Dietary fibre and the modulation of CRC risk

Dietary fibre has been defined as carbohydrate polymers with ten or more monomeric units which are not hydrolysed by the endogenous enzymes in the small intestine of human subjects^{( 5 )}. It encompasses non-digestible carbohydrates (NDC) such as resistant starch (RS) and polydextrose. RS is the portion of dietary starch that avoids enzymatic digestion and absorption in the small intestine and consequently reaches the large bowel undigested⁽ Reference Cummings and Stephen ^{6 )}. Here, it is the target for fermentation by resident colonic bacteria to produce SCFA such as acetate, propionate and butyrate⁽ Reference Topping and Clifton ^{7 )}. Four types of RS have been described: type 1 physically inaccessible RS, e.g. that are found in seeds; type 2 resistant granules, e.g. in uncooked potato or unripe banana; type 3 retrograded RS, found in cooked and cooled foods such as bread; type 4 chemically modified RS which is used widely for technological purposes in processed foods⁽ Reference Topping and Clifton ^{7 )}.

Based on clinical observations in Africa, Burkitt was one of the first to report an association between higher consumption of dietary fibre and reduced CRC incidence⁽ Reference Burkitt ^{8 )}. Since then, many epidemiological studies have quantified links between dietary fibre intake and CRC risk. A recent meta-analysis of the results from twenty-five prospective studies confirmed an inverse correlation between dietary fibre intake and CRC risk⁽ Reference Aune, Chan and Lau ^{9 )}. The authors concluded that CRC risk is 10 % lower for every 10 g/d increase in dietary fibre intake.

Early attempts to explain the protective effects of dietary fibre focused on the increased faecal bulk, reduced transit time and dilution of faecal content which were expected to reduce exposure of the large bowel to carcinogens⁽ Reference Hylla, Gostner and Dusel ^{10 ,} Reference Lipkin, Reddy and Newmark ^{11 )}. More recently, the focus has shifted to the beneficial effects of SCFA, in particular butyrate, which are produced by colonic fermentation of NDC⁽ Reference Williams, Coxhead and Mathers ^{12 )}. Butyrate is produced predominantly by bacterial species of the Clostridia, Roseburia and Eubacteria genera⁽ Reference Pryde, Duncan and Hold ^{13 )}. Butyrate is the preferred energy source for colonocytes and plays an important role within the large bowel mucosa where it regulates key cellular processes including proliferation, differentiation and apoptosis that contribute to the maintenance of homeostasis⁽ Reference Hamer, Jonkers and Venema ^{14 )}. Furthermore, butyrate has been reported to positively modulate WNT signalling⁽ Reference Bordonaro, Lazarova and Sartorelli ^{15 )}, a pathway that is frequently aberrantly activated in CRC, suggesting that this may be one of the mechanisms through which butyrate, and other NDC, are protective against CRC.

The WNT signalling pathway

WNT signalling comprises three different pathways: the canonical or β-catenin pathway, the non-canonical planar cell polarity pathway and the WNT/calcium pathway. The principal pathway, that is most relevant in human health and disease, is the canonical pathway. The canonical WNT signalling pathway is observed in several tissue and cell types⁽ Reference Clevers ^{16 )}, including colonic crypts, where it regulates cell proliferation, migration and differentiation⁽ Reference Bienz and Clevers ^{17 )}.

In the inactive state, or when activation of the pathway is inhibited by antagonists such as secreted frizzled-related proteins (SFRP), the β-catenin destruction complex, comprising adenomatous polyposis coli, AXIN, glycogen synthase kinase 3β and casein kinase 1, phosphorylates β-catenin (Fig. 1a)⁽ Reference Bienz and Clevers ^{17 ,} Reference Schneikert and Behrens ^{18 )}. Phosphorylated β-catenin is recognised by β-transducin repeat-containing protein which targets β-catenin for ubiquitination by the E3 ubiquitin ligase complex and, consequently, proteasomal degradation⁽ Reference Clevers ^{16 ,} Reference Schneikert and Behrens ^{18 )}. With low cytoplasmic concentrations of β-catenin, there is no translocation to the nucleus, transcription is not activated and the T cell factor and lymphoid enhancer factor-1 transcription factors are bound to, and inhibited by, Groucho and transducin-like enhancer of split. Activation of the canonical pathway occurs when a WNT ligand binds to a frizzled receptor, resulting in an interaction with co-receptors of the LDL receptor-related protein family (Fig. 1b). The β-catenin destruction complex is inactivated following inhibition of AXIN by dishevelled. Consequently, cytoplasmic levels of β-catenin rise and the protein is translocated to the nucleus where it binds to, and activates, T cell factor/lymphoid enhancer factor-1 transcription factors⁽ Reference Sierra, Yoshida and Joazeiro ^{19 )}, which switch on transcription of target genes such as c-MYC and c-JUN.

