Broccoli & Signalling Pathways 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 mM SF for 4 h at 37uC. In (A) and (B) m/z 1183.9 corresponds to insulin-SF MH55+ 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. doi:10.1371/journal.pone.0002568.g005 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 TGFb1 with a physiological appropriate concentration of SF (2 mM for 30 minutes), followed by dialysis for 4 h to simulate SF pharmacokinetics, results in enhanced Smad- mediated transcription. As TGFb1/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 TGFb 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 TGFb1, 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 PLoS ONE | www.plosone.org 10 July 2008 | Volume 3 | Issue 7 | e2568