In vitro analysis of potential anticancer effects associated with watercress

Epidemiological studies indicate that there is an inverse relationship between consumption of cruciferous vegetables and risk of cancer. As a result there is much interest in understanding the anticancer potential not only of cruciferous vegetables themselves but also of the key phytochemicals contained in them. This project focuses on the potential anticancer properties of watercress, a cruciferous vegetable which is cultivated around the world and eaten raw as a salad vegetable as well as in cooked dishes. Watercress is the most abundant source of gluconasturtiin, a precursor to the phytochemical phenethyl isothiocyanate (PEITC), and is also a rich source of indole-3- carbinol (13C) and quercetin. This project addressed the hypothesis that the in vitro anticancer activity of watercress can be enhanced by altering the growth environment. To investigate this it was necessary to develop assays that can be used to assess the in vitro anticancer activity of watercress-derived compounds, to explore the mechanisms by which PEITC exhibits its anticancer effects, and to determine potential effects of altered growth conditions on in vitro anticancer properties. The activity of watercress-derived phytochemicals was analysed in a series of in vitro assays. Based on these results, inhibition of MCF7 cell growth and activation of Nrf2-dependent transcription were selected as potential assays for subsequent analysis of watercress extracts. Mechanistic studies demonstrated that PEITC inhibited the transcriptional activity of hypoxia inducible factor (HIF), a key positive regulator of angiogenesis in malignant cells. Inhibition of HIF function was associated with inhibition of mammalian target of rapamycin complex 1 (mTORC1) activity and decreased HIF1 a mRNA translation, and was dependent on the presence of the mTORC1 regulator, tuberous sclerosis complex 2 (TSC2). I also demonstrated that, in addition to effects on HIF1 a mRNA translation, PEITC inhibited general protein synthesis and modulated two other key regulators of translation, eukaryotic initiation factor 2 (eIF2) and eukaryotic elongation factor 2 (eEF2). In proof-of-principle experiments, I demonstrated that growth inhibition and Nrf2 activation assays were suitable for the analysis of crude watercress extracts and that a natural variety of "red" watercress displayed approximately 10-fold more potent in vitro anticancer activity than standard, commercial "green" watercress. However, in a series of field trials, modulation of time of harvest, water availability or sulfur fertilisation did not alter the in vitro effects of watercress extracts in growth inhibition/Nrf2 activation assays. In conclusion, this study suggests that, rather than altering environmental factors, selective breeding might be a better approach to increase the in vitro anticancer activity of watercress. Moreover, my findings have increased our understanding of the mechanisms underlying the anticancer activity of PEITC, uncovering a novel role for PEITC in inhibiting HIF1 a and total protein synthesis, and identifying several key regulators of these processes that are modulated by PEITC