Review
Mechanisms of primary cancer prevention by butyrate and other products formed during gut flora-mediated fermentation of dietary fibre

In respectful memory of Prof. Dr. Beatrice L. Pool-Zobel.
https://doi.org/10.1016/j.mrrev.2009.04.001Get rights and content

Abstract

Dietary fibres are indigestible food ingredients that reach the colon and are then fermented by colonic bacteria, resulting mainly in the formation of short-chain fatty acids (SCFA) such as acetate, propionate, and butyrate. Those SCFA, especially butyrate, are recognised for their potential to act on secondary chemoprevention by slowing growth and activating apoptosis in colon cancer cells. Additionally, SCFA can also act on primary prevention by activation of different drug metabolising enzymes. This can reduce the burden of carcinogens and, therefore, decrease the number of mutations, reducing cancer risk. Activation of GSTs by butyrate has been studied on mRNA, protein, and enzyme activity level by real-time RT-PCR, cDNA microarrays, Western blotting, or photometrical approaches, respectively. Butyrate had differential effects in colon cells of different stages of cancer development. In HT29 tumour cells, e.g., mRNA GSTA4, GSTP1, GSTM2, and GSTT2 were induced. In LT97 adenoma cells, GSTM3, GSTT2, and MGST3 were induced, whereas GSTA2, GSTT2, and catalase (CAT) were elevated in primary colon cells. Colon cells of different stages of carcinogenesis differed in post-transcriptional regulatory mechanisms because butyrate increased protein levels of different GST isoforms and total GST enzyme activity in HT29 cells, whereas in LT97 cells, GST protein levels and activity were slightly reduced. Because butyrate increased histone acetylation and phosphorylation of ERK in HT29 cells, inhibition of histone deacetylases and the influence on MAPK signalling are possible mechanisms of GST activation by butyrate. Functional consequences of this activation include a reduction of DNA damage caused by carcinogens like hydrogen peroxide or 4-hydroxynonenal (HNE) in butyrate-treated colon cells. Treatment of colon cells with the supernatant from an in vitro fermentation of inulin increased GST activity and decreased HNE-induced DNA damage in HT29 cells. Additional animal and human studies are needed to define the exact role of dietary fibre and butyrate in inducing GST activity and reducing the risk of colon cancer.

Introduction

Now a decade has passed since the publication of the first report of the World Cancer Research Fund on “Food, Nutrition and the Prevention of Cancer: A Global Perspective” [1]. With this report a new dimension of the field came into view because the accumulated evidence was showing that cancer and diet were strongly interrelated. Moreover, on account of the possibility to adjust diet, it was deemed also possible in the long run to prevent diet-related cancers. Because the amount of available literature on this topic has significantly increased within 10 years, just recently the second edition of this expert report was published [2]. While many of the described aspects have been refined in the update due to the large amount of new literature, the general picture that adjustment of diet could prevent some cancers is maintained. Of particular interest in this context was, and still is, colorectal cancer, of which a significant proportion is diet or exposure related. Avoidable risk factors are considered to be diets rich in meat, especially in conjunction with genetic predisposing genotypes [3], [4], [5], [6]. Opposed to this, healthy behaviours such as the consumption of high quantities of vegetables, fruits, or dietary fibres, coupled to physical exercise have been shown to be associated with reduced risk [7], [8]. The epidemiological evidence in humans is supported by animal studies which have shown e.g. that dietary haem increased faecal cation content, cytolytic activity of faecal water and colonic epithelial proliferation in rats [9], a property that can be inhibited by dietary calcium [10]. Furthermore, Pierre et al. reported that diets with meat promoted the formation of aberrant crypt foci (ACF) in azoxymethane treated rats on low calcium diets and that this was associated with high concentrations of lipoperoxides in the faeces and faecal water cytotoxicity [11]. In F344 female rats fed with N-nitroso-N-methylurea it was shown that the simultaneous feeding of a fat diet and haem-iron caused a significant increase in the incidence of colon cancer compared to a diet without haemoglobin [12]. On the other hand, Xu and Dashwood summarised results from ACF and tumour bioassays which demonstrated that constituents of tea, green vegetables, cruciferous vegetables and dairy products all have a potential to protect against meat-related heterocyclic amines which are colon carcinogens [13], [14]. Additionally, the results of Vogel et al. showed that green vegetables in the diet of rats significantly reduce the risk of meat-induced colorectal cancer [15]. Reviews on the potential of dietary and other agents to act chemopreventive in animal systems are available by Bruce and Corpet, both pioneers in the development of appropriate experimental models [16], [17].

