Flavonoids as anticarcinogens

Flavonoids are amongst the most thoroughly studied anticarcinogens, but much of the evidence has been derived from in vitro studies, using compounds such as quercetin aglycone, which is rarely found in the diet, and never in the human body. Under physiological conditions, a relatively small fraction of the quercetin glycosides found in foods is hydrolysed at the intestinal surface, and the released aglycone is absorbed and rapidly metabolised, largely to glucuronides, which are then transferred to the circulation⁽Reference Day, Gee, DuPont, Johnson and Williamson⁸⁾. Unabsorbed polyphenols eventually become available for bacterial fermentation, yielding a mixture of phenolic acids in the colon⁽Reference Scalbert, Morand, Manach and Remesy⁹⁾. This section is a general overview of interactions between flavonoids and potentially anticarcinogenic mechanisms.

Flavonoids and colorectal carcinoma

There is moderately good epidemiological evidence for protective effects of fruits and vegetables against colorectal cancer, and this has focused attention on the possibility that these effects might be due to the biological activity of flavonoids acting at one or more stages in the development of the disease. Most colorectal carcinomas in developed countries develop from initially benign adenomatous polyps via the adenoma-carcinoma sequence⁽Reference Winawer³¹⁾. This step-wise pathway involves both morphological changes associated with the emergence and growth of the localised lesion, and a progressive disruption of the genome, including acquisition of somatic mutations⁽Reference Vogelstein, Fearon, Hamilton, Kern, Preisinger, Leppert, Nakamura, White, Smits and Bos³²⁾ and silencing of genes through changes to the epigenetic marks regulating gene expression. The most widely studied epigenetic mechanism is the hypermethylation of the cytosine residues in CpG-rich sequences (CpG-islands) located within the promoter regions of expressed genes⁽Reference Jubb, Bell and Quirke³³⁾. These sequences are normally unmethylated, but once methylated during the development of cancer they become progressively silenced by mechanisms involving recruitment of protein complexes, compaction of adjacent chromatin and suppressed transcription⁽Reference Bestor³⁴⁾. Cancers of the alimentary tract exhibit more aberrant methylation than cancers of the lung, ovary and bladder⁽Reference Esteller, Corn, Baylin and Herman³⁵⁾, and silencing of DNA-repair and tumour-suppressor genes makes a major contribution to the genomic dysfunction associated with colorectal carcinoma⁽Reference Issa³⁶⁾.

Although food-borne flavonoids undoubtedly modulate molecular signals involved in the development of colorectal cancer under experimental conditions, the concentrations of these compounds in food are relatively low and there is little epidemiological evidence to support a protective role against cancer for flavonoids derived from food⁽Reference Lin, Zhang, Wu, Willett, Fuchs and Giovannucci³⁷⁾. There is better evidence however that flavonoids from tea may play a protective role at the population level. Tea is an infusion prepared from the leaves of the plant Camellia sinensis and is one of the major sources of flavonoids in many western and oriental diets. The polyphenolic constituents of the tea infusion depend upon the processing of the leaves. Green tea is steamed immediately after picking to denature the polyphenol oxidases in the leaves, and hence stabilise the low molecular weight compounds, which are principally the catechins: epicatechin (EC), ( − )-epigallocatechin gallate (EGCG), ( − )-epigallocatechin (EGC) and ( − )-epicatechin-3-gallate (ECG). In contrast, black tea and Oolong tea are crushed and allowed to oxidise prior to drying. This leads to polymerisation of the polyphenols, forming high levels of theaflavins and thearubigens, with a corresponding reduction in the levels of catechins.

Black tea polyphenol consumption shows protective effects against oxidative DNA damage to the colon in animal models⁽Reference Lodovici, Casalini, De Filippo, Copeland, Xu, Clifford and Dolara³⁸⁾, and black tea significantly reduced the formation of aberrant crypt foci in a rats with preneoplastic lesions induced by azoxymethane. In the early stages GST and glutathione peroxidase (GPx) gene expression were up-regulated, suggesting that black tea effects might be mediated by antioxidative mechanisms⁽Reference Sengupta, Ghosh and Das³⁹⁾. EGCG reduces the invasive activity and growth of murine colon 26-L5 cell line in vitro, and significantly reduced lung metastasis in BALB/c mice inoculated with 26-L5 cells⁽Reference Ogasawara, Matsunaga and Suzuki⁴⁰⁾.

