Antioxidant response element (ARE), also termed as the electrophile response element, is a cis-regulatory element or enhancer sequence, which is found in the promoter region of several genes encoding detoxification enzymes and cytoprotective proteins. The core sequence of ARE includes 5′-TGACNNNGC-3′ and responds mainly to oxidative stress inducers (Rushmore et al., 1991). Under conditions of oxidative stress, Nrf2 dissociates from Keap1, translocates into the nucleus, forms heterodimer with small Maf family of transcription factors, and binds to the ARE to transcriptionally activate antioxidant genes. Bach1 (BTB and CNC homology1) is a transcriptional repressor of ARE. Under normal physiological conditions, Bach1 forms a dimer with Maf protein and prevents Nrf2 binding to ARE. Also Bach1 undergoes rapid nuclear export and proteasomal degradation in response to Nrf2/ARE inducers.
Reactive oxygen species (ROS) and reactive nitrogen species acting on the antioxidant response elements (AREs) modulate the expression of cytoprotective enzymes including quinone reductase (QR1), UDP-glucuronyl transferase and glutathione S-transferase. Many transcription factors are redox sensitive including activator protein-1(AP-1), NF-κB, Nrf2, p53 and the glucocorticoid receptor. Gene expression responses to oxidative stress are necessary to ensure cell survival and are largely attributed to specific redox-sensitive transcription factors . AP-1 is responsive to low levels of oxidants resulting in AP-1/DNA binding and an increase in gene expression. AP-1 activation is due to the induction of JNK activity by oxidants resulting in the phosphorylation of serine 63 and serine 73 in the c-Jun transcription domain. With high concentration of oxidants, AP-1 is inhibited and genes expression is impeded.
Similarly, NF-κB contains a redox-sensitive critical cysteine residue (cysteine 62) in the p50 subunit that is involved in DNA binding. NF-κB is normally sequestered in the cytoplasm by IκB, but under oxidative conditions, IκB is phosphorylated by IκB kinase (IKK), ubiquitinated and subsequently degraded. ROS production appears to be necessary to initiate the events leading to the dissociation of the NF-κB/IκB complex, but excessive ROS production (oxidative stress) results in the oxidation of cysteine 62 which does not affect its translocation to the nucleus but rather interferes with DNA binding and decreases gene expression .
Pivotal to the antioxidant response typical in mammalian homeostasis and oxidative stress is the important transcription factor Nrf-2. Nrf-2 is indispensible to cellular defense against many chemical insults of endogenous and exogenous origin, which play major roles in the etiology pathogenesis of many cancers and inflammation-related diseases such as inflammatory bowel disease and Parkinson’s disease. Under homeostatic conditions, Nrf-2 is mainly sequestered in the cytoplasm by a cytoskeleton-binding protein called Keap-1. The ARE controls the expression of cytoprotective enzymes including quinone reductase (QR1), UDP-glucuronyl transferases, and glutathione S-transferases and is regulated, at least in part, by levels of the transcription factor Nrf-2.
The array of genes coordinately modulated through Nrf-2 included phase I drug-metabolizing enzymes, phase II detoxification and antioxidant genes, and phase III transporter genes. The regulation of these genes could have significant effects on the prevention of tumor initiation by enhancing the cellular defense system, activation of procarcinogens/reactive intermediates, and increasing the excretion/efflux of reactive carcinogens or metabolites. In an elegant study, WFA has been shown to activate mitogen-activated protein kinase ERK1/2 and RSK and subsequently leading to activation of ETS-like transcription factor-1 (ELK1)-CHOP and upregulation of the death receptor 5 (DR5) . Accordingly, WFA treatment inhibited breast tumor formation in an animal model of breast cancer. However, mechanism of WFA-mediated ERK1/2 phosphorylation is not known.
Widodo et al. have recently showed that WFA promoted selective killing of cancer cells involving ROS stress and mitochondrial damage. Additionally, treatment of human myeloid leukemia cells with WFA increased cell death. A recent study reported that WFA induced apoptosis in human head and neck cancer cells through ROS production. WFA also induced cyclooxygenase-2 (COX-2) expression and production of prostaglandin E2. However, inhibition of COX-2 by NS-398, a selective inhibitor of COX-2, did not abrogate WFA-mediated apoptotic events. WFA inhibited DNA binding of NF-κB and promoted nuclear cleavage of p65/Rel by activated caspase-3. N-acetylcysteine reversed all these events suggesting thereby a prooxidant effect of WFA. Novel norwithanolides, extracted from Deprea subtriflora D’Arcy (Solanaceae) have also been reported to induce QR expression. Furthermore, tubocapsenolide A (TA) induced apoptosis in breast cancer cells partly due to transient increase in ROS and decrease in cytoplasmic glutathione content. Thiol antioxidant, N-acetylcysteine, prevented all of the TA-induced effects, while nonthiol antioxidants such as trolox and vitamin C were ineffective to prevent TA-mediated cell death. Other withanolides have displayed ARE-inducing activity. Structure–activity relationship analysis revealed that jaborosalactone 5 promoted antiproliferative activity and induction of quinone reductase 1 activity.