Mechanism of Action of Phenethylisothiocyanate and Other Reactive Oxygen Species-Inducing Anticancer Agents

Abstract

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 ...

Mechanism of Metformin-dependent Inhibition of Mammalian Target of Rapamycin (mTOR) and Ras Activity in Pancreatic Cancer

“…Thus, Sp transcription factors are not only important as prognostic factors for pancreatic cancer patients but also regulate multiple pro-oncogenic pathways/genes in pancreatic cancer cells. Moreover, knockdown of Sp1 in pancreatic cancer cells decreases growth and invasion and induces apoptosis, confirming the pro-oncogenic functions of this factor (49). These results suggest that drugs such as metformin and other agents (31)(32)(33)(34)(35)(36)39) that target Sp1, Sp3, and Sp4 represent a class of new mechanism-based drugs that can be used in combination therapies for treating this deadly disease.…”

“…The importance of targeting Sp transcription factors in pancreatic cancer was confirmed by knockdown of Sp1, which resulted in inhibition of growth and invasion and induction of apoptosis (49). Moreover, high Sp1 expression in pancreatic tumors is a prognostic factor for decreased pancreatic cancer patient survival (50).…”

Benzyl Isothiocyanate (BITC) Induces Reactive Oxygen Species-dependent Repression of STAT3 Protein by Down-regulation of Specificity Proteins in Pancreatic Cancer

“…Drugs that target Sp TFs gave responses similar to those observed after Sp knockdown, and this includes decreased expression of several pro-oncogenic genes, including bcl-2, survivin, p65NFB, ␤-catenin, multiple receptor tyrosine kinases, vascular endothelial growth factor (VEGF), and its receptors (22)(23)(24)(25)(26)(27)(28)(29)(30). We recently reported that many of the responses/ genes affected by PEITC in pancreatic cancer cells were due to induction of ROS and ROS-dependent down-regulation of Sp1, Sp3, Sp4, and pro-oncogenic Sp-regulated genes (30). These results complement studies showing that hydrogen peroxide and t-butylhydroperoxide also down-regulate Sp proteins (22,23,27).…”

Targeting Apoptosis Pathways in Cancer—Letter

“…For example, the authors have summarized the effects of various proapoptotic agents that act through two interrelated pathways, namely "mitochondrial-mediated" and "reactive oxygen species (ROS)-mediated" apoptosis because drugs targeting mitochondria often induce ROS. Previous studies in this laboratory have demonstrated that natural products such as curcumin, betulinic acid, phenethyl isothiocyanate (PEITC), piperlongumine, and celastrol, which induce ROS and apoptosis, also downregulate specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, which are highly expressed in cancer cells (2)(3)(4)(5)(6). For most of these studies, compound-induced apoptosis and compound-induced downregulation of Sp transcription factors were reversed in cancer cells after cotreatment with antioxidants.…”

“…The high expression of Sp transcription factors in cancer cells is due, in part, to suppression of the transcriptional (Sp) repressors ZBTB10/ZBTB34 and ZBTB4 by microRNA-27a (miR-27a) and miR-20a/miR-17-5p, respectively (6)(7)(8). These microRNAs are members of the miR-23a 27a 24-2 and miR-17-92 clusters, which are overexpressed in multiple cancer cells and tumors.…”