Orner et al. ⁽Reference Orner, Dashwood, Blum, Diaz, Li and Dashwood⁴¹⁾ used the Apc(min) mouse model to compare the anti-tumorigenic effects of green tea with those of “white” tea, a product that contains even higher levels of flavonoids. Mice treated with both types of tea had significantly lower numbers of tumours than the untreated controls, and there were significant reductions in beta-catenin and beta-catenin/Tcf-4 regulated proteins Cyclin D⁽Reference Parkin, Bray, Ferlay and Pisani¹⁾ and c-Jun in the apparently normal intestines of mice treated with both white tea and sulindac. Similarly, Ju et al, working with Apc(min) mice showed that treatment with EGCG reduced the high levels of nuclear beta-catenin in these mice, and inhibited aberrant gene expression⁽Reference Ju, Hong, Zhou, Pan, Bose, Liao, Yang, Liu, Hou, Lin, Ma, Shih, Carothers and Yang⁴²⁾.

A rigorous meta-analysis of epidemiological studies on the relationship between tea consumption and colorectal cancer was recently published by Sun et al. ⁽Reference Sun, Yuan, Koh and Yu⁴³⁾, who identified 25 papers describing studies conducted in European, North American and Asian populations. Eight studies provided evidence for a protective effect of green tea such that the highest had an odds ratio for colon cancer of 0·82 (95 % CI 0·69–0·98) compared to the lowest consumers. There was no evidence of a protective effect against rectal cancer, nor was there evidence for a protective effect of black tea against cancer at either site. The same group has recently compared urinary metabolites of tea polyphenols with subsequent risk of colorectal during 16 years of follow-up in a cohort of 18 244 Chinese men. Subjects with higher levels of EGC and 4′-0-methyl-EGC in their urine had a significantly lower risk of colon cancer, but not of rectal cancer.

Wine is another commonly consumed beverage capable of delivering high concentrations of polyphenols to the alimentary tract. Polyphenols derived from red wine reduced colon tumour yield and oxidative DNA damage, induced in rat colon mucosa by the model carcinogen 1,2-dimethylhydrazine. Functional pathway analysis carried out on microarray gene expression data showed that wine polyphenol consumption down-regulated the inflammatory response and steroid metabolism⁽Reference Dolara, Luceri, Filippo, Femia, Giovannelli, Caderni, Cecchini, Silvi, Orpianesi and Cresci⁴⁴⁾.

Flavonoids and gastric carcinoma

Two distinct histological sub-types of gastric cancer have been identified: intestinal-type and diffuse-type⁽Reference Lauren⁴⁵⁾. Unlike colorectal cancer, the incidence of gastric cancer is in worldwide decline, with the sharpest reductions over the last few decades occurring in the industrialised world. However, whereas distal, intestinal-type gastric cancers have tended to decline in industrialised countries, the incidence of proximal cancers of the gastric cardia has increased recently⁽Reference Crew and Neugut⁴⁶⁾. The bacterium Helicobacter pylori is the principal known risk factor for gastric carcinogenesis⁽Reference Peek and Blaser⁴⁷⁾. Infection rates are inversely associated with affluence, both within and between countries, but the interaction between H. pylori and other environmental factors remains to be established.

As with colorectal cancer, a multistage sequence in the development of intestinal-type gastric carcinoma has been identified, beginning with chronic gastritis, and proceeding to mucosal atrophy, and intestinal metaplasia⁽Reference Correa⁴⁸⁾. The latter stage involves the development of a cellular phenotype similar to that of the intestine, which is associated with the ectopic expression of the protein CDX2, a transcription factor normally expressed by intestinal epithelial cells. The final stage of malignant transformation leads to the appearance of a discreet tumour with glandular histology. APC and K-RAS mutations do occur in some tumours, and methylation of the CpG-island of tumour-suppressor and DNA-repair genes is widely reported. Diffuse-type gastric cancers do not form discreet lesions but develop as small groups of cells distributed through the mucosal tissue. This pathway is associated specifically with mutations or epigenetic silencing of E-cadherin. H. pylori infection predisposes to both types of gastric cancer⁽Reference Huang, Sridhar, Chen and Hunt⁴⁹⁾, but its mechanism of action is best understood in the case of chronic gastritis-atrophy-metaplasia sequence of intestinal-type carcinogenesis. Recently Ruggiero et al. ⁽Reference Ruggiero, Tombola, Rossi, Pancotto, Lauretti, Del Giudice and Zoratti⁵⁰⁾ reported that certain polyphenols ameliorate the adverse effects of H. pylori infection on the gastric mucosa in a mouse model, probably by exerting antitoxic activity.

