Carcinogenesis Advance Access originally published online on July 24, 2009
Carcinogenesis 2009 30(10):1754-1762; doi:10.1093/carcin/bgp182
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1-Cyano-2,3-epithiopropane is a novel plant-derived chemopreventive agent which induces cytoprotective genes that afford resistance against the genotoxic 
,β-unsaturated aldehyde acrolein
Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK
1 Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
2 Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai 980-8577, Japan
3 Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba 305-8577, Japan
4 Exploratory Research for Advanced Technology Environmental Response Project, Japan Science and Technology Corporation, Tsukuba 305-8577, Japan
* To whom correspondence should be addressed. Tel: +44 1382 632788; Fax: +44 1382 669993; Email: j.d.hayes{at}dundee.ac.uk
Epithionitriles represent a previously unrecognized class of cancer chemopreventive phytochemical generated from alkenyl glucosinolates in cruciferous vegetables. In rat liver RL-34 epithelial cells, 1-cyano-2,3-epithiopropane (CETP), 1-cyano-3,4-epithiobutane (CETB) and 1-cyano-4,5-epithiopentane (CETPent) were shown to induce cytoprotective enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione (GSH) S-transferase A3 and the glutamate–cysteine ligase modifier subunit; CETP was more potent in this regard than were either CETB or CETPent, with 50 µM CETP eliciting a remarkable 
10-fold induction of NQO1. Furthermore, 50 µM CETP stimulated a 2.0-fold overproduction of GSH in RL-34 cells. Transfection experiments demonstrated that epithionitriles induced gene expression through an antioxidant response element (ARE) and that transactivation of an Nqo1-luciferase reporter plasmid was dependent on NF-E2 p45-related factor 2 (Nrf2), a cap'n'collar basic region leucine zipper transcription factor. Evidence is presented that CETP affected Nrf2-mediated induction of ARE-driven transcription by inhibiting Kelch-like ECH-associated protein 1 (Keap1), a ubiquitin ligase substrate adaptor that negatively regulates Nrf2. We found that Nqo1 was expressed constitutively at high levels in Keap1–/– mouse embryonic fibroblasts (MEFs) and it was not further induced by CETP. However, knock-in of mouse Keap1 or zebrafish Keap1a into Keap1–/– MEFs repressed Nqo1-luciferase reporter gene activity, but repression by the murine or zebrafish proteins was antagonized by CETP. Pre-treatment of Nrf2+/+ MEFs, but not Nrf2–/– MEFs, with 15 µM CETP for 24 h conferred 2.4-fold resistance against subsequent exposure to the 
,β-unsaturated aldehyde acrolein, indicating that the phytochemical exerts chemopreventive properties against genotoxic xenobiotics.
Abbreviations: AITC, allyl ITC; ARE, antioxidant response element; CETB, 1-cyano-3,4-epithiobutane; CETP, 1-cyano-2,3-epithiopropane; CETPent, 1-cyano-4,5-epithiopentane; CEXB, 1-cyano-3,4-epoxybutane; CEXP, 1-cyano-2,3-epoxypropane; CEXPent, 1-cyano-4,5-epoxypentane; ESP, epithiospecifier protein; GCL, glutamate–cysteine ligase; GCLM, GCL modifier; GSH, glutathione; GST, GSH S-transferase; ITC, isothiocyanate; Keap1, Kelch-like ECH-associated protein 1; LD50, lethal dose 50 (i.e. a dose of xenobiotic that kills 50% of cells); MEF, mouse embryonic fibroblast; mRNA, messenger RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NQO1, NAD(P)H:quinone oxidoreductase 1; Nrf2, NF-E2 p45-related factor 2; PKC, protein kinase C; RT-PCR, real-time polymerase chain reaction; SFN, sulforaphane; Sin, sinigrin
Received May 29, 2009; revised July 1, 2009; accepted July 21, 2009.