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Broccoli Consumption May Prevent or Reduce Risk of Protate Cancer
 
 
  Broccoli Consumption Interacts with GSTM1 to Perturb Oncogenic Signalling Pathways in the Prostate
 
PLoS One July 2008
 
".....This study provides, for the first time, experimental evidence obtained in humans to support observational studies that diets rich in cruciferous vegetables may reduce the risk of prostate cancer and other chronic disease......In conclusion, we consider that our study has provided a mechanistic basis for the reduction in risk of prostate cancer through broccoli consumption, as suggested by epidemiological studies. Further studies with larger cohorts, combined with the assessment of clinical endpoints, are warranted......it is of considerable interest that broccoli intervention is associated with perturbation of TGF_1β, EGF and insulin signalling, each of which has been associated with prostate carcinogenesis.....in addition to carcinogenesis at other sites, and inflammation associated with myocardial infarction......It is likely that the major bioactive products derived from broccoli are the isothiocyanates, sulforaphane and iberin. These have been shown to have a multitude of biological activities in cell models consistent with anticarcinogenic activity....."
 
Maria Traka1, Amy V. Gasper1, Antonietta Melchini1,2, James R. Bacon1, Paul W. Needs1, Victoria Frost3, Andrew Chantry3, Alexandra M. E. Jones4, Catharine A. Ortori5, David A. Barrett5, Richard Y. Ball6, Robert D. Mills6, Richard F. Mithen1*
 
1 Phytochemicals and Health Programme, Institute of Food Research, Norwich Research Park, Norwich, United Kingdom, 2 Department Farmaco-Biologico, School of Pharmacy, University of Messina, Messina, Italy, 3 School of Biological Sciences, University of East Anglia, Norwich, United Kingdom, 4 The Sainsbury Laboratory, Norwich Research Park, Norwich, United Kingdom, 5 Centre for Analytical Bioscience, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom, 6 Department of Histopathology/Cytopathology, Norfolk and Norwich University Hospital, Norwich, United Kingdom
 
ABSTRACT
 
Background

 
pidemiological studies suggest that people who consume more than one portion of cruciferous vegetables per week are at lower risk of both the incidence of prostate cancer and of developing aggressive prostate cancer but there is little understanding of the underlying mechanisms. In this study, we quantify and interpret changes in global gene expression patterns in the human prostate gland before, during and after a 12 month broccoli-rich diet.
 
Methods and Findings
 
Volunteers were randomly assigned to either a broccoli-rich or a pea-rich diet. After six months there were no differences in gene expression between glutathione S-transferase mu 1 (GSTM1) positive and null individuals on the pea-rich diet but significant differences between GSTM1 genotypes on the broccoli-rich diet, associated with transforming growth factor beta 1 (TGF_1β) and epidermal growth factor (EGF) signalling pathways. Comparison of biopsies obtained pre and post intervention revealed more changes in gene expression occurred in individuals on a broccoli-rich diet than in those on a pea-rich diet. While there were changes in androgen signalling, regardless of diet, men on the broccoli diet had additional changes to mRNA processing, and TGF_1β, EGF and insulin signalling. We also provide evidence that sulforaphane (the isothiocyanate derived from 4-methylsuphinylbutyl glucosinolate that accumulates in broccoli) chemically interacts with TGF_1β, EGF and insulin peptides to form thioureas, and enhances TGF_1β/Smad-mediated transcription.
 
Conclusions
 
These findings suggest that consuming broccoli interacts with GSTM1 genotype to result in complex changes to signalling pathways associated with inflammation and carcinogenesis in the prostate. We propose that these changes may be mediated through the chemical interaction of isothiocyanates with signalling peptides in the plasma. This study provides, for the first time, experimental evidence obtained in humans to support observational studies that diets rich in cruciferous vegetables may reduce the risk of prostate cancer and other chronic disease.
 
Introduction
 
Prostate cancer is the most frequently diagnosed non-cutaneous cancer within the male population of western countries [1]. Epidemiological studies have suggested that diets rich in cruciferous vegetables, such as broccoli, may reduce the risk of prostate cancer [2], in addition to cancers at other sites [3]-[9] and myocardial infarction [10]. Some studies have specifically demonstrated that consuming one or more portions of broccoli per week can reduce the incidence of prostate cancer [11], and also the progression from localized to aggressive forms of prostate cancer [12]. The reduction in risk may be modulated by glutathione S-transferase mu 1 (GSTM1) genotype, with individuals who possess at least one GSTM1 allele (i.e. approximately 50% of the population) gaining more benefit than those who have a homozygous deletion of GSTM1 [11]. Our study investigates the mechanistic basis to the protective effect of broccoli and the interaction with GSTM1 genotype.
 
