Nrf2/ARE activation assay

The ability of the extracts to induce Nuclear factor erythroid 2 (NF-E2)-related factor like-2 (Nrf2) activity was determined in a luciferase reporter assay by using the AREc32 cells (stable human mammary MCF-7-derived reporter cell line with a luciferase reporter gene construct that is under the control of the rat Glutathione-S-Transferase (Gsta2) Antioxidant Response Element (ARE) promoter, with eight copies of the ARE in the promoter region [14]). The transcriptional regulatory element ARE is involved in the activation of genes that code for a number of antioxidant proteins and enzymes that are protective against oxidative insults. The induction of the genes is controlled by the transcription factor Nrf-2, whose activity is usually repressed by the inhibitory factor Kelch-like ECH associated protein 1 (Keap1) that facilitates its degradation, while electrophilic agents prevent Keap1 from targeting Nrf2 for degradation [14]. The AREc32 cell line is, therefore, used to examine whether an anti-cancer drug or drug candidate can induce ARE-driven gene expression, as induction of ARE causes luciferase activity.

AREc32 cells were seeded into 96-well plates at a density of 1.2 × 10⁴ cells per well. After 24 h, the medium was discarded and 100 μl of medium containing each plant extract was added at a range of concentrations into receiving wells. Then, after another 24 h of incubation, medium was discarded, cells were washed with phosphate-buffered saline (PBS) and 20 μl of luciferase lysis buffer was added to each well, followed by a freeze-thaw cycle to achieve complete lysis. The cell lysate was then aspirated and dispensed into a white 96-well plate. 100 μl of a luciferase reporter substrate was then added to each well and luminescence measured (CLARIOstar Microplate Reader, BMG Labtech, UK) immediately. The level of luciferase activity for each treatment was compared to the basal level of luciferase activity in control cells and presented as a fold increase. Tert-butylhydroquinone (tBHQ; 25 μM) served as positive control. Each experiment was repeated three times, with three replicates in each experiment.