Keywords

1 Introduction

“Let food be your medicine and medicine be your food”. With this statement, almost 2500 years ago Hippocrates offered to human population a beautiful recommendation dressed up like a medical prescription that still appears contemporary in our present era. Without having to disturb the first physicians and scientists in the history which, however, understood the medicinal properties of foods (Fenwick and Heaney 1983), it should be noted that, in the last decades, the scientific research paid particular attention on the impact of diet, in general, or such foods, specifically, on both several pathological conditions or maintenance of the wellness. In this sense, we could mention the first studies on the Mediterranean diet and the prevention of cardiometabolic diseases, as well as the previous studies on the bioactive compounds contained in red wine that led to the well-known French paradox. All this is to say that, besides the extraordinary researches in the pharmaceutical field, a relevant piece of evidence supports the potential benefits of natural products, mainly food-deriving bioactive compounds, in both prevention and management of various pathological conditions.

Notably, in the 1989 Professor Stephen DeFelice finalized the concept of the strong interplay between diet and health coining the term “nutraceutical”, as portmanteau of the words “nutrition” and “pharmaceutical”, identifying the pharmacological potential of food-derived compounds (Santini and Novellino 2014). It should be stressed that this is a science studying the effects of bioactive compounds from animal or vegetal foods on human health, following a pharmacological approach. It means that nutraceutical should not be confused with further alternative or non-conventional practices, since the single components of foods are investigated with a meticulous scientific rigour, properly designing in vitro and in vivo studies and providing strong evidence. Interestingly, nutraceuticals are considered as supplements containing food-derived extracts, concentrated and administered in a suitable pharmaceutical form, highlighting once more the pharmaceutical meaning of nutrition. Overall, thus, the careful analysis of the available literature in this field produced in the last 10–15 years, allows considering the nutraceutical as a useful tool to contrast the development of several diseases, acting “Beyond the diet, before the drugs” as stated by Professor Ettore Novellino in 2012 (Santini and Novellino 2014).

This current shift of the scientific interest from the mere study of the drugs to the evaluation of further and eventually natural therapeutic approaches should not be interpreted as a new direction operated by the researchers, but rather as a novel route that run alongside. And it should not scare off, since this just represents the natural progress of the science. We would like to give an example that might sounds like as an off-topic, but may help to understand the importance to keep an open mind when approaching the scientific research.

Until not many decades ago it was thought that Universe consisted solely of ordinary matter, which constitutes stars, planets, gasses and cosmic dust. During ‘70s, several studies and researches provided a novel interpretation of the Universe, proving the existence of both dark matter and dark energy. Interestingly, it was proved that dark matter and dark energy constitute the majority of the Universe, whereas ordinary matter only represent a limited part. These findings opened the way to new fields of application in theoretical physic and a novel comprehension of the complexity of the Universe. This fascinating travel into the galaxy perfectly suggests that static certainties are not admitted in sciences, and that research is a dynamic process that continually remodels the previous discoveries, providing novel interpretations. In this context, medical science is not exempt from this continuous progress.

Coming back to the focus of this chapter, also the research in the tumour field deeply investigated the protective role of such diets or foods in prevention of various cancers (Verhoeven et al. 1996), specifically focusing on vegetable consumption. Among these, cruciferous gained a particular interest since their regular consumption has been linked to reduced development of different kinds of cancers, including colorectal, breast, kidney, and upper digestive tract cancers (Bosetti et al. 2012). Although the exact mechanisms by which these vegetables are able to counteract the processes of carcinogenesis are still unclear, many studies clarified the possible signalling pathways in which the bioactive compounds of cruciferous vegetables may be involved.

In the following sections of the present chapter we will offer you an initial overview on the cruciferous vegetables, in terms of botanical and chemical features, with a focus on the human metabolism of their bioactive compounds. Subsequently, we will discuss the relationship occurring among these vegetables and cancer, reporting the main putative anticancer mechanisms of action and signalling pathways.

2 Cruciferous Vegetables

In this section we will explore the Cruciferous vegetable world, describing in detail the botanical features and their bioactive compounds, in terms of chemical features and human metabolism after ingestion.

