Broccoli Consumption Interacts with GSTM1 to Perturb Oncogenic Signalling Pathways in the Prostate 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. Mithen 1 * 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, 7 Department of Urology, Norfolk and Norwich University Hospital, Norwich, United Kingdom Abstract Background: Epidemiological 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 individuals on the pea-rich diet but significant differences between GSTM1 genotypes on the broccoli-rich diet, associated with transforming growth factor beta 1 (TGFb1) 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 TGFb1, EGF and insulin signalling. We also provide evidence that sulforaphane (the isothiocyanate derived from 4-methylsuphinylbutyl glucosinolate that accumulates in broccoli) chemically interacts with TGFb1, EGF and insulin peptides to form thioureas, and enhances TGFb1/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. Trial Registration: ClinicalTrials.gov NCT00535977 Citation: Traka M, Gasper AV, Melchini A, Bacon JR, Needs PW, et al. (2008) Broccoli Consumption Interacts with GSTM1 to Perturb Oncogenic Signalling Pathways in the Prostate. PLoS ONE 3(7): e2568. doi:10.1371/journal.pone.0002568 Editor: Peter Nelson, Fred Hutchinson Cancer Research Center, United States of America Received February 20, 2008; Accepted May 19, 2008; Published July 2, 2008 Copyright: 2008 Traka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Biotechnology and Biological Sciences Research Council, UK. The sponsors had no role in the design and conduct of the study, analysis and interpretation of the results and did not review the manuscript prior to publication. Competing Interests: Richard Mithen is a co-inventor on patents (US:6340784 and Europe P1069819B1) associated with high glucosinolate broccoli. * E-mail: Richard.Mithen@bbsrc.ac.uk 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 individ- uals 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-methylsul- phinylpropyl glucosinolates in its florets, which are converted to the isothiocyanates (ITCs) sulforaphane (SF) and iberin (IB), respectively, either by plant thioglucosidases (‘myrosinases’) following tissue damage or, if the myrosinases have been denatured by cooking or blanching prior to freezing, by microbial PLoS ONE | www.plosone.org 1 July 2008 | Volume 3 | Issue 7 | e2568