Fig. 1. The canonical WNT signalling pathway. (a) In the inactive state, when there are no WNT ligands bound to frizzled receptors or when WNT antagonists, such as WIF1, inhibit activation of the WNT pathway, the β-catenin destruction complex (adenomatous polyposis coli (APC), AXIN, casein kinase (CK1) and glycogen synthase kinase 3β (GSK3β)) binds to and phosphorylates β-catenin. This targets β-catenin for ubiquitination by β-transducin 117 repeat-containing protein (β-TrCP) and, consequently, proteasomal degradation. In the nucleus, lymphoid enhancer factor-1 (LEF) and T cell factor (TCF) transcription factors are repressed by Groucho. (b) When the WNT pathway is activated by binding of WNT ligands to frizzled receptors, the β-catenin destruction complex is inhibited by dishevelled (Dvl). Levels of dephosphorylated, active β-catenin rise and β-catenin translocates to the nucleus where it binds to LEF and TCF transcription factors to activate transcription of target genes such as c-MYC and c-JUN.

The WNT signalling pathway is also implicated in several cancers including CRC⁽ Reference Luo, Chen and Deng ^{20 )}, where it is constitutively activated in approximately 90 % of sporadic cases⁽ Reference Klaus and Birchmeier ^{21 )}. Aberrant expression of the WNT pathway results mainly from the loss of the APC tumour suppressor gene⁽ Reference Kinzler and Vogelstein ^{22 )}. However, genetic and epigenetic mechanisms also contribute to altered expression of other components of the pathway e.g. activating mutations in β-catenin⁽ Reference Morin, Sparks and Korinek ^{23 )} and down-regulation of SFRP1 due to promoter hypermethylation⁽ Reference Caldwell, Jones and Gensberg ^{24 )} and, therefore, to tumour development.

The effects of resistant starch on colorectal carcinogenesis

Studies investigating the effects of RS on colorectal carcinogenesis have yielded inconsistent results. While some studies have indicated a chemoprotective effect of RS, others have reported no effect and some have observed adverse effects.

In rats treated with 1,2-dimethylhydrazine dihydrochloride (DMH; a carcinogen used widely in experimental studies of CRC formation), incidence of tumours was reduced when the animals were fed Novelose 330 (a type 3 RS) compared with those fed a standard diet⁽ Reference Bauer-Marinovic, Florian and Muller-Schmehl ^{25 )}. High-amylose maize starch (HAMS; a type 2 RS) has also been shown to reduce incidence and the number of adenocarcinomas in rats when the HAMS diet was provided for 4 weeks prior to injection with the carcinogen azoxymethane⁽ Reference Le Leu, Brown and Hu ^{26 )}.

The formation of aberrant crypt foci (ACF), which are the earliest detectable pre-neoplastic lesions in CRC⁽ Reference Alrawi, Schiff and Carroll ^{27 )}, is a marker of early-stage colorectal carcinogenesis and has been used as a surrogate endpoint in several studies investigating colorectal carcinogenesis in animal models. Using an azoxymethane-induced model of ACF, rats given raw potato starch (a type 2 RS) for 3 weeks post carcinogen treatment had significantly reduced ACF formation compared with controls⁽ Reference Liu and Xu ^{28 )}. In contrast, rats fed moderate and high doses of RS prior to injection with azoxymethane had a significantly greater number of ACF compared with those on the control diet and those on the low dose of RS. These results suggest that RS may be protective if given during the promotion stage of colorectal carcinogenesis i.e. following carcinogen treatment, but may have adverse effects at the pre-initiation stage⁽ Reference Liu and Xu ^{28 )}. Consistent with findings from this study, two earlier animal studies reported protective effects of RS given during the promotion stage⁽ Reference Thorup, Meyer and Kristiansen ^{29 ,} Reference Cassand, Maziere and Champ ^{30 )}. Thorup et al. ⁽ Reference Thorup, Meyer and Kristiansen ^{29 )} reported that there were significantly fewer ACF in azoxymethane-treated rats fed a potato starch-containing (RS) diet compared with those fed control or corn starch-containing diets. Similarly, rats fed RS (with or without a vitamin A supplement) for 12 weeks post-DMH injection had significantly reduced ACF formation⁽ Reference Cassand, Maziere and Champ ^{30 )}.