Broccoli accumulates 4-methylsulphinylbutyl and 3-methylsulphinylpropyl glucosinolates in its florets, which are converted to the isothiocyanates (ITCs) sulforaphane (SF) and iberin (IB), respectively, either by plant thioglucosidases (emyrosinasesf) following tissue damage or, if the myrosinases have been denatured by cooking or blanching prior to freezing, by microbial thioglucosidases in the colon (Figure 1) [13]. These ITCs do not have the pungent flavour qualities associated with other dietary ITCs, such as those from mustards, rockets and watercress. SF and IB are passively absorbed by enterocytes, conjugated with glutathione and transported into the systemic circulation to be metabolized via the mercapturic acid pathway and excreted predominantly as N-acetylcysteine conjugates in the urine [14]. We previously demonstrated that following broccoli consumption, 45% of SF in the plasma occurs as free SF, as opposed to thiol conjugates, and that the peak concentration of SF and its thiol conjugates is less than 2 μM, falling to low (nM) levels within a few hours [14]. It was also shown that GSTM1 null individuals excrete a higher proportion of SF via mercapturic acid metabolism than GSTM1 positive individuals, and it was speculated that the remaining SF may be metabolized via an unknown pathway, and that this may account for the anticarcinogenic activity of broccoli.
 
Cell and animal studies have shown that SF is a potent inducer of phase 2 enzyme gene transcription, and, at higher concentrations, of cell cycle arrest and apoptosis, all consistent with anticarcinogenic activity [15]. However, these phenomena occur when cultured cells are exposed to considerably higher levels of SF (i.e. 10-100 μM for 24 h) than those found transiently in plasma after broccoli consumption (i.e. <2 μM for less than one hour). At these physiological concentrations, it is likely that any SF entering cells would be immediately conjugated with glutathione due to the relatively high intracellular glutathione concentration, with no effect on, for example, the Keap1-Nrf2 complex [16], [17]. Consistent with this hypothesis, there was no evidence for induction of phase 2 gene transcription in the gastric mucosa after an acute broccoli intervention [18].
 
In this study, we initially adopt an empirical approach in humans to elucidate the mechanisms that underlie the beneficial effects of a broccoli-rich diet, and explore the interaction with GSTM1. We compare and interpret global gene expression profiles in human prostate biopsy tissue before, during and after a 12 month broccoli-rich diet and a 12-month pea-rich diet. Subsequently, we provide a mechanistic explanation for how the observed changes in gene expression may be induced by normal dietary broccoli consumption.
 
Discussion
 
To our knowledge, this is the first dietary intervention study to analyse global gene expression profiles within a target tissue before and after a 12 month intervention, and to stratify gene expression profiles by genotype. While we do not observe any consistent changes in plasma PSA levels over the 12 month period of the intervention, we were able to quantify extensive changes in gene expression. We find little evidence to support potential mechanisms derived from animal and cell models to explain the observational data that consuming broccoli may reduce risk of cancer, but considerable evidence for the perturbation of several signalling pathways that are associated with carcinogenesis and inflammation (Table 2b and c). It is possible that the net effect of perturbation of these pathways may reduce the risk of cell proliferation, and maintain cell and tissue homoeostasis. However, whilst quantification of gene expression and pathway analyses provides information concerning which pathways may be modified by time or diet, it can provide little information about the precise nature of how these pathways are perturbed. This requires further analysis of mRNA and protein turnover, and post translational protein modifications such as phosphorylation, associated with components of the signal transduction pathway and downstream targets. Furthermore, the effect of pathway perturbation may depend upon the precise cell type, with potentially differential effects occurring in epithelial and stromal cells. Despite these reservations, it is of considerable interest that broccoli intervention is associated with perturbation of TGF_1β, EGF and insulin signalling, each of which has been associated with prostate carcinogenesis [31]-[35], in addition to carcinogenesis at other sites [28], [36], [37], and inflammation associated with myocardial infarction [38]. It is noteworthy that broccoli consumption was also associated with alterations in mRNA processing, and this is being further explored.
 
It is likely that the major bioactive products derived from broccoli are the isothiocyanates, sulforaphane and iberin. These have been shown to have a multitude of biological activities in cell models consistent with anticarcinogenic activity [15]. However, these studies largely involve exposing cells to concentrations of SF and IB far in excess of those which occur transiently in the plasma after broccoli consumption, and are mediated by the intracellular activity of the ITCs by, for example, perturbing intracellular redox status, depletion of glutathione and perturbation of the Keap1-Nfr2 complex. We question whether these processes would occur in vivo, as any of the ITCs entering cells would immediately be inactivated through conjugation with glutathione that would be present in relatively high concentration. Thus, we explored whether the biological activity of ITCs may be mediated through their chemical interaction with signalling peptides within the extracellular environment of the plasma, which has a low glutathione concentration. We demonstrated that ITCs readily form thioureas with signalling proteins in the plasma through covalently bonding with the N-terminal residue. It is likely that ITCs chemically react with other plasma proteins and a global analysis of plasma protein modifications by ITCs is warranted. It is also possible that other types of chemical modification of plasma proteins by ITCs may occur, such as covalent bonding through cysteine and lysine residues [39], [40].
 
