Original Contribution
4-Ketopinoresinol, a novel naturally occurring ARE activator, induces the Nrf2/HO-1 axis and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling

https://doi.org/10.1016/j.freeradbiomed.2011.12.012Get rights and content

Abstract

The Nrf2/ARE pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and has been considered a potential target for cancer chemoprevention because it eliminates harmful reactive oxygen species or reactive intermediates generated from carcinogens. The objectives of this study were to identify novel Nrf2/ARE activators and to investigate the mechanistic signaling pathway involved in the activation of Nrf2-mediated cytoprotective effects against oxidative-induced cell injury. A stable ARE-driven luciferase reporter cell line was established to screen a potentially cytoprotective compound. 4-Ketopinoresinol (4-KPR), the (α–γ) double-cyclized type of lignan obtained from adlay (Coix lachryma-jobi L. var. ma-yuen Stapf), activates ARE-driven luciferase activity more effectively than the classical ARE activator tert-butylhydroquinone. 4-KPR treatment resulted in a transient increase in AKT phosphorylation and subsequent phosphorylation and nuclear translocation of Nrf2, along with increased expression of ARE-dependent cytoprotective genes, such as heme oxygenase-1 (HO-1), aldo-keto reductases, and glutathione synthetic enzyme. 4-KPR suppresses oxidative stress-induced DNA damage and cell death via upregulation of HO-1. Inhibition of PI3K/AKT signaling by chemical inhibitors or RNA interference not only suppressed 4-KPR-induced Nrf2/HO-1 activation, but also eliminated the cytoprotective effect against oxidative damage. These observations in an ARE-regulated gene system suggest that 4-KPR is a novel Nrf2/ARE-mediated transcription activator, activates the Nrf2/HO-1 axis, and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling.

Graphical abstract

Highlights

► 4-Ketopinoresinol (4-KPR), the (α–γ) double-cyclized type of lignan, is a novel Nrf2/ARE-mediated transcription activator. ► 4-KPR suppresses oxidative stress-induced DNA damage and cell death via the Nrf2/HO-1 axis. ► 4-KPR induces Nrf2/HO-1-mediated cytoprotective effects against oxidative damage via PI3K/AKT signaling.

Section snippets

Materials

4-KPR was purified from methanol extracts of the adlay hull, and its chemical structure was analyzed using 1H NMR, 13C NMR, infrared, and EI–MS spectra [15]. HPLC and EI–MS established the purity of 4-KPR as greater than 99% (data not shown). Hydrogen peroxide (H2O2), t-BHQ, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2′,7′-chlorofluorescein diacetate (DCFH-DA), zinc protoporphyrin-IX (ZnPPIX), LY294002, wortmannin, SB203580, PD98059, U0126, H-7, and Ro-31-8220 were

Establishment of a stable ARE-driven luciferase reporter cell line for screening of novel Nrf2 modulators

To generate an optimized luciferase reporter system for compound screening, the DNA fragments containing 2, 4, 9, or 15 copies of the ARE (GTGACAAAGCACCC) were subcloned into the pGL3 vector. Several human oral cell lines, including KB, DOK, Fadu, HSC-3, OECM-1, SCC-15, SCC-25, Tu183, and UMSCC-1, were used to test these reporter constructs, and a well-known Nrf2 activator, t-BHQ [21], represented a positive control. After transient transfection and 50 μM t-BHQ exposure, HSC-3 cells showed a

Discussion

A potential chemopreventive strategy involving the induction of a battery of cytoprotective genes has become a recent focus of attention in cancer research. These proteins protect cells against the toxic and carcinogenic effects of reactive chemical metabolites via several mechanisms, which generally share an ability to detoxify electrophiles [2], [3], [4], [7], [8]. Numerous studies have suggested, and to differing extents demonstrated, that ARE sequences present in the upstream promoter

Acknowledgments

The study was supported by grants from the National Health Research Institutes (CA-100-PP-05), the National Science Council (NSC98-2320-B-400-003-MY3), and the Department of Health (DOH99-TD-C-111-004), Taiwan, Republic of China. We thank Dr. Jeffrey S. Chang and Ms. Chia-Rung Tsai for the statistics support.

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