Some studies have also reported no effect of RS on CRC in animal models. Although DMH-treated rats fed RS (3 or 10 %) had significantly increased butyrate concentrations compared with those fed control diets, there were no effects of RS on the development of CRC⁽ Reference Sakamoto, Nakaji and Sugawara ^{31 )}. Similarly, there was no effect on ACF formation in rats fed HAMS for 4 weeks prior to DMH injection⁽ Reference Maziere, Meflah and Tavan ^{32 )}.

In contrast, adverse effects of RS in promoting colorectal carcinogenesis have also been reported. Increased ACF density and colonic tumour formation were observed by Young et al. in DMH-injected rats fed potato starch for 31 weeks⁽ Reference Young, McIntyre and Albert ^{33 )}. It has been argued that carcinogen-treatment in animals may not be the best model for studies of human colorectal carcinogenesis and that genetic models which recapitulate aberrant WNT signalling because of Apc mutation may be more appropriate. Using the Apc1638N mouse model of sporadic CRC⁽ Reference Smits, Kartheuser and Jagmohan-Changur ^{34 )}, we observed that feeding RS (a 1:1 blend of raw potato starch and Hylon VII, both type 2 RS) for 5 months increased tumour formation within the small intestine⁽ Reference Williamson, Kartheuser and Coaker ^{35 )}. However, this adverse effect was prevented with the synergistic administration of aspirin, which suggests that reducing inflammation may reduce CRC risk.

The effects of resistant starch and butyrate on the expression of WNT pathway components

The effects of RS on the expression of WNT pathway components have been investigated in only one published study in which carcinogen-treated rats were fed diets containing three corn maize varieties: an Argentinian strain, a Guatemalan strain and a hybrid of the two⁽ Reference Cray ^{36 )}. Those animals fed the Argentinian strain, which yielded the lowest RS content, had enhanced β-catenin expression compared with those rats fed the hybrid diet. In addition, expression of the WNT antagonist, SFRP4, was significantly lower in rats fed the diets (Guatemalan strain and hybrid) with the highest RS content. Altogether, these results suggest that RS reduced WNT pathway activity in carcinogen-treated rats. However, this study also found that expression of two other negative regulators of WNT signalling, AXIN2 and WISP1, was significantly reduced following the consumption of the two higher RS diets, which would be indicative of reduced WNT pathway inhibition with RS. These observations underline the complexity of regulation of WNT signalling and the difficulty in drawing unequivocal conclusions.

In contrast with the paucity of studies with RS and WNT signalling, there are several studies, notably from the Bordonaro group, which have investigated the effects of butyrate on the expression of WNT pathway components. These investigators observed that treatment of SW620 colon carcinoma cells with 5 mm butyrate (a physiological concentration) induced apoptosis and that this was associated with increased activity of the T cell factor transcription factor indicating that the apoptotic effect may have been mediated through effects on WNT signalling⁽ Reference Bordonaro, Mariadason and Aslam ^{37 )}. Furthermore, butyrate treatment increased formation of β-catenin–T cell factor complexes, which would activate transcription of target genes. A later study by the same group found that butyrate increased the levels of unphosphorylated, and therefore active, β-catenin following butyrate treatment in eight CRC cell lines⁽ Reference Bordonaro, Lazarova and Sartorelli ^{38 )}.

Other studies have investigated effects of butyrate on another functional outcome of the WNT pathway i.e. differentiation. There is an evidence that butyrate treatment of LIM2537 colon cancer cells induces WNT signalling and that this is paralleled by greater differentiation of the, usually, poorly differentiated tumour cells⁽ Reference Vincan, Leet and Reyes ^{39 )}. The activity of glycogen synthase kinase 3β, a member of the β-catenin destruction complex, was significantly reduced in the butyrate-treated cells and, indeed, this led to stabilised pools of β-catenin within the cytoplasm which would be expected to increase WNT activity. There was an inverse correlation between glycogen synthase kinase 3β expression and differentiation, suggesting that the induction of differentiation by butyrate resulted from an increase in WNT signalling. Unexpectedly, this was not paralleled by an increase in the expression of two target genes of the WNT pathway, c-MYC and CCND1.