Previous studies have shown that isothiocyanate-derived thioureas modify the physicochemical and enzymatic properties of the parental proteins [26], [27]. Thus, it is possible that the perturbation of signalling pathways in the prostate is mediated by protein modifications that occur in the extracellular environment. We provide further evidence for this hypothesis by demonstrating that pre incubation of TGF_1β with a physiological appropriate concentration of SF (2 μM for 30 minutes), followed by dialysis for 4 h to simulate SF pharmacokinetics, results in enhanced Smad-mediated transcription. As TGF_1β/Smad-mediated transcription inhibits cell proliferation in non-transformed cells [31], [41], the enhancement of Smad-mediated transformation by SF would be consistent with the anticarcinogenic activity of broccoli, in addition to reduced risk of myocardial infarction [10], [38]. In certain circumstances, enhancement of TGF_ signalling has been associated with tumour progression within already initiated cells, although the precise pathways by which this is mediated have not been fully resolved [42]. To what extent a broccoli-rich diet may influence these processes requires further studies. However, we consider it likely that it is the net effect of changes in several pathways, as opposed to just TGF_1β, which may underlie the observed reduction in both cancer and myocardial infarction through broccoli/crucifer consumption.
 
A previous study has demonstrated that isothiocyanates can inhibit EGF signalling, but without a mechanistic explanation [43]. In the current study, we show that SF will bind to the EGF ligand, and this may underlie our results and those reported previously [43]. Moreover, chemical modification of signalling proteins by ITCs may be complemented by modification of receptor proteins, as has previously been shown for the TRPA1 receptor [39], [40].
 
Perturbation of signalling pathways is additionally determined by GSTM1 genotype. The interaction between diet and GSTM1 on gene expression may partially explain the contradictory results from those case control studies which lack dietary assessment and which have or have not associated the GSTM1 null genotype with enhanced risk of prostate cancer [44]-[47]. GSTM1 enzyme activity catalyses both the formation and the cleavage of SF - glutathione conjugates [48]. We suggest that following transport into the plasma from enterocytes, GSTM1 activity (originating either from hepatic cell turnover [49] or leakage from peripheral lymphocytes [50]) catalyses the cleavage of the SF-glutathione conjugate within the low glutathione environment of the plasma [51] to determine the extent of free SF that is available for protein modification, as discussed above, and which is not excreted via mercapturic acid metabolism (Figure 1). Thus low levels of SF, as would be expected from normal dietary consumption of broccoli, may lead to subtle changes in cell signalling, which, over time, result in profound changes in gene expression. In this manner, consuming one portion of broccoli per week if one is GSTM1 positive, or more if one is GSTM1 null [14], may contribute to a reduction in cancer risk.
 
In addition to the insight this study provides to the effect of broccoli consumption on gene expression, we consider that our study may have broader implications. First, we demonstrate that routine prostate needle biopsies can be used for global gene expression analyses in addition to histological assessment, and that it is possible to monitor changes in expression with time. It is notable that men within both dietary arms of the study had significant changes in the androgen receptor pathway. It is possible that these changes in androgen signalling are associated with aging and independent of diets, or they may have been induced by a common component of both the broccoli-rich and pea-rich diet. To our knowledge there is no data on the rate of change on androgen signalling in men of this age with HGPIN. This observation suggests further study is warranted. Analysis of the rate of change of gene expression of men diagnosed with either HGPIN or localized prostate cancer through sequential biopsies may provide reliable biomarkers to measure the likelihood of both carcinogenesis and progression to aggressive cancer, and complement histological examination of needle biopsies and measurement of plasma PSA levels. Secondly, stratification of global gene expression profiles by genotype has been informative, and this approach could be extended to other genes to dissect patterns of gene expression in prostate or other tissues. Lastly, it is conceivable that other dietary phytochemicals, such as polyphenolic derivatives, could also chemically interact with signalling peptides in the plasma, in a similar manner to the suggested mechanism of action of isothiocyanates.
 
In conclusion, we consider that our study has provided a mechanistic basis for the reduction in risk of prostate cancer through broccoli consumption, as suggested by epidemiological studies. Further studies with larger cohorts, combined with the assessment of clinical endpoints, are warranted.
 
 
 
 
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