Reactive oxygen species (ROS), including radicals such as superoxide, nitric oxide, and hydroxyl radicals and nonradical species such as hydrogen peroxide, ozone, and peroxynitrates, function in normal cells to maintain homeostasis via redox pathways (1-3). In some cancer cell lines, a modest increase in forms of ROS can enhance cell proliferation, survival, and drug resistance; however, further increases in ROS that cannot be attenuated by intracellular redox systems result in cell death (3). ROS levels are higher in cancer than in noncancer cells, and drug-induced elevation of ROS is a "way to selectively kill cancer cells without causing toxicity to normal cells" (3). Drug-induced ROS in cancer cells may be due to inhibition or inactivation of redox pathway enzymes or due to direct effects on mitochondria, which include opening of the permeability transition pore complex, resulting in decreased mitochondrial membrane potential (MMP) and activation of proapoptotic cascades (3-5).Several anticancer drugs that induce ROS, including arsenic trioxide, the methyl ester of 2-cyano-3,12-dioxo-oleana-1,9-dien-28-oic acid (CDDO-Me), curcumin, betulinic acid, a synthetic nonsteroidal anti-inflammatory drug (NSAID) (GT-094), and celastrol also downregulate specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 and prooncogenic Sp-regulated genes (6-11). Similar effects have been reported for H 2 O 2 , t-butylhydroperoxide, and pharmacologic doses of ascorbate that induce H 2 O 2 (12). Moreover, the effects of ROS inducers and prooxidants on growth inhibition, induction of apoptosis, and downregulation of Sp proteins and Sp-regulated genes are attenuated after cotreatment with antioxidants (6-12). Induction of ROS by CDDO-Me, GT-094, betulinic acid, and celastrol decreases expression of Sp transcription factors through downregulation of microRNA 27a (miR-27a) and/or miR-17/miR-20a and induction of the miR-regulated Sp repressors ZBTB10 and ZBTB4, respectively (6-9). The relationship between drug-induced ROS and disruption of miR-dependent suppression of ZBTB transcriptional repressors suggests that this may be an important underlying mechanism of action for other ROS-inducing anticancer agents.Phenethylisothiocyanate (PEITC) and other isothiocyanates (ITCs) have been linked to the chemoprevention and anticancer activity associated with consumption of cruciferous vegetables (13)(14)(15)(16)(17). PEITC and other ITCs inhibit cancer cell growth, survival, and angiogenesis, and these activities are accompanied by downregulation of total and activated STAT3 protein (18), suppression of NF-B (and p65 downregulation) (16, 17), and decreased expression of epidermal growth factor receptor (EGFR) (18), genes involved in migration/invasion (19,20), and bcl-2 and cyclins (21-23). Silencing of Sp1 and Sp1/Sp3/Sp4 (combined) by RNA interference (RNAi) shows that many of these same genes are regulated by Sp transcription factors in cancer cell lines (8)(9)(10)(11)24). PEITC and the related benzyl analog (BITC) also induce ROS ...

NR4A1 (Nur77, TR3) is an orphan nuclear receptor that is overexpressed in pancreatic cancer and exhibits pro-oncogenic activity. RNAi interference of NR4A1 expression in Panc-1 cells induced apoptosis and subsequent proteomic analysis revealed the induction of several markers of endoplasmic reticulum (ER) stress including glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), and activating transcription factor-4 (ATF-4). Treatment of pancreatic cancer cells with the NR4A1 antagonist 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH), gave similar results. Moreover, both NR4A1 knockdown and DIM-C-pPhOH induced reactive oxygen species (ROS), and induction of ROS and ER stress by these agents was attenuated after co-treatment with antioxidants. Manipulation of NR4A1 expression coupled with gene expression profiling identified a number of ROS metabolism transcripts regulated by NR4A1. Knockdown of one of these transcripts, thioredoxin domain containing 5 (TXNDC5), recapitulated the elevated ROS and ER stress; thus, demonstrating that NR4A1 regulates levels of ER stress and ROS in pancreatic cancer cells to facilitate cell proliferation and survival. Finally, inactivation of NR4A1 by knockdown or DIM-C-pPhOH decreased TXNDC5, resulting in activation of ROS/ER stress and pro-apoptotic pathways.