Germann et al. observed that 4·5 mm butyrate treatment of CC531 rat colon carcinoma cells resulted in positive modulation of expression of four WNT pathway target genes (CCND1, c-MYC, FOSL1 and FST), which have been reported to be up-regulated in CRC⁽ Reference Wong and Pignatelli ^{40 –} Reference Germann, Dihlmann and Hergenhahn ^{42 )}. However, the effects of butyrate on c-MYC expression are complex. While increased c-MYC transcription is observed with butyrate-induced WNT signalling, this treatment may result in less c-MYC expression through inhibition of elongation causing a transcriptional block⁽ Reference Wilson, Velcich and Arango ^{43 )}.

Studies using LT97 (a cell line representative of an early stage in the progression from the normal mucosa to adenomas) showed greater up-regulation of WNT signalling and, consequently, induction of apoptosis with butyrate treatment suggesting that early-stage neoplasms may be more responsive to the effects of butyrate⁽ Reference Lazarova, Lee and Wong ^{44 )}.

A recent study by the Bordonaro group utilised total human microarray analyses to identify a total of 1587 direct and indirect targets of the WNT pathway whose expression was modulated by a physiologically-relevant concentration of butyrate (5 mm) in HCT-116 CRC cells⁽ Reference Lazarova, Chiaro and Bordonaro ^{45 )}. The differentially-expressed genes included those encoding proteins that are involved in several key processes including differentiation, migration and DNA replication.

Modulation of WNT signalling by resistant starch and butyrate via epigenetic mechanisms

Gene expression may be regulated by epigenetic mechanisms, such as histone modifications, DNA methylation and the expression of microRNA (miRNA)⁽ Reference Mathers, Strathdee and Relton ^{46 )}. These mechanisms result in heritable changes in gene expression and function without alterations in the DNA sequence itself⁽ Reference Dupont, Armant and Brenner ^{47 )}.

The effects of resistant starch and butyrate on colonic crypt cell proliferation

Studies on the effects of RS and butyrate on crypt cell proliferation have yielded conflicting results. This divergence in findings is due, in part, to differences in the health status of the tissue under study. In some cases, mucosal cells from the normal colon respond to butyrate (and other SCFA) by increasing proliferation. In contrast, there is almost universal agreement that butyrate treatment of cancer cells suppresses proliferation⁽ Reference Williams, Coxhead and Mathers ^{12 )}. Furthermore, differences in RS or butyrate dose, type of RS, participant/cell characteristics and length of treatment may influence responses and contribute to difficulties in drawing unambiguous conclusions from available data. For example, we have shown that the DNA mismatch repair status of cells determines their cell proliferative/apoptotic response to butyrate treatment⁽ Reference Dronamraju, Coxhead and Kelly ^{63 )}.

Findings from studies that have investigated the effects of RS on colonic crypt cell proliferation are summarised in Table 1. The majority of the studies that have investigated the effects of RS supplementation on cell proliferation in the colon of healthy subjects have found no effect on proliferation. This includes the study by Wacker et al. ⁽ Reference Wacker, Wanek and Eder ^{64 )} which administered the largest dose of RS (up to 59·7 g /d). In contrast, a number of studies have reported reduced cell proliferation in the colorectum of individuals with neoplasia⁽ Reference Bauer-Marinovic, Florian and Muller-Schmehl ^{25 ,} Reference Le Leu, Brown and Hu ^{26 )}. Importantly, we have also shown that supplementation of CRC patients with a 1:1 blend of Novelose 240 and Novelose 330 (RS types 2 and 3) reduced the proportion of mitotic cells in the top half of the crypt⁽ Reference Dronamraju, Coxhead and Kelly ^{65 )}. Study of the distribution of mitotic cells within the crypt (rather than measuring total proliferation within the whole crypt) may be a better indicator of CRC risk because alterations in the distribution of mitotic cells within the crypt have been observed to be one of the earliest detectable pre-malignant alterations in the apparently-normal mucosa of those at higher risk⁽ Reference Mills, Shepherd and Hall ^{66 ,} Reference Terpstra, van Blankenstein and Dees ^{67 )}.

Table 1. Studies that have investigated the effects of resistant starch (RS) on colonic crypt cell proliferation

HAMS, high amylose maize starch; HAMSB, butyrylated high amylose maize starch.

