To confirm whether digoxin-mediated reversing of gemcitabine resistance in SW1990/Gem cells-derived xenografts was Nrf2 dependent, SW1990/Gem-shControl cells and SW1990/Gem-shNrf2 cells were injected into the subdermal space of nude mice on the right flanks. As shown in Fig. 8A–C, SW1990/Gem-shControl cells-derived xenografts in the combination group had smaller tumor volume and lighter tumor weight than that in the gemcitabine group. In contrast, SW1990/Gem-shNrf2 cells-derived xenografts responded to gemcitabine and combination treatment similarly (Fig. 8E–G). Interesting, the body weight of nude mice was no significant changed in this experiment (Fig. 8D, H). Moreover, compared with SW1990/Gem-shControl cells-derived xenografts, SW1990/Gem-shNrf2 cells-derived xenografts had lower protein levels of Nrf2, NQO1, HO-1, GCLC and ABCC5 (Fig. 8I–J and Supplementary Fig. S4G–H). In addition, digoxin could markedly decrease the protein levels of Nrf2, NQO1, HO-1, GCLC and ABCC5 in SW1990/Gem-shControl cells-derived xenografts, but no significantly change of these protein levels were observed in SW1990/Gem-shNrf2 cells-derived xenografts (Fig. 8I–J and Supplementary Fig. S4G–H). Furthermore, compared with gemcitabine group, the combined treatment of gemcitabine with digoxin markedly increased cell apoptosis only in SW1990/Gem-shControl cells-derived xenografts, but not in SW1990/Gem-shNrf2 cells-derived xenografts which had a high rate of cell apoptosis with gemcitabine treatment (Fig. 8J). Interestingly, gemcitabine and digoxin in combination significantly inhibited cell proliferation compared with gemcitabine treatment alone in SW1990/Gem-shControl cells-derived xenografts (Fig. 8J). These results showed that digoxin could sensitize gemcitabine-resistant pancreatic cancer cell xenografts to gemcitabine through inhibiting Nrf2 signaling.
Fig. 8 Digoxin sensitized SW1990/Gem cells-derived xenografts to gemcitabine treatment by inhibiting Nrf2 signaling. (A–D) Digoxin sensitized SW1990/Gem-shControl cells-derived xenografts to gemcitabine treatment. (E–H) Digoxin could not sensitize SW1990/Gem-shNrf2 cells-derived xenografts to gemcitabine treatment. (I) Effects of digoxin on the protein levels of Nrf2, NQO1, HO-1, and GCLC in tumor tissues. (J) Tumor tissues were subjected to IHC-Nrf2, IHC-NQO1, IHC-HO-1, IHC-GCLC, IHC-Ki67 and TUNEL staining. All images were shown at ×200. Data were expressed as mean ± SD, n = 6. Significant differences were indicated as ***P < 0.001 vs. vehicle group, ###P < 0.001 vs. gemcitabine group. N.S., no significant.