As with colorectal cancer, there are numerous studies showing that a variety of flavonoids inhibit the proliferation of gastric carcinoma cells⁽Reference Yoshida, Sakai, Hosokawa, Marui, Matsumoto, Fujioka, Nishino and Aoike⁵¹⁾ and induce apoptosis⁽Reference Yoshimizu, Otani, Saikawa, Kubota, Yoshida, Furukawa, Kumai, Kameyama, Fujii, Yano, Sato, Ito and Kitajima⁵²⁾ but relatively little evidence for protective effects of flavonoids at the population level. Sun et al. ⁽Reference Sun, Yuan, Lee, Yang, Gao, Ross and Yu⁵³⁾ conducted a nested case–control study to explore the relationship between urinary markers of exposure to tea polyphenols and subsequent risk of gastric cancer in a cohort of over 18 000 Chinese men. There was a strong inverse association between urinary EGC and risk of gastric and oesophageal cancer (odds ratio 0·52, 95 % CI 0·28–0·97), which suggests that tea polyphenols may exert anticarcinogenic effects in this part of the gut, as well as the colon. It is less clear whether similar protective effects of polyphenols occur in populations consuming conventional Western diets. Lagiou et al. ⁽Reference Lagiou, Samoli, Lagiou, Peterson, Tzonou, Dwyer and Trichopoulos⁵⁴⁾ examined the relationship between dietary intake of six classes of flavonoids (flavanones, flavan-3-ols, flavonols, flavones, anthocyanidins and isoflavones) and vitamin C in the aetiology of stomach cancer in a small case-control study conducted in Greece, and observed a statistically significant protective effect of flavanones. They commented that this might account for the apparently beneficial effects of fruit reported in some epidemiological studies, but given the small size of the study and the difficulty of estimating flavonoid intakes from dietary records, further research would be needed to explore this possibility.

It is worth noting that flavonoids also exert mutagenic effects in vitro and for a long period before the growth of interest in phytochemicals as anticarcinogens they were regarded as possible carcinogens. Gaspar et al. ⁽Reference Gaspar, Laires, Monteiro, Laureano, Ramos and Rueff⁵⁵⁾ used the Ames test to show that the mutagenicity of red wines correlated well with their quercetin content, but it has not since been established that these adverse effects are directly relevant to human health.

Flavonoids and oesophageal carcinoma

Oesophageal cancer occurs in two histological subtypes, squamous cell carcinoma (OSCC), and adenocarcinoma (OA). Overall the disease occurs somewhat more frequently in less developed countries than in the industrialised west⁽Reference Ferlay, Bray, Pisani and Parkin⁵⁶⁾ but some of the steepest contrasts in reported incidence occur within countries in Africa and Asia, where squamous carcinoma is the predominant form. Such variations appear to be associated with a combination of micronutrient deficiencies and high exposure to environmental mutagens⁽Reference Roth, Strickland, Wang, Rothman, Greenberg and Dawsey⁵⁷⁾. Over the last 30 years, the incidence of OSCC has decreased in many industrialised countries, whereas an unexplained increase in the rates of OA has occurred in the United States and Western Europe⁽Reference Newnham, Quinn, Babb, Kang and Majeed⁵⁸⁾.

Typically, adenocarcinoma develops from Barrett's oesophagus, an intestinal-type metaplasia that replaces the normal squamous mucosa in the lower third of the oesophagus⁽Reference Prach, MacDonald, Hopwood and Johnston⁵⁹⁾, apparently in response to chronic irritation by gastric reflux. In 10–15 % of cases there is progression via low- and high-grade dysplasia to adenocarcinoma, accompanied by somatic mutations of genes including p53 and K-RAS, chromosomal losses and aneuploidy, and methylation of the CpG-islands of APC, CDKN2A, ESR1 and E-Cadherin ⁽Reference Wild and Hardie⁶⁰⁾.

Gastro-oesophageal reflux disease (GERD) is the strongest identified risk-factor for OA⁽Reference Lagergren, Bergstrom, Lindgren and Nyren⁶¹⁾, and the disease shows a strong positive relationship with obesity⁽Reference Lagergren, Bergstrom and Nyren⁶²⁾. Several epidemiological studies show evidence for an inverse correlation between fruit and vegetable consumption and risk of both OA and OSCC⁽Reference Bosetti, La Vecchia, Talamini, Simonato, Zambon, Negri, Trichopoulos, Lagiou, Bardini and Franceschi^63, Reference Zhao, Cao, Ma and Liu⁶⁴⁾. Engel and colleagues estimated that the population attributable risk (PAR) associated with low fruit and vegetable consumption was of 29 % for OSCC and 15 % for OA⁽Reference Engel, Chow, Vaughan, Gammon, Risch, Stanford, Schoenberg, Mayne, Dubrow, Rotterdam, West, Blaser, Blot, Gail and Fraumeni⁶⁵⁾.