2.1 Botanical Features

Cruciferous are vegetables belonging to the botanical family Brassicaceae , order Capparales, in which sixteen families are included (National Resource Conservation Services 2020). The Brassicaceae family is very large, containing about 3000 species in 350 genera, including a large number of edible plants, such as B. oleracea (i.e. cabbage, cauliflower, broccoli, Brussels sprouts), B. rapa and B. napus (including Chinese cabbage and rape), and other genera including radish and cress. The main cruciferous vegetables used in the human diet are listed in Table 7.1.

Table 7.1 Main Cruciferous vegetables used in the human diet
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Generally, the terms Cruciferae or Cruciferaceae refer to the so-called “cabbage family” or “mustard family”, as the latin term “Brassica” means cabbage. The petals of the Brassicaceae family present a peculiar cruciform arrangement, from which it derives its name. These plants can be annuals, biennals or perennials. Due to their good adaption to mild temperatures ranging 16–18 °C, these plants can grow in temperate areas, during the cool season. In particular, Brassicaceae are cultivated in the Mediterranean region, Europe, North America and Southwest and Central Asia (National Resource Conservation Services 2020).

2.2 Chemistry of Bioactive Compounds

Similar to other vegetables, Cruciferous vegetables are a good nutrients and bioactive compounds source, including fibre, folate, carotenoids and chlorophyll, which chemopreventive properties have been demonstrated. Among these, glucosinolates deserve to be discussed more in detail.

2.2.1 Glucosinolates

Glucosinolates are the most abundant phytochemicals contained in Cruciferous vegetables, and they are responsible for their pungent aroma and spicy taste (Drewnowski and Gomez-Carneros 2000). In plants, they derive from valine, alanine, leucine, phenylalanine, isoleucine, methionine, tryptophan and tyrosin (IARC 2004) with a complex biosynthetic process involving the formation of aldoximes as intermediate, the oxidation of the N-hydroxy-amino acids and the subsequent decarboxylation and glucosidation reactions of the thiol function. The so-produced glucosinolates may be involved in further transamination, condensation, isomerisation and decarboxylation reactions, resulting in the synthesis of different structures (Popolo et al. 2017). The chemical structures of the main cruciferous vegetables are represented in Fig. 7.1.

Fig. 7.1
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Chemical structures of Cruciferous vegetables bioactive compounds

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Glucosinolates are also subject to hydrolytic processes operated by such enzymes, mainly myrosinases, leading to the formation of further bioactive compounds, including indoles and isothiocyanates (Holst and Williamson 2004). In particular, myrosinase is a β-thioglucosidase catalysing the hydrolysis of the S-glucoside giving an aglycone and the D-glucose; the aglycone, in turn, rearranges giving sufate and other compounds, such as nitriles, isothiocyanates, thiocyanates and epithionitriles (Hayes et al. 2008). Among these, one of the most widely distributed glucosinolates is the indole-3-ylmethylglucosinolate, starting from which both indole-3-acetonitrile and indole-3-carbinol (I3C) are produced. Although these reactions occur spontaneously, glucosinolates and myrosinases are naturally stored in different compartments of the plant cell, and they can meet only when the cell structure is ruptured. This physical separation is an evolutionary adaptation aimed to preserve the plant integrity protecting from cell damages, since the glucosinolate breakdown is toxic to both herbivores and pathogens (Katz et al. 2018).

Interestingly, the glucosinolate profile may be severely affected by such cooking procedures (Palermo et al. 2014), including boiling that inactivates the myrosinases (Campbell and Slominski 1990). Moreover, since most glucosinolates are thermally stable and the myrosinases can be inactivated by high temperature, the formation of isothiocyanates and indoles might be avoided by such cooking procedures; however, both chopping and chewing cause a cell rupture leading glucosinolates and myrosinases come in contact (Higdon et al. 2007). In this context, it should be taken into account that glucosinolates are water-soluble, thus they may be lost into the cooking water. 9–15 min-boiling process of cruciferous vegetables, indeed, causes a total glucosinolates content decrease around the 18–59% (McNaughton and Marks 2003); in this sense, the use of such cooking methods including steaming or microwaving should be encouraged in order to reduce the glucosinolate losses (Higdon et al. 2007).

More than a hundred glucosinolates have been identified in plants, leading to unique hydrolysis products. As examples, broccoli is a rich source of glucoraphanin and glucobrassicin, which are precursors of sulforaphane and I3C, respectively. On the other hand, watercress is a source of gluconasturtiin, the precursor of phenethyl isothiocyanate (Higdon et al. 2007; Popolo et al. 2017).

