Increased mRNA translation drives carcinogenesis and is an attractive target for the development of new anti-cancer drugs. In this work, we investigated effects of phenethylisothiocyanate (PEITC), a phytochemical with chemopreventive and anti-cancer activity, on mRNA translation. PEITC rapidly inhibited global mRNA translation in human breast cancer-derived MCF7 cells and mouse embryonic fibroblasts (MEFs). In addition to the known inhibitory effects of PEITC on mTORC1 activity, we demonstrate that PEITC increased eIF2α phosphorylation. PEITC also increased formation of stress granules which are typically associated with eIF2α phosphorylation and accumulation of translationally stalled mRNAs. Analysis of genetically modified MEFs demonstrated that optimal inhibition of global mRNA translation by PEITC was dependent on eIF2α phosphorylation, but not mTORC1 inhibition. We extended this study into primary leukemic B cells derived from patients with chronic lymphocytic leukaemia (CLL). CLL cells were stimulated in vitro with anti-IgM to mimic binding of antigen, a major driver of this leukemia. In CLL cells, PEITC increased eIF2α phosphorylation, inhibited anti-IgM-induced mTORC1 activation and decreased both basal and anti-IgM-induced global mRNA translation. PEITC also inhibited transcription and translation of MYC mRNA and accumulation of the MYC oncoprotein, in anti-IgM-stimulated cells. Moreover, treatment of CLL cells with PEITC and the BTK kinase inhibitor ibrutinib decreased anti-IgM-induced translation and induced cell death to a greater extent than either agent alone. Therefore, PEITC can inhibit both global and mRNA specific translation (including MYC) via effects on multiple regulatory pathways. Inhibition of mRNA translation may contribute to the chemopreventive and anti-cancer effects of PEITC.

Phenethyl isothiocyanate (1) is a natural dietary phytochemical with cytostatic, cytotoxic, and antitumor activity. The effects of 1 were investigated on the activity of mTOR, a kinase that enhances the translation of many RNAs encoding proteins critical for cancer cell growth, including the angiogenesis regulator HIF1α. Compound 1 effectively blocked HIF1α RNA translation in MCF7 breast cancer cells, and this was associated with reduced phosphorylation of 4E-BP1 and p70 S6K, well-characterized downstream substrates of the mTOR-containing mTORC1 complex. Compound 1 also inhibited mTORC1 activity in mouse embryonic fibroblasts (MEFs). The 1-mediated inhibition of mTORC1 activity appeared to be independent of the upstream regulators PTEN, AKT, ERK1/2, and AMPK. By contrast, 1-mediated inhibition of mTORC1 activity was dependent on the presence of TSC2, part of a complex that regulates mTORC1 activity negatively. TSC2-deficient MEFs were resistant to 1-mediated inhibition of p70 S6K phosphorylation. TSC2-deficient MEFs were also partially resistant to 1-mediated growth inhibition. Overall, the present results confirm that 1 inhibits mTORC1 activity. This is dependent on the presence of TSC2, and inhibition of mTORC1 contributes to optimal 1-induced growth inhibition. Inhibition of RNA translation may be an important component of the antitumor effects of phenethyl isothiocyanate.

Phenethyl isothiocyanate (PEITC) is a naturally occurring electrophile which depletes intracellular glutathione (GSH) levels and triggers accumulation of reactive oxygen species (ROS). PEITC is of considerable interest as a potential chemopreventive/chemotherapeutic agent, and in this work, we have investigated the effects of PEITC on human breast cancer cell lines. Whereas PEITC readily induced apoptosis in MDA-MB-231 cells (associated with rapid activation of caspases 9 and 3, and decreased expression of BAX), MCF7 cells were relatively resistant to the apoptosis promoting effects of PEITC. The relative resistance of MCF7 cells was associated with high basal expression of NRF2, a transcription factor that coordinates cellular protective responses to oxidants and electrophiles and raised intracellular levels of GSH. This raised basal expression of NRF2 appeared to be a response to on-going production of ROS, since treatment with the antioxidant and GSH precursor N-acetylcysteine (NAC) reduced NRF2 expression. Moreover, pre-treatment of MDA-MB-231 cells with NAC rendered these cells relatively resistant to PEITC-induced apoptosis. In summary, our data confirm that PEITC may be an effective chemopreventive/therapeutic agents for breast cancer. However, differences in the basal expression of NRF2 and resultant changes in GSH levels may be an important determinant of sensitivity to PEITC-induced apoptosis.