Increasingly, however, doubt is cast on the strength of reported inverse relationships, since recent human studies are not showing, for instance, that fruit and vegetable intakes, the major sources of dietary fibre, are associated with a reduced cancer incidence in general and in particular with reduced colorectal cancer risk [18], [19]. Moreover, intervention with a diet low in fat and high in dietary fibre was not shown to decrease the intermediate marker of polyp recurrence [20]. Based on the knowledge of mechanisms of action of fibre from experimental and animal studies, it is therefore becoming apparent that more detailed studies are needed on how different types of foods and dietary fibres contribute to gut health and how they may act on a molecular basis. In particular, it is of interest to better understand overall metabolism, the roles of the products formed during the process of gut fermentation and how these products interact with each other in a chemopreventive mode of action with the colon mucosa. Consequently, it is logical to postulate that beneficial effects are possibly not only mediated by “vegetables and fruits” or “dietary fibres from plant foods” in general, but also by specific types of dietary fibres that are beneficially fermented by the gut flora to yield products that contribute to chemoprotection. In other words, it is now recognised that the traditional chemoprotective role of dietary fibre, which formerly consisted of faecal bulking [21], rapid transit [22] and enhanced defaecation, may have added benefits. These purported health promoting properties could include the so called prebiotic activity [23], putatively encompassing cell protective effects of particular antioxidants that can be liberated in the colon after fermentation by the gut flora [24], [25]. A prebiotic was first defined as “a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health” [23]. After the first definition of prebiotics, a lot of research on these carbohydrates has been done leading to clear criteria that characterise prebiotics, namely (1) resistance to gastric acidity, to hydrolysis by mammalian enzymes, and to gastrointestinal absorption; (2) fermentation by intestinal microflora; and (3) selective stimulation of the growth and/or activity of those intestinal bacteria that contribute to health and well-being [26]. Hence, prebiotics can have an impact on gut health in general, and are believed to play an important role in the prevention of colorectal cancer, as has been highlighted by a recent review [27].

In this context, results of a recent study by Schatzkin et al. are of interest [28]. The authors aimed to investigate the relation between dietary fibre and whole-grain food intakes and the incidence of invasive colorectal cancer in the prospective National Institutes of Health—AARP Diet and Health Study. Key results were that total dietary fibre intake was not associated with colorectal cancer, whereas whole-grain intake was inversely associated with colorectal cancer risk. This indicates a particular role of whole grains as source of dietary fibres (and of other gut health promoting ingredients) for which it will be of interest to better understand molecular mechanisms of activities [29]. Examples of the outstanding prebiotic types of fibres in this context are inulin, oligofructoses and related fructans—in short, inulin-type fructans. It is however important to note that it depends on the definition whether these inulin-type fructans can be classified as dietary fibres. Originally dietary fibres were defined as the remnants of the plant cell wall that are not hydrolysed by the alimentary enzymes of man, which included mainly non-starch polysaccharides and does not include inulin. Since this definition is still accepted in some parts of the world, inulin is not classified as dietary fibre worldwide. Anyway, since newer definitions in many parts of the world are based on physiological properties and thus include more or less all carbohydrates (typically with a degree of polymerisation ≥3) that are resistant to digestion, inulin-type fructans are classified as dietary fibres in many countries. Inulin-type fructans (β(2-1)fructans) are extracted from chicory roots (Cichorium intybus) and prepared to be added to various types of food. They are also present in a number of foods, such as garlic, onion, artichoke and asparagus as natural ingredients. Their average consumption in the normal human diet has been evaluated to amount to several grams per day [30]. They are defined to be prebiotic food ingredients since they selectively increase growth of bifidobacteria, which have anticancer potential [31], or enhance formation of short-chain fatty acids (SCFA), such as acetate, butyrate, and propionate. Of these, butyrate, and propionate also have beneficial properties [32], [33]. In non-transformed cells, butyrate is utilised as an energy source [34] whereas in tumour cells butyrate reduces survival by inducing apoptosis and inhibiting proliferation [35]. Thus butyrate most likely acts on secondary chemoprevention by reducing the number of cells in cancerous lesions and thereby slowing or inhibiting formation of malignant tumours.

Cancer chemoprevention is characterised by the use of natural, synthetic, or biologic (from a living source) substances to reverse, suppress, or prevent the development of cancer [36]. The underlying principles of chemoprevention are summarised in Fig. 1. Basically three different phases of prevention can be distinguished, namely primary prevention, secondary prevention and therapy. Primary prevention describes the inhibition of initiation, the first step of tumourigenesis by reduction of toxification or induction of detoxification. This can be accomplished e.g. by preventing the formation of ultimate carcinogens or reactive oxygen species as well as by antioxidative effects and is also called blocking activity. The promotion of initiated cells to preneoplastic cells is inhibited by secondary prevention, e.g. by reduction of cell growth or enhancement of differentiation and apoptosis in initiated cells. Agents that affect secondary prevention are suppressing agents. Blockage of progression of preneoplastic cells to neoplastic cells, which occurs comparably late during carcinogenesis, is termed tertiary chemoprevention and includes therapeutic approaches.

The role of butyrate in secondary chemoprevention is subject of numerous studies and has been extensively reviewed [37], [38]. Another mechanism of chemoprotection by fermentation products, especially by butyrate, has been hypothesised to be the induction of glutathione-S-transferases (GSTs) and other stress response genes [39]. This action of complex fermentation products and butyrate on primary chemoprevention is the subject of the present review.