Likewise, the findings from investigations of the effects of butyrate on colonic crypt cell proliferation are inconsistent. While there is an evidence that butyrate may increase cell proliferation in the healthy colon in specific circumstances ⁽ Reference Scheppach, Bartram and Richter ^{68 ,} Reference Kien, Blauwiekel and Bunn ^{69 ,} Reference Mortensen, Langkilde and Joergensen ^{70 )}, based on our earlier observations in rats, we concluded that there is no convincing evidence that SCFA (or butyrate, specifically) are responsible for raising crypt cell proliferation above normal. In those instances where greater SCFA supply has been associated with increased crypt cell proliferation, the increase may be (1) from a hypoproliferative state towards a normal proliferative state, (2) a transient phenomenon accompanying tissue hypertrophy or (3) a homeostatic response to increased cell loss by cell sloughing or apoptosis⁽ Reference Key, McClean and Mathers ^{71 )}.

The differential responses of normal and cancer cells to butyrate treatment are referred to as the butyrate paradox. Comalada et al. ⁽ Reference Comalada, Bailon and de Haro ^{72 )} compared the effects of butyrate treatment on healthy fetal human colon cells and on HT-29 colorectal adenocarcinoma cells. Butyrate inhibited cell proliferation of the HT-29 cells but had no effect on the normal cells. Significantly reduced cell proliferation of HT-29 cells treated with 5 mm butyrate for 48 h has also been reported by Hodin et al. ⁽ Reference Hodin, Meng and Archer ^{73 )}.

Bordonaro et al. ⁽ Reference Bordonaro, Lazarova and Sartorelli ^{15 )} have suggested that the differences in the effects of butyrate observed on proliferation in healthy compared with cancerous cells may be due to the sensitivity and responsiveness of these cells to butyrate. They proposed that, in cancerous cells where the WNT pathway is hyperactive, butyrate further induces WNT signalling and consequently apoptosis. However, in healthy cells where normal, moderate levels of WNT activity are found, butyrate contributes to the normal regulation of processes within the colon by the WNT pathway, such as the induction of proliferation. However, the latter is likely to increase only if starting from an abnormally low level⁽ Reference Key, McClean and Mathers ^{71 )}.

The effects of resistant starch and butyrate on apoptosis in the large bowel

A number of studies, mostly in vivo, have shown that increased WNT activity is associated with induction of apoptosis⁽ Reference Bordonaro, Mariadason and Aslam ^{37 ,} Reference Romagnolo, Berrebi and Saadi-Keddoucci ^{74 ,} Reference Wong, Huelsken and Birchmeier ^{75 )}. Furthermore, the Bordonaro group have reported a linear relationship between WNT activity and levels of apoptosis in ten CRC cell lines⁽ Reference Lazarova, Bordonaro and Carbone ^{76 )}. However, several in vitro studies have reported the opposite effect⁽ Reference Groden, Joslyn and Samowitz ^{77 –} Reference Zhang, Otevrel and Gao ^{79 )}.

In sixteen pigs supplemented with raw potato starch for 16 weeks, Nofrarias et al. ⁽ Reference Nofrarias, Martinez-Puig and Pujols ^{80 )} observed a significant reduction in apoptosis within the crypts of pigs fed the RS diet compared with controls. A similar earlier study also reported a reduction in apoptosis in the colon of pigs fed a potato starch diet and, interestingly, these researchers reported significantly higher faecal butyrate concentrations following the RS diet⁽ Reference Claus, Losel and Lacorn ^{81 )}.

The majority of studies have agreed that butyrate induces apoptosis⁽ Reference Armstrong and Mathers ^{82 )}, primarily via induction of the intrinsic and extrinsic apoptotic pathways. Furthermore, butyrate has been reported to modulate the expression of apoptotic genes including down-regulation of the anti-apoptotic gene BCL-2 ⁽ Reference Avivi-Green, Polak-Charcon and Madar ^{83 )} and up-regulation of the pro-apoptotic gene BAX ⁽ Reference Purwani, Iskandriati and Suhartono ^{84 )}. In vivo, increased apoptosis has been observed in both the distal and proximal colon of carcinogen-treated rats fed a diet with RS type 3⁽ Reference Bauer-Marinovic, Florian and Muller-Schmehl ^{25 )}. Furthermore, significantly greater levels of apoptosis have been found in the distal colon of carcinogen-treated rats fed a butyrylated high amylose maize starch, which produces significantly greater concentrations of butyrate in the colon, compared with HAMS and a low RS diet⁽ Reference Clarke, Young and Topping ^{85 )}. In addition, this increase in apoptosis correlated positively with distal colonic luminal butyrate concentrations, suggesting that the enhanced apoptosis was a consequence of the extra butyrate production. However, some studies have reported no effect of butyrate on levels of apoptosis in the colon⁽ Reference Winter, Young and Hu ^{86 ,} Reference Le Leu, Hu and Brown ^{87 )}.