2.3 Metabolism of Bioactive Compounds

Once ingested throughout the diet, cruciferous vegetable-derived bioactive compounds follow the physiological gastrointestinal digestion, thus they undergo several physic-chemical and biochemical conditions naturally occurring during these processes, which may severely affect both their chemical features and absorption rate. Overall, the metabolic fate of these compounds can be summarised as following described at different levels:

  • Stomach : at the gastric acid pH, glucosinolate-derived products can combine with each other forming complexes of bioactive compounds, also known as acid condensation products (Shertzer and Senft 2000), including dimer 3,3′-diindolylmethane and a cyclic trimer, which biological activities differ from those of glucosinolate-derived products (Bjeldanes et al. 1991; Anderton et al. 2004a)

  • Upper intestine: at the intestinal neural pH, the major glucosinolate hydrolysis products are stable isothiocyanates; in contrast, β-hydroxy-isothiocyanates are unstable and undergo spontaneous processes leading to the formation of further compounds, including oxazolidine-2-thiones or alcohols (Higdon et al. 2007)

  • Lower intestine: the enzymatic activities of the human microbiota may contribute to further metabolise glucosinolates, forming hydrolysis products (Shapiro et al. 1998).

Additionally, the main pharmacokinetic features may be summarised with the so-called ADMET scheme (abbreviation for absorption, distribution, metabolism, excretion and toxicity).

  • A – absorption: glucosinolate-derived products are quickly absorbed; isothiocyanates can be absorbed by both upper and lower intestine (Popolo et al. 2017)

  • D – distribution: after the absorption, glucosinolate-derived products are distributed in high-vascularised tissues, such as heart, kidney, lung, liver and brain (Anderton et al. 2004b)

  • M – metabolism: after the intestinal absorption, glucosinolate-derived products undergo complex metabolic processes. Firstly, isothiocyanates are conjugated to glutathione, forming complexes that are further metabolized to mercapturic acid through the activities of various enzymes, including γ-glutamyltranspeptidase, cysteinylglycinase and N-acetyltransferase (Higdon et al. 2007)

  • E – excretion: glucosinolates and their hydrolysis products have a low half-life, due to the quick renal clearance, the rapid inactivation, the low molecular weight and the low water solubility. However, glucosinolate-derived metabolites have been detected in urine (Popolo et al. 2017)

  • T – toxicity: at high dose may be laxative. As a major concern, prolonged exposure to elevated amounts of glucosinolates may cause cytotoxicity and genotoxicity, resulting in dysfunctions at renal, thyroid gland, liver and pancreas level (Stoewsand 1995; Kassie et al. 1999; Holst and Williamson 2004). These toxic doses, however, are not reachable with the dietary consumption.

3 Cruciferous Vegetables and Cancer

Over the years, cruciferous vegetables gained great interest by the scientific research due to their potential chemopreventive effect. In this sense, one of the main challenges in investigating the relationship existing between the consumption of cruciferous vegetable and the risk of cancer in humans is related to the need to separate the benefits deriving by a general vegetable rich diet from those deriving directly by those rich in cruciferous vegetables (McNaughton and Marks 2003). However, evidence reports that the regular cruciferous vegetable consumption is linked to reduced rates of incidence of cancer (IARC 2004). In particular, epidemiologic studies published in the last century evidenced an inverse association between the consumption of some type of cruciferous vegetables and the risk of developing such types of cancer (Verhoeven et al. 1996), including lung, digestive tract, breast and prostate cancers (Higdon et al. 2007; Popolo et al. 2017). In general, these chemopreventive effects are attributable to the phytochemicals contained in this class of vegetables, acting via different mechanism of action, as will be discussed in the next section. In Table 7.2 the main evidences reporting the association between the consumption of cruciferous vegetables and cancer risk are summarised.