Isothiocyanates (ITCs) are electrophilic compounds derived from plants and are thought to play a major role in the potential chemopreventive effects associated with high intake of cruciferous vegetables. ITCs are also being evaluated for chemotherapeutic activity in early phase clinical trials. In addition to their effects on carcinogen metabolism and cancer cell survival and proliferation, ITCs have been shown to effectively interfere with angiogenesis in vitro and in vivo. Angiogenesis is the development of a new blood supply from existing vasculature and is required for tumours to develop beyond a small size limit determined by the diffusion limit for oxygen. Inhibition of angiogenesis may play a key role in the potential chemopreventive/chemotherapeutic activity of ITCs. In this review we highlight recent data demonstrating that ITCs have anti-angiogenic activity and identify potential molecular targets for these effects, including hypoxia-inducible factor (HIF), nuclear factor κB (NF-κB), activator protein 1 (AP1) and tubulin. We also discuss these findings in light of the potential chemopreventive/chemotherapeutic effects of ITCs.

The ferrocene analogue 4b had an IC50 = 4 nM (HDAC1), 180 nM (HDAC8) and was effective in a Xenopus model of tubulin deacetylation. Analogue 4a displayed mainly μM IC50 values against HDACs apart from HDAC6 (IC50 = 69 nM).

Dietary intake of isothiocyanates (ITC) has been associated with reduced cancer risk. The dietary phenethyl ITC (PEITC) has previously been shown to decrease the phosphorylation of the translation regulator 4E binding protein 1 (4E-BP1). Decreased 4E-BP1 phosphorylation has been linked to the inhibition of cancer cell survival and decreased activity of the transcription factor hypoxia-inducible factor (HIF), a key positive regulator of angiogenesis, and may therefore contribute to potential anti-cancer effects of PEITC. In the present study, we have investigated the in vitro and in vivo effects of watercress, which is a rich source of PEITC. We first demonstrated that, similar to PEITC, crude watercress extracts inhibited cancer cell growth and HIF activity in vitro. To examine the effects of dietary intake of watercress, we obtained plasma and peripheral blood mononuclear cells following the ingestion of an 80 g portion of watercress from healthy participants who had previously been treated for breast cancer. Analysis of PEITC in plasma samples from nine participants demonstrated a mean maximum plasma concentration of 297 nM following the ingestion of watercress. Flow cytometric analysis of 4E-BP1 phosphorylation in peripheral blood cells from four participants demonstrated significantly reduced 4E-BP1 phosphorylation at 6 and 8 h following the ingestion of watercress. Although further investigations with larger numbers of participants are required to confirm these findings, this pilot study suggests that flow cytometry may be a suitable approach to measure changes in 4E-BP1 phosphorylation following the ingestion of watercress, and that dietary intake of watercress may be sufficient to modulate this potential anti-cancer pathway.

Phenethyl isothiocyanate (PEITC), a natural dietary isothiocyanate, has anti-cancer activity in various in vitro and in vivo models. PEITC inhibits angiogenesis but the molecular mechanisms that underlie this effect are not known. We have now demonstrated that PEITC is an effective inhibitor of hypoxia inducible factor (HIF), a transcription factor that plays an important role in expression of pro-angiogenic factors. PEITC inhibited the activation of a HIF-dependent reporter construct following incubation of cells in hypoxia, or treatment with the hypoxia mimetic cobalt chloride. PEITC also interfered with the accumulation of HIF1α protein and induction of the endogenous HIF target genes, CAIX, GLUT1, BNIP3 and VEGF-A. The ability of PEITC to inhibit HIF activity was independent of the activity of prolyl hydroxylases, the Von-Hippel–Landau protein and the proteasome, all of which are required for the normal rapid turnover of HIF1α in normoxia. Decreased expression of HIF1α in PEITC treated cells was not associated with changes in the levels of HIF1α RNA suggesting that PEITC may inhibit HIF activity by decreasing translation of the HIF1α RNA. Consistent with this, PEITC decreased phosphorylation of the translation regulator 4E-BP1. Our data demonstrate that PEITC is an effective inhibitor of HIF activity. This may contribute to the anti-angiogenic and anti-cancer effects of PEITC.