Original Contribution4-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
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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