Table 7.2 Evidence reporting the role of Cruciferous vegetable in cancer
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In addition to these studies investigating the effect of cruciferous consumption on the cancer risk, interesting evidence reported the existence of such human genetic differences that may play a role in modulating the chemopreventing potential of these vegetables (Lampe and Peterson 2002). In particular, genetic polymorphisms affecting the enzymatic activities of glutathione S-transferases (GST ; a family of enzymes metabolizing various compounds, including isothiocyanates, resulting in enhancing their excretion) may severely affect the metabolism of glucosinolate-deriving products. More specifically, homozygous individuals for the null variants of the GSTM1 and GSTT1 genes are not able to produce the GSTs (Coles and Kadlubar 2003), resulting in reduced elimination and increased exposure to isothiocyanates following the consumption of cruciferous vegetables (Seow et al. 1998). In this context, studies reported that in GSTM1-null and/or GSTT1-null individuals the inverse association between cruciferous vegetable-derived isothiocyanate intake and the risk of lung (Lewis et al. 2001; Spitz et al. 2000; London et al. 2000; Zhao et al. 2001) or colorectal cancer (Slattery et al. 2000; Seow et al. 2002; Turner et al. 2004) was more pronounced, suggesting that these genetic polymorphisms may enhance the chemoprotective effects of cruciferous vegetables via slowing the elimination rate of the bioactive compounds.

4 Main Putative Anticancer Mechanisms of Action

As aforementioned, there is evidence suggesting the chemompreventive potential of cruciferous vegetable-deriving bioactive compounds. In the following section we will discuss the main putative mechanisms of action related to this effect. In general, the anticancer activity of this class of phytochemicals is exerted through various signalling pathways involving hormone regulation, DNA repairing, cell division and growth, angiogenesis, apoptosis and inflammation (Wang and Jiang 2012), that are altered in cancer cells (Popolo et al. 2017).

4.1 Cell Cycle Arrest

The cell cycle consists of a complex process in which cells go from a resting phase (G0) to a subsequent proliferation, following distinct phases named G1, S, G2 and M. Cyclins are key proteins regulating the progression toward the single phases of the cell cycle. Cyclins may form complexes with cyclin-dependent kinases (CDKs), that are inhibited by specific enzymes, including p21 and p27. This mechanism that control the cell proliferation is deregulated in cancer cells (Diaz-Moralli et al. 2013). It has been demonstrated that isothiocyanates may modulate the expression levels of both cyclins and CDKs and up-regulate the p21 and p27 expression, resulting in induced cell cycle arrest. This effect has been reported in various studies conducted on different cancer cell lines, including human colon carcinoma, cervical cancer cells, bladder cancer cells, oral carcinoma cells, prostate cancer cells and human pancreatic cancer cells (Watson et al. 2013; Mitsiogianni et al. 2019).

4.2 Apoptosis

Apoptosis is a process inducing a programmed cell death that, in physiological conditions, is finely regulated, contributing to the maintenance of the normal cell number. Differently, in cancer cells the ability to respond to apoptosis signals is lost and cells proliferate rapidly and constantly. The pro-apoptotic effect of cruciferous vegetable-deriving phytochemicals, mainly I3C, was investigated in different cancer cell lines, including prostate, cervical and breast cancer cells, reporting that these compounds act at different levels, including (a) shifting in the expression levels of Bax and Bcl2 toward a ratio promoting the cell death, (b) PARP cleavage, (c) DNA laddering and (d) decreasing the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activation (Higdon et al. 2007; Watson et al. 2013).

4.3 Signalling Pathways Involving Akt

One of the most frequent targets of sporadic cancers is the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) signalling pathway (Ahmad et al. 2013). PI3Ks are lipid kinases that phophorylate various targets, including phosphatidylinositol 4,5-bisphosphate (PIP2), leading to the formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), a second messenger involved in crucial cell pathways (Zhao et al. 2017). PIP3 binds to further kinases, including Akt that in turn, once activated, regulates various cell processes, such as progression of the cell cycle, promotion of cell survival and cell growth by phosphorylating the mTOR complex 1 (Popolo et al. 2017). One of the crucial targets of Akt is NF-κB, which role in cancer development is well-established. In particular, NF-κB represents a link between inflammatory status and cancer, regulating the expression of genes involved in inhibition of the apoptotic processes and cell cycle progression, resulting in promoting angiogenesis and metastasis (Karin 2006; Hussain and Harris 2007). I3C was reported to be able to block the Akt/NF-κB signalling pathways, resulting in exerting an anticancer effect via inhibition of (a) progression of the cell cycle, (b) survival and (c) metastasis; these effects have been demonstrated in different cell lines, including B-cell precursor acute lymphoblastic leukemia and lung cancer cells (Popolo et al. 2017). In particular, the modulation of the NF-κB activation mediated by I3C results in the inhibition of the expression of both proliferative and anti-apoptotic genes, including c-Myc, IAP1, Bcl-xL, Bcl-2 and XIAP (Safa et al. 2015). Moreover, it has been reported that in Hep-2 cells, I3C affect the expression of key proteins involved in the PIP3/Akt signalling pathway, including PI3K p110α, PI3K p110β, PI3K class III, GSK3-β, p-PDK1, Akt, p-Akt and p-c-Raf (Popolo et al. 2017). Similarly, in breast and prostate cancer cells, the loss in the activity of Akt mediated by I3C resulted in alteration of various growth factors, including FOXO3a (Rahman et al. 2004). Furthermore, I3C was shown to inhibit the prostate cancer cell proliferation down-regulating the IGF1/Akt signalling pathway (Li et al. 2014).

