As upregulation of Nrf2 protein levels were observed in SW1990/Gem and Panc-1/Gem cells, we evaluated the inhibitory effect of digoxin on the protein levels of Nrf2. We found that total and nuclear Nrf2 protein levels were markedly decreased in SW1990/Gem and Panc-1/Gem cells treated with 20, 40, and 80 nM of digoxin for 24 h (Fig. 3A–B). Subsequently, we observed the effect of 80 nM digoxin on Nrf2 protein level in SW1990/Gem and Panc-1/Gem cells at different time points. We found that the protein level of Nrf2 began to reduce from 6 h after digoxin treatment (Fig. 3C–D). Endogenous Nrf2 immunostaining further proved the inhibitory effects of digoxin on the protein expression of Nrf2 in SW1990/Gem and Panc-1/Gem cells (Fig. 3E–F). Interestingly, digoxin at the doses of 20, 40 and 80 nM could not significantly inhibit total and nuclear Nrf2 protein levels in SW1990 and Panc-1 cells (Supplementary Fig. S2C–D). Next, we investigated whether digoxin could inhibit the expressions of Nrf2 target genes in SW1990/Gem and Panc-1/Gem cells. We found that Nrf2 target genes encoding antioxidant enzymes, enzymes of glutathione synthesis and conjugation, drug metabolizing enzymes and transporters, metabolic enzymes were markedly decreased in SW1990/Gem and Panc-1/Gem cells treated with 80 nM of digoxin (Fig. 4A–B). Meanwhile, 80 nM of digoxin significantly suppressed protein expressions of NQO1, HO-1, GCLC and ABCC5 (Fig. 4C–D and Supplementary Fig. S4A–B). These results indicated that digoxin could inhibit Nrf2 signaling in SW1990/Gem and Panc-1/Gem cells.
Fig. 3 Digoxin inhibited the Nrf2 signaling in SW1990/Gem and Panc-1/Gem cells. (A–B) Effects of digoxin on the protein level of Nrf2 in SW1990/Gem and Panc-1/Gem cells. (C–D) Effects of digoxin on the protein level of Nrf2 in SW1990/Gem and Panc-1/Gem cells at different time points. (E–F) Immunostaining of endogenous Nrf2 in SW1990/Gem and Panc-1/Gem cells. All images were shown at ×200. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as **P < 0.01, ***P < 0.001 vs. control group. N.S., no significant.
Fig. 4 Digoxin inhibited the expressions of Nrf2 target genes in SW1990/Gem and Panc-1/Gem cells. (A–B) Effects of digoxin on the expressions of Nrf2 target genes in SW1990/Gem and Panc-1/Gem cells. The colors of the heatmap reflect log2-expression levels of Nrf2 target genes in SW1990/Gem and Panc-1/Gem cells. (C–D) Effects of digoxin on protein levels of NQO1, HO-1 and GCLC in SW1990/Gem and Panc-1/Gem cells. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as **P < 0.01, ***P < 0.001 vs. control group. N.S., no significant.