Broccoli & Signalling Pathways Table 2. Pathway analyses of prostate biopsy tissue. Table 3. Differentially expressed probes in prostate tissue. Fold change P,0.05* P,0.005* P,0.0005* (a) Differences between benign and malignant TURP tissue Benign v Malignant 3810 (353) 683 (7) (b) Differences between GSTM1 positive and genotypes Benign (TURP) Malignant (TURP) Pre-intervention 6 month peas .1.0 .1.5 .2.0 .1.0 .1.5 .2.0 .1.0 .1.5 .2.0 .1.0 .1.5 .2.0 .1.0 .1.5 .2.0 .1.0 .1.5 .2.0 .1.5 .2.0 .1.0 .1.5 .2.0 6 month broccoli 7976 (351) 434 (4) (c) Differences between paired samples 0–12 months broccoli .1.0 0–12 months peas 1081 (59) 277 (7) 661 (538) 160 (186) 50 (40) 686 (431) 244 (152) 44 (33) 730 (484) 252 (79) 43 (9) 2790 (91) 316 (8) 220 (220) 33 (41) 5 (10) 2857 (96) 1243 (12) 213 (0) 1199 (42) 495 (18) 81 (1) 400 (2) 140 (0) 19 (17) 7 (14) 1 (3) 8 (13) 4 (8) 1 (3) 26 (9) 16 (3) 8 (1) 268 (2) 31 (0) 6 (4) 5 (3) 1 (1) 151 (0) 141 (0) 62 (0) 19 (0) 19 (0) 4 (0) 124 (0) 104 (0) 54 (0) 17 (0) 14 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0) 0 (0) 0 (0) 0 (0) 1 (0) 1 (0) 1 (0) 0 (0) 0 (0) 0 (0) Pathway a. Benign compared with malignant TURP tissue Genes changed/ Genes on MAPP Adjusted P-value* Focal adhesion TGFb receptor Circadian exercise Fatty acid metabolism Prostaglandin synthesis regulation 14/31 Actin binding GPCRs Class A rhodopsin-like b. GSTM1 positives compared with GSTM1 nulls post six month broccoli intervention c. Paired samples pre and post 12 month dietary intervention EGFR1 Adipogenesis TGF-beta receptor 0–12 month peas Androgen receptor 0–12 month broccoli ** mRNA processing Androgen receptor TGFb receptor Insulin signalling Delta-notch Wnt signalling EGFR1 IL-2 57/187 47/151 20/48 24/80 53/213 15/262 76/177 52/130 58/151 18/112 40/125 33/112 39/151 38/159 24/ 85 28/109 40/177 21/ 76 ,0.001 0.002 0.006 0.012 0.026 0.028 0.05 ,0.001 0.026 0.039 0.042 ,0.001 ,0.001 0.004 0.014 0.019 0.02 0.02 0.036 EGFR, epidermal growth factor receptor; GPCRs, G-protein coupled receptors; IL- 2, interleukin 2; TGFb, transforming growth factor beta; TURP, transurethral resection of the prostate; Wnt, wingless-type MMTV integration site. Pathways in GenMAPP that are enriched in the gene lists that differentiate groups are shown. Only pathways with adjusted P values #0.05 are shown. Also, the number of genes changing between groups that belong to these pathways is shown alongside the total number of genes that constitute the pathway. Pathway analysis was performed on gene lists generated in dChip that were statistically significant (P#0.05, Welch modified two-sample paired or unpaired t-test) between the two groups. No fold cutoff was used. For details on gene lists see Table 3. *P-values were calculated in GenMAPP using a non-parametric statistic based on 2000 permutations of the data and further adjusted for multiple testing by Westfall-Young adjustment. **GSTM1 positive volunteers, n = 4. doi:10.1371/journal.pone.0002568.t002 Variation in plasma PSA levels PSA levels prior to the intervention were in similar range to that previously reported for men of an equivalent age range diagnosed with HGPIN [25]. There was no significant association with GSTM1 genotype, and no consistent changes in PSA levels after six or 12 months within either arm of the intervention study (Table 1). Differences in global gene expression between GSTM1 positive and individuals We initially genotyped the resected TURP tissue samples and compared gene expression profiles between GSTM1 positive and genotypes within the benign and malignant samples. We found few differences between genotypes, with similar high median false Probe numbers that have satisfied the comparison criteria of fold change and P- value cutoffs are shown. Numbers in parentheses represent the median false discovery rate calculated in dChip after 100 permutations of the samples. *P-values were calculated in dChip by a Welch modified two-sample t-test. n = 18 for Benign, n = 14 for Malignant; n = 4 for GSTM1(+) Benign, n = 14 for GSTM1(2) Benign; n = 5 for GSTM1(+) Malignant, n = 9 for GSTM1(2) Malignant; n = 7 for GSTM1(+) Pre-intervention, n = 3 for GSTM1(2) Pre- intervention; n = 6 for GSTM1(+) 6-month broccoli, n = 5 for GSTM1(2) 6-month broccoli; n = 3 for GSTM1(+) 6-month pea and n = 5 for GSTM1(2) 6-month pea intervention. **P-values were calculated in dChip by a Welch modified two-sample paired t- test, n = 4 for each of the diet interventions. doi:10.1371/journal.pone.0002568.t003 discovery rates (Figure 3a, Table 3b). Likewise, we compared gene expression profiles obtained from needle biopsies of the prostate from GSTM1 positive and men who had previously been diagnosed with HGPIN and found few differences. We then compared gene expression profiles between GSTM1 genotypes in needle biopsy tissue of twenty-one men who had been recruited into the dietary intervention study. Eight of the men within this study had been asked to consume 400 g of steamed frozen peas per week, and the other thirteen were requested to consume 400 g of steamed frozen broccoli per week, but otherwise to consume their normal diet. Diet was assessed with seven-day diet diaries prior to the intervention and after six months. No significant differences were found in diet components, apart from the consumption of broccoli and peas (Table 4). We found many differences in the prostate gene expression between GSTM1 PLoS ONE | www.plosone.org 6 July 2008 | Volume 3 | Issue 7 | e2568