4.4 AHR Signalling Pathway

The aryl hydrocarbon receptor (AHR) is a transcription factor regulating the expression of genes involved in various processes, including inflammation, detoxification and cancer. More specifically, acting as a xenobiotic chemical sensor, AHR binds to xenobiotic responsive elements in the promoters of targeted genes, regulating their expression. It has been demonstrated that indole derivatives are able to modulate the AHR activity (Popolo et al. 2017). In particular, I3C inhibits the binding between AHR and COX-2 promoter, which over-expression plays a crucial role in the development of various cancers, including mammary (Banerjee et al. 2002) and esophangeal cancers (Li et al. 2002). Moreover, the chemopreventive effects mediated by the interaction between I3C and AHR has been also reported in in vitro studies on colorectal cancer (Bonnesen et al. 2001; Frydoonfar et al. 2002; Hudson et al. 2002; Lee et al. 2005; Neave et al. 2005; Díaz-Díaz et al. 2016; Megna et al. 2016). Further evidence reported the capability of I3C to down-regulate the expression of the estrogen receptors (ER)-α (that is involved in promoting the cell proliferation) and activate the ER-β (that is associated with reduced cellular proliferation) (Firestone and Sundar 2009).

4.5 Carcinogen Metabolising Enzymes

Metabolising enzymes, including phase I and phase II enzymes, play a pivotal role in cancer development. Among the phase I enzymes, there is the cytochrome p450 (CYP450), while among the phase II enzymes there are GST, quinone reductase (QR), and NAD(P)H quinone oxidoreductase-1 (NQO1) (Leone et al. 2017). Phase I enzymes increase the hydrophilicity of carcinogenic compounds via various reactions (i.e. oxidation, reduction, hydrolysis), resulting in increasing their reactivity and the consequent DNA damage. On the other hand, phase II enzymes cause the reactive intermediates conjugation, promoting their urinary excretion through the mercapturic acid pathway (Talalay and Fahey 2001; Keum et al. 2009). It has been reported that cruciferous vegetable-deriving bioactive compounds are able to inhibit the activity of phase I enzymes and activate the phase II enzymes; in particular, this activation seems to be exerted through increased transcription of these enzymes (Abbaoui et al., 2018).

4.6 Epigenetic Effects

Besides the aforementioned effects of both indoles and isothiocyanates in signalling pathways directly involved in cancer development, evidence suggests a role of these phytochemicals in epigenetic processes, in particular, altering the epigenetic modulators expression, such as enzymes controlling histone methylation and microRNAs. Human in vivo studies, indeed, demonstrated that the supplementation with idole-deriving products increased the expression of Let family microRNA and decreased the expression of the histone methyltransferase EZH2; moreover, these compounds may restore the expression levels of the silenced miR-34a, that is recognized as a regulator of cancer suppression (Watson et al. 2013).

5 Conclusion

Cruciferous vegetables may be considered as good sources of bioactive compounds which chemopreventive effect has been widely reported and the mechanisms of action well-described, as schematically summarised in Fig. 7.2. Overall, this evidence supports the observational data showing the direct association between reduced cancer risk and regular consumption of this kind of vegetables reported in various studies. Such promising, this evidence may serve to drive the scientific research in the nutraceutical field toward the further investigation of this beneficial potential of Cruciferous vegetable-derived bioactive compounds, formulating proper nutraceuticals aimed to prevent the risk to develop various cancers. This in turn, may be a start point for designing animal-based studies and clinical trials aimed to substantiate these effects in vivo.

Fig. 7.2
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Main mechanisms of action for the anticancer activity of Cruciferous phytochemicals

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