Aspirin and other synthetic COX-2 enzyme inhibitors are protective against OA⁽Reference Vaughan, Dong, Blount, Ayub, Odze, Sanchez, Rabinovitch and Reid¹⁵⁾. A number of flavonoids are COX-2 inhibitors⁽Reference Baumann, von Bruchhausen and Wurm¹⁸⁾ and some (e.g. apigenin, chrysin, and kaempferol) can suppress COX-2 transcription by mechanisms that include activation of the peroxisome proliferator-activated receptor (PPAR) gamma transcription factor⁽Reference Liang, Tsai, Tsai, Lin-Shiau and Lin¹⁹⁾, and inhibition of NF-κB expression⁽Reference Liang, Huang, Tsai, Lin-Shiau, Chen and Lin⁶⁶⁾. COX-2 transcription is inhibited in vitro not just by quercetin aglycone, but also by the metabolites quercetin 3-glucuronide, quercetin 3′-sulphate, and 3′-methylquercetin 3-glucuronide. These compounds are found in human plasma, and both quercetin and quercetin 3′-sulphate also inhibit COX-2 enzyme activity⁽Reference O'Leary, de Pascual-Tereasa, Needs, Bao, O'Brien and Williamson⁶⁷⁾. Quercetin aglycone inhibits COX-2 expression and induces apoptosis in the oesophageal adenocarcinoma cell line OE33 in vitro ⁽Reference Cheong, Ivory, Doleman, Parker, Rhodes and Johnson⁶⁸⁾.

EGCG is another flavonoid which is of particular interest in the context or oesophageal cancer. In studies carried out on N-nitrosomethylbenzylamine (NMBA)-induced OSCC in rats, EGCG reduced the incidence of tumours in a dose-dependent manner, and significantly down-regulated the gene expression of both cyclin D1 and COX-2⁽Reference Li, Shimada, Sato, Maeda, Itami, Kaganoi, Komoto, Kawabe and Imamura⁶⁹⁾. Perhaps the most intriguing property of EGCG in this context is its ability to inhibit DNA methyltransferase (DNMT) activity. Epigenetic silencing of genes involved in cell cycle regulation, like p16 and retinoic acid receptor beta (RARβ), and DNA repair, like O6-methylguanine-DNA methyltransferase (MGMT) and human mutL homologue 1 (hMLH1), are important events in OSCC development⁽Reference Nie, Liao, Zhao, Song, Yang, Wang and Yang⁷⁰⁾. The EGCG-induced inhibition of DNMT reversed the methylation status of the promoters of these genes in an OSCC cell line leading to their re-expression⁽Reference Fang, Wang, Ai, Hou, Sun, Lu, Welsh and Yang⁷¹⁾. Similar results were obtained using the same in vitro model treated with genistein, a soy-derived isoflavone⁽Reference Fang, Jin, Wang, Liao, Yang, Wang and Yang⁷²⁾. However the inhibition of DNMT1 activity by genistein was relatively weak, suggesting that it might act by a different pathway⁽Reference Fang, Jin, Wang, Liao, Yang, Wang and Yang⁷²⁾. Other flavonoids (myricetin, quercetin, hesperetin, naringenin, epigenin and luteolin) have also been shown to inhibit DNMT activity in OSCC nuclear extracts, although less efficiently than EGCG.

Flavopiridol is a synthetic flavone identical to one obtained from the Indian plant Dysoxylum binectiferum, which has attracted a great deal of attention as a possible chemotherapeutic agent for the treatment of gastrointestinal and other solid tumours. The primary action of flavopiridol is to inhibit cyclin dependent kinases (CDK) 1, 2 and 4⁽Reference Carlson, Dubay, Sausville, Brizuela and Worland⁷³⁾. Indeed it is the first pan-cyclin-dependent kinase (CDK) inhibitor to enter clinical trials. Flavopiridol has been shown to induce G1 or G2/M cell cycle arrest and apoptosis and reduce protein levels of cyclin D1 and Retinoblastoma (Rb), which are crucial regulators of G0/G1 cell cycle checkpoint, in several OA and OSCC cell lines⁽Reference Schrump, Matthews, Chen, Mixon and Altorki⁷⁴⁾. Promising results have also been obtained using in vivo models of OA and OSCC. Unfortunately however, flavopiridol has adverse side effects that appear to be due to its ability to act as a general suppressor of gene transcription⁽Reference Blagosklonny⁷⁵⁾. Some recent studies showed that flavopiridol treatment enhanced the sensitivity to radiation of OA cell lines, and xenografts in nude mice⁽Reference Raju, Ariga, Koto, Lu, Pickett, Valdecanas, Mason and Milas^76, Reference Sato, Kajiyama, Sugano, Iwanuma and Tsurumaru⁷⁷⁾. Further investigations are needed to assess whether flavopiridol can be used safely in combination with other therapeutic agents or radiation for clinical purposes.