Triterpenoid electrophiles (avicins) activate the innate stress response by redox regulation of a gene battery

Abstract

Avicins are proapoptotic and anti-inflammatory triterpene electrophiles isolated from an Australian desert tree, Acacia victoriae. The presence of two alpha,beta unsaturated carbonyl groups (Michael reaction sites) in the side chain of the avicin molecule prompted us to study its effects on NF-E2-related factor 2 (Nrf2), a redox-regulated transcription factor that controls the expression of a battery of detoxification and antioxidant proteins via its binding to antioxidant response element (ARE). Avicin D-treated Hep G2 cells showed translocation of Nrf2 into the nucleus and a time-dependent increase in ARE activity. These properties were sensitive to DTT, suggesting that avicins affect one or more critical cysteine residues, probably on the Keap1 molecule. Downstream of ARE, an activation of a battery of stress-induced proteins occurred. The implications of these findings were evaluated in vivo in mouse skin exposed to an ancient stressor, UV light. Avicins inhibited epidermal hyperplasia, reduced p53 mutation, enhanced apoptosis, decreased generation of 8-hydroxy-2'-deoxyguanosine, and enhanced expression of NADPH:quinone oxidoreductase 1 and heme oxygenase-1. These data, combined with our earlier published work, demonstrate that avicins represent a new class of plant stress metabolites capable of activating stress adaptation and suppressing proinflammatory components of the innate immune system in human cells by redox regulation. The relevance for treatment of clinical diseases in which stress responses are dysfunctional or deficient is discussed.

Figure 1

Nuclear localization of Nrf2 and activation of ARE-mediated gene expression. (a) Hep G2 cells transfected with EGFP-Nrf2 were either untreated or treated with avicin D (2 μg/ml) for 1 hour at 37°C. Nuclear localization of Nrf2 was studied by fluorescence microscopy (H&E staining; ×400). (b) Hep G2 cells were treated with avicin D (2 μg/ml) for 0–4 hours at 37°C. Western blot analysis of cytoplasmic and nucleaE-LUC plasmid, and LUC activity was measured as described in Methods. Dose response of ARE activation was studied using 0–2 μg/ml of avicin D for 2 hours at 37°C (c), and kinetics of ARE activation was studied using 2 μg/ml of avicin D for 0–16 hours at 37°C (d). (e) Effect of DTT (100 μM) on ARE activation was studied by application of DTT to cells for 2 hours, before, after, or along with avicin D (2 μg/ml).

Figure 2

Effect of avicin D on NQO1 and HO-1 enzymes. (a) Expression of NQO-1 was studied in Hep G2 cells treated with 0–2 μg/ml of avicin D for 2 hours, by Western blot analysis. (b and c) Dicoumarol-sensitive NQO1 activity was assayed in Hep G2 cells treated with 0–2 μg/ml of avicin D for 2 hours (b) and 2 μg/ml of avicin D for 0–4 hours (c), as described in Methods. NQO1 activity was expressed in nanomoles of dichlorophenolindohenol (DCPIP) reduced per minute per milligram protein. (d) Hep G2 cells treated with avicin D (2 μg/ml) for 0–16 hours were analyzed for changes in levels of HO-1 by Western blot analysis. (e) Hep G2 cells treated with avicin D (2 μg/ml) for 0–24 hours were assayed for bilirubin levels as described in Methods. (f) Synthesis of ferritin was studied in Hep G2 cells treated with avicin D (2 μg/ml) for 16–24 hours.

Figure 3

Effect of avicin D on TRX_red (a), GSH (b), GPx (c), and GST levels (d). Hep G2 cells were treated with avicin D (2 μg/ml) for 0–300 minutes, and cells were lysed by the freeze-thaw method. Levels or activity of the different proteins were measured as described in Methods. The results shown for each of the proteins are representative of three different assays.

Figure 4

Representative microphotographs showing the effect of AE on epidermal hyperplasia (a and b; H&E staining; ×200), p53 mutations (c and d; H&E staining; ×200), and apoptosis (e and f; H&E staining; ×400) in SKH-1 hairless mouse skin. Mice treated with acetone (a, c, and e) or 0.4 mg of AE (b, d, and f) were irradiated with UVB as described in Methods.

Figure 5

(a) Effect of AE on 8-OH-dG formation. Skin sections from mice treated with AE after or before exposure to UVB were analyzed for 8-OH-dG levels as described in Methods. (b) Effect of AE on expression of NQO1 in mouse skin. Mice treated with acetone (control) or AE (0.4 mg) were exposed to UV. Immunohistochemistry of NQO1 (H&E staining; ×400) was done as described in Methods.

Figure 6

A proposed model of avicin-induced enhancement of the innate stress response. By redox regulation of thiols, avicins have opposing effects on NF-κB–mediated and Nrf2-mediated transcription, resulting in downregulation of proinflammatory components of the innate immune response and upregulation of the intrinsic stress response. The early and late induction of Nrf2-regulated proteins enables a prolonged protective effect. Avicins’ proapoptotic response to stress, reported earlier, is functionally separate but linked to the enhancement of innate stress response. 4-HNE, 4-hydroxy-2-nonenal. OH•, hydroxyl radical.