GSTs [EC Nr 2.5.1.18] are enzymes of biotransformation that detoxify many carcinogens [40]. The increased cellular levels of such enzyme systems has been shown to protect against food-derived genotoxic compounds such as 4-hydroxynonenal (HNE) in tumour-derived cell lines [41]. Similar mechanisms occurring in non-transformed cells may very well reduce cancer initiation and thus be considered an effective means of primary cancer chemoprevention [42], since GSTs are capable of detoxifying endogenous and exogenous (food- or smoking-derived) carcinogens like HNE or benzo(a)pyrene [43].

There have already been a number of reviews on SCFA and their role in gut health. Cummings has reported of SCFA occurrence in the colonic lumen and in hepatic tissues decades ago [32], [44]. Csordas wrote a comprehensive compilation on the toxicology of these compounds [45], while other examples of reviews on SCFA and gut health were by Mortensen and Claussen [46], Topping and Clifton [47], as well as by Augenlicht et al. [48]. Some of the findings have led to a number of critical editorials highlighting controversies that pertain to opposing effects of butyrate in vitro and in vivo or to findings on its different types of activities in non-transformed versus transformed colon cells [49], [50], [51]. The main purpose of the following review is to compile own laboratory experiments on the effects of butyrate, and if available, on gut fermentation products from dietary fibres (especially from inulin), in different colonocyte cultures in vitro. Hereby the focus is on recent findings of modulated gene or protein expression, in particular of genes and proteins related to drug metabolism and to stress response. Newer publications on effects of butyrate by other authors will also be reviewed in the context of these objectives. Thus, this review will gather data on the butyrate-mediated induction of GSTs and other drug metabolising enzymes. Excellent reviews on GSTs have been published by Hayes [40], [43], [52] and others [53], [54], [55], [56]. More specific articles on the roles of GSTs in nutrition and their expression levels in human cells or their particular relation to factors of colon cancer are also available [39], [57], [58], [59], [60], [61], [62], [63], [64], [65], as are reviews on the anticancer properties of inulin-type fructans [66], [67], which is another focus of the present report.

Section snippets

Experimental approaches to study effects of butyrate in human colon cells in vitro

It has been of interest to explore whether butyrate could contribute to chemoprotection in colon cells not only by reducing growth of tumour cells [35], committing them to more rapidly go into apoptosis [68], serving as survival factor for normal non-transformed colon cells [34], [50] or enhancing mucin synthesis [69], but also via the mechanism of favourably altering patterns of drug metabolism [70]. For this, several of our studies have first dealt with finding tolerable and toxic

GST-gene activation by butyrate may be mediated by different mechanisms

A number of different promoter elements and transcription factors are involved in regulation of GST gene activation. The best studied mechanism of GST gene activation involves the antioxidant/electrophile-responsive element (ARE/ERE), a promoter element which can be found in the 5′ upstream region of many rodent GST genes, e.g. mouse GSTA1, rat GSTA2 and rat GSTP [87]. Different members of the helix-loop-helix basic leucine zipper (bZIP) family of transcription factors, including Nrf, Jun, Fos,

DNA damage caused by H2O2 and HNE was reduced by butyrate pre-treatment

The prevention or inhibition of genotoxic effects by DNA damaging agents is collectively called “antigenotoxicity”. To explore this experimentally in cell culture, different mechanisms and experimental protocols are feasible. We used a new treatment protocol to depict different types of effects on genetic damage and DNA repair [112]. For this, the putative chemoprotective is added to 3 phases of an experimental protocol originally developed for molecular epidemiological studies [113]. Phase a:

Effects of complete gut fermentation samples better reflect in vivo exposure conditions than butyrate alone

Dietary fibres reach the colon unaltered, where they are fermented by the gut flora to yield products such as SCFA. Of these, butyrate is physiologically relevant to the colonic epithelium in which it serves as a principle energy source [131]. Interest in its role as a possible protective agent has arisen from its properties to inhibit survival of cells in vitro [35], including colon tumour cell lines [68], [132]. Other findings show that it also protects from H2O2-induced genetic damage in

Summary and conclusions

It is well known that nutrition leads to a considerable burden of toxic and genotoxic factors in the gut lumen. Faecal samples for instance have been shown to contain bile acids, amines, sulfates, bacterial toxins as well as additional products of bacterial biotransformation, non-digested food residues, excretable metabolites, and toxic compounds.

Beneficial effects of fibres are seen in their ability to shorten the transit time of the faeces in the gut and thus reduce exposure to the toxins.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

Work on primary cells was supported by the German Research Council, Deutsche Forschungsgemeinschaft, Germany (DFG PO 284/8-1). The work on LT97 cells was supported by the German Research Council, BMBF, Germany (FKZ 01EA0103) and the work on HT29 cells by ORAFTI, Tienen, Belgium (PRECANTOO). The intervention study with synbiotics was supported by EU (QCRT-1999-00346). Research on prebiotics was supported by the German Research Council, BMBF (FKZ 01EA0503 + PTJ-BIO/0313829C/D’O), Deutsche

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