Broccoli consumption interacts with GSTM1 to perturb oncogenic signalling pathways in the prostate

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 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 (TGFbeta1) 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 TGFbeta1, EGF and insulin signalling. We also provide evidence that sulforaphane (the isothiocyanate derived from 4-methylsuphinylbutyl glucosinolate that accumulates in broccoli) chemically interacts with TGFbeta1, EGF and insulin peptides to form thioureas, and enhances TGFbeta1/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.

Figure 1. Metabolism of 4-methylsulphinylbutyl glucosinolate and sulforaphane.

Upon entry into enterocytes sulforaphane (SF) is rapidly conjugated to glutathione, exported into the systemic circulation and metabolized through the mercapturic acid pathway. Within the low glutathione environment of the plasma the SF-glutathione conjugate may be cleaved, possibly mediated by GSTM1, leading to circulation of free SF in the plasma. This free SF can modify plasma proteins including signalling molecules, such as TGFβ, EGF and insulin.

Figure 2. LDA of an independent prostate microarray data set.

Linear discriminant analysis (LDA) using the benign (B) and malignant (M) TURP prostate tissue for this study as training samples to classify the laser-capture microdissected (LCD) epithelial prostate cell samples (GEO Accession:GDS1439), consisting of benign (Be), primary cancer (PCa) and metastatic cancer (MCa) samples. LDA was performed on a gene list that distinguished the benign and malignant TURP samples as described in Methods. Here, the first linear discriminant (LD1) is shown.

Figure 3. Effect of dietary intervention on gene transcription.

a, Number of probes that differ between GSTM1 positive and null genotypes (P≤0.005, Welch modified two-sample t-test) in TURP tissue from benign (Ben) and malignant (Mal) prostates, and TRUS-guided biopsy tissue from volunteers at pre-intervention (Pre), post 6 months broccoli-rich diet (Broc) and post 6 months pea-rich diet (Peas). b, Number of probes that differ between pre-intervention TRUS-guided biopsy samples and after 6 months broccoli (6B)-, 6 month pea (6P)-, 12 month broccoli (12B)- and 12 month pea (12 P)-rich diets (P≤0.005, Welch modified two-sample paired t-test). Shading correspond to different fold cutoffs applied. See Table 2 for full details of probe numbers, P-values and median false discovery rates.

Figure 4. LC-MS of insulin incubated with and without SF in human plasma.

Extracted ion LC-MS chromatograms (m/z 1183.6–1184.1) of insulin-SF MH₅ ⁵⁺ in (A) unmodified insulin (20 µg/ml) in human plasma control and (B) human plasma incubated with insulin (20 µg/ml) and 50 µM SF for 4 h at 37°C, showing the appearance of two different insulin-SF conjugates at retention times of 6.46 and 7.08 min. The enhanced product ion (EPI)-MS spectra of these two insulin-SF conjugates are shown in Figure 5.

Figure 5. Enhanced product ion (EPI)-MS spectra of the two insulin-SF conjugates.

MS² product ion spectra of (A) 6.46 min and (B) 7.08 min retention time peaks from LC-MS analysis of human plasma incubated with bovine insulin and 50 µM SF for 4 h at 37°C. In (A) and (B) m/z 1183.9 corresponds to insulin-SF MH₅ ⁵⁺ and in (A) m/z 235.0 corresponds to Gly-SF, the N-terminal amino acid of insulin A chain and in (B) m/z 325.2 corresponds to Phe-SF, the N-terminal amino acid of insulin B chain.

Figure 6. LC-MS of TGFβ1 incubated with and without SF.

Extracted ion chromatograms (MS) of precursor masses representing the unmodified N-terminal peptide of TGFβ1 (m/z 768.5) and the modified N-terminal peptide (m/z 877.2) A of m/z 768.2–769.2 from DMSO treated TGFβ1, B of m/z 768.2–769.2 from SF treated TGFβ1, C of m/z 876.7–877.7 DMSO treated TGFβ1 and D of m/z 876.7–877.7 SF treated TGFβ1.

Figure 7. N-terminal modification of TGFβ1 by SF.

MS/MS spectra of m/z 768.7 representing the unmodified N-terminal peptide of TGFβ1 at retention time 23.43 min (A) and m/z 877.2 representing a modified form of TGFβ1 seen only in SF treated samples at retention time 30.85 minutes (B). Note that the y ion series remains the same while the b ion series shifts (Δ) indicating an N-terminal modification of mass 217±0.8 Da. Figure S2 provides an explanation of the mass addition of 217, as opposed to 177.

Figure 8. Activation of TGFβ1/Smad mediated transcription by SF.

NIH3T3 cells containing a CAGA12-luc plasmid were treated with TGFβ1 alone, TGFβ1 and 10 mM DTT, which disrupts the active TGFβ1 dimer, or TGFβ1 and 2 µM SF. All samples were pre-incubated for 30 minutes and further dialyzed for 4 h so that the final concentration of SF was 34 nM. As an additional negative control cells received no treatment or only 34 nM SF, both of which failed to induce luciferase. Chemiluminescence was normalized to the protein concentration of each sample (for details see Methods). This is a representative experiment of a total of four similar experiments performed. Data shown are mean (s.e.m) of three replicates.