soya, and the flavo- has recently been shown to interfere with small intestinal car- nol quercetin found in apples and onions have been the focus cinogenesis in the ApcMin mouse (4), a model of the human of considerable research interest as they can prevent malig- heritable condition familial adenomatous polyposis coli (5). nancies in rodent models of prostate, breast, or gastrointesti- Tricin inhibited prostaglandin E-2 (PGE-2) generation in colo- nal carcinogenesis (1–3). More than 4,000 flavonoids have rectal cells in vitro and in ApcMin mice in vivo (4). PGE-2 is gen- been identified to date, and only a handful of which, including erated from arachidonic acid via the cyclooxygenase (COX) epigallocatechin-3-gallate, genistein, and quercetin, have been pathway. Inhibition and/or down-regulation of components investigated in depth. The putative cancer chemopreventive of this pathway, especially of the isoenzyme COX-2, are con- properties of most flavonoids are virtually unknown. Knowl- sidered to be important mechanisms of colorectal cancer che- edge of molecular features that impart anticarcinogenic activ- moprevention (6). Recently, methoxy moieties have been characterized as phar- macophor imparting anticarcinogenic properties onto fla- vones. 5,7-Dimethoxyflavone and 5,7,4′-trimethoxyflavone Authors' Affiliations: 1Cancer Biomarkers and Prevention Group, Department of Cancer Studies and Molecular Medicine and 2Department of Biochemistry, were superior to their hydroxy congeners in terms of systemic University of Leicester, Leicester, United Kingdom availability in rats and ability to inhibit oral cancer cell prolif- Received 3/19/09; revised 4/18/09; accepted 5/5/09; published OnlineFirst 7/28/09. eration in vitro (7). The latter finding implies an intrinsic advan- Grant support: Cancer Research UK grant C325/A6691. Note: H. Cai and S. Sale contributed equally to this work. tage of methoxylated flavones over hydroxylated ones in terms Requests for reprints: Andreas J. Gescher, Department of Cancer Studies of growth-inhibitory properties. In the light of all this evidence, and Molecular Medicine, University of Leicester, Robert Kilpatrick Clinical we wished to explore the role of phenyl methyl ether moieties Sciences Building, Leicester LE2 7LX, United Kingdom. Phone: 44- in the adenoma-preventive efficacy of flavones in the ApcMin 1162231856; Fax: 44-1162231855; E-mail: [email protected]. ©2009 American Association for Cancer Research. mouse. Two analogues of tricin differing in O-methylation doi:10.1158/1940-6207.CAPR-09-0081 status were selected, apigenin (4′,5,7-trihydroxyflavone) and www.aacrjournals.org 743 Cancer Prev Res 2009;2(8) August 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on September 28, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst July 28, 2009; DOI: 10.1158/1940-6207.CAPR-09-0081 Cancer Prevention Research ApcMin mice that received flavones with their diet for the fi- nal month of their life. Third, the ability of the flavones to compromise PGE-2 generation, a potential mechanism of their chemopreventive action, was compared in human colo- rectal cancer cells in vitro. In addition, differences between the three flavones in affinity for COX enzymes, which cata- lyze arachidonic acid metabolism to generate PGE-2, were explored in silico. Overall, the work was designed to help de- fine criteria that aid prioritization of flavones for colorectal cancer chemoprevention agent development. Materials and Methods Materials PMF was synthesized as described before (13) by condensing 2′-hy- droxy-4′,6′-dimethoxyacetophenone with 3,4,5-trimethoxybenzoyl chloride in pyridine. The resulting trimethoxybenzoate was subjected to Baker-Venkataraman migration to obtain a β-diketone, which was cyclized to the flavone. The identity of PMF was confirmed by the ap- propriate melting point and spectrometric/spectroscopic data, and it was >99% pure by high-performance liquid chromatography analysis (14). Apigenin was purchased from Apin Chemical Co. Tricin was cus- tom synthesized by Syncom. For the cell culture experiments, flavones were dissolved in DMSO, and the final DMSO concentration in cellu- lar incubations was 0.1%. All other chemicals were purchased from Sigma-Aldrich Chemical Co. Animals and interventions Breeding colonies were established in the Leicester University Bio- medical Services facility using C57BL/6J Min/+ (ApcMin) mice origi- nally obtained from The Jackson Laboratory. Ear tissue from newborn mice was genotyped for the presence of the mutation using PCR as described previously (15). Experiments were carried out under animal project license PPL 80/2167, granted to Leicester University by the UK Home Office. The experimental design was vetted by the Leicester University Local Ethical Committee for Animal Experimentation Fig. 1. Chemical structures of flavones used in this study. and met the standards required by the United Kingdom Coordinating Committee on Cancer Research guidelines (16). ApcMin mice (14-25 per group) received standard American Institute of Nutrition-93G (AIN- 3′,4′,5′,5,7-pentamethoxyflavone (PMF; for structures, see 93G) diet or AIN-93G diet supplemented with flavone. In the case of Fig. 1), and their effects were compared with those of tricin. the long-term intervention study, animals received apigenin at a die- Apigenin, des-dimethoxy tricin, occurs in many leafy vegeta- tary concentration of 0.2%, or PMF at 0.05% or 0.2%, from week 4 to bles and fruits and is reported to possess cancer chemopreven- the end of the their life (week 16). Both agents were investigated in tive properties (8). PMF, fully O-methylated tricin, has been two separate experiments with their own controls. In the study to as- found in the leaves of Murraya paniculata,aconstituentof sess the effect of flavones on proliferation of established adenomas, traditional Indonesian herbal medicines (9), and fruits of ApcMin mice received standard AIN-93G diet until week 12 after the Brazilian Neoraputia magnifica (10), both plants being weaning and then diet containing apigenin, tricin, or PMF (0.2%) up members of the Rutaceae plant family. The only pharmaco- to week 16. Dietary doses of 0.2% and 0.05% equate to approximately logic property of PMF reported thus far is its ability to re- 300 and 75 mg/kg murine body weight, respectively. Animals were culled by cardiac exsanguination under halothane anesthesia in week verse P-glycoprotein–mediated multidrug resistance in 16. The intestinal tract was removed and flushed with PBS. Intestinal cancer cells (11). The hypothesis was tested that apigenin is tissue was cut open longitudinally, and multiplicity, location, and size less, and PMF more, capable than tricin of interfering with of adenomas were recorded as described previously (15) using a mag- adenoma development, as a consequence of the relative nifying glass (×5). Polyp volume was derived