Figures & data
Figure 1 Schematic representation of the Nrf2/Keap1 intracellular pathway.
Notes: Under normal conditions, Keap1 binds Nrf2 in the cytoplasm and promotes both the Cullin-3 containing ubiquitin E3 ligase ubiquitination of Nrf2 and its targeting for degradation by the proteasome. When Nrf2 is stabilized through electrophiles, oxidants, or other agents that can interact with Keap1 cysteine thiols, or by agents that increase kinase-dependent phosphorylation of Nrf2, it can accumulate in the nucleus, form heterodimers with small Maf proteins, and bind with the ARE of target genes.
Abbreviations: Nrf2, nuclear factor (erythroid-derived 2)-like 2; ARE, antioxidant response element; Keap1, Kelch-Like ECH-Associated Protein 1.
Abbreviations: Nrf2, nuclear factor (erythroid-derived 2)-like 2; ARE, antioxidant response element; Keap1, Kelch-Like ECH-Associated Protein 1.
Figure 2 Typical ARE sequence logo. An ARE sequence logo was generated using predicted Nrf2 binding sites in the regulatory regions of the highest upregulated genes from Hybertson et al,Citation40 in which cultured human umbilical vein endothelial cells were treated with a phytochemical Nrf2 activator mixture.
Notes: Using oPOSSUM 3.0 web-based software (http://opossum.cisreg.ca/oPOSSUM3/) we evaluated Nrf2 binding sites in the 25 genes with the highest fold-induction from our previous phytochemical Nrf2 activation study using Protandim® (LifeVantage, Inc., Sandy, UT, USA; a mixture of extracts of milk thistle, bacopa, ashwagandha, green tea, and turmeric),Citation40 examining 10,000 bases upstream and 5,000 bases downstream of the transcription start site and aligning potential Nrf2 binding sites.Citation43,Citation44 Thirty one Nrf2 binding sites were identified in 14 of the 25 genes that were upregulated by the Nrf2 activator.Citation40
Abbreviations: Nrf2, nuclear factor (erythroid-derived 2)-like 2; ARE, antioxidant response element.
Abbreviations: Nrf2, nuclear factor (erythroid-derived 2)-like 2; ARE, antioxidant response element.
Table 1 Potential Nrf2 inhibitors
Figure 3 Luciferase activity versus concentration of two nutritional supplements.
Notes: Both of the tested nutritional supplements Protandim® (Lifevantage, Inc, Sandy, UT, USA) – reported by the manufacturer to be an Nrf2 activator – and Supercritical Antioxidants™ (New Chapter, Inc, Brattleboro, VT, USA), not reported by the manufacturer to be an Nrf2 activator – upregulated the Nrf2-driven luciferase reporter gene expression in the AREc32 cell line, depicted as relative light units. Briefly, AREc32 cells were grown by standard methods, then trypsinized, counted and seeded at 20,000 cells/well on 24-well plates. After 24 hours the cells were reattached and growing, and were treated with varied concentrations of the test material extracts in duplicate. Protandim® was obtained by BG as a gift from Professor Joe McCord; Supercritical Antioxidants™ was obtained by retail purchase. The agents were extracted overnight with 95% ethanol in 15 mL tubes on a rocking table. The extract was obtained by centrifugation and then added to the wells in an appropriate concentration range by dilution into phosphate buffer solution, with final volumes added to culture wells ranging from 1 to 10 μL/well. An ethanol blank at the highest 10 μL/well level was used as the zero control. The cells were incubated for 24 hours, then washed and lysed, after which the lysate was assayed for luciferase activity by measuring luciferin-dependent chemiluminescence, reported as relative light units for the contents of each well. Each assay was performed in duplicate. Luciferase activity correlates directly in this case with Nrf2 activation.
Abbreviations: Nrf2, nuclear factor (erythroid-derived 2)-like 2; ARE, antioxidant response element.
Abbreviations: Nrf2, nuclear factor (erythroid-derived 2)-like 2; ARE, antioxidant response element.
