MTT assay was used to test the toxicity of digoxin in SW1990/Gem and Panc-1/Gem cells. As shown in Fig. 2A, 20 and 40 nM of digoxin had no significant cytotoxicity, and 80 nM of digoxin exhibited only slight cytotoxicity. To eliminate the effect of digoxin on SW1990/Gem and Panc-1/Gem cells, nontoxic doses (20, 40 and 80 nM) of digoxin were used in the following experiments. Digoxin at the doses of 20, 40 and 80 nM enhanced the cytotoxicity of gemcitabine in SW1990/Gem and Panc-1/Gem cells (Fig. 2B–C). Digoxin increased the sensitivity of SW1990/Gem cells to gemcitabine by 5.27-fold, 13.97-fold and 35.83-fold at the doses of 20, 40 and 80 nM, while it increased the sensitivity of Panc-1/Gem cells to gemcitabine by 3.91-fold, 7.17-fold and 20.43-fold at the doses of 20, 40 and 80 nM, respectively (Table 3). Meanwhile, our results demonstrated that gemcitabine in combination with digoxin dramatically inhibited cell colony formation and increased the number of cell undergoing apoptosis when compared with gemcitabine alone (Fig. 2D–I). Interestingly, digoxin at the doses of 20, 40 and 80 nM could not significantly enhance the cytotoxicity of gemcitabine in SW1990 and Panc-1 cells (Supplementary Fig. S2A–B). These results indicated that digoxin could enhance the chemosensitivity of gemcitabine in SW1990/Gem and Panc-1/Gem cells, but not in their parental cells. In addition, we evaluated the effect of digoxin on the sensitivity of SW1990/Gem and Panc-1/Gem cells to other anticancer agents including etoposide, paclitaxel, cisplatin, 5-FU, ara-C and doxorubicin and found that digoxin could significantly enhance the sensitivity of SW1990/Gem and Panc-1/Gem cells to these anticancer agents (Table 3 and Supplementary Fig. S3).
Fig. 2 Digoxin enhanced the sensitivity of SW1990/Gem and Panc-1/Gem cells to gemcitabine. (A) The cytotoxicity of digoxin to SW1990/Gem and Panc-1/Gem cells. (B–C) Reverse effects of digoxin on drug resistance of gemcitabine in SW1990/Gem and Panc-1/Gem cells. (D–F) Colony formation assay. (G–I) Flow cytometric analysis of apoptosis. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as ***P < 0.001 vs. control group, ###P < 0.001 vs. gemcitabine group.
Table 3 Effects of digoxin on the sensitivity of SW1990/Gem and Panc-1/Gem cells to different anticancer drugs.
Group SW1990/Gem Panc-1/Gem
IC50 RF IC50 RF
Gemcitabine (μM) Control 519.59 ± 31.14 1 283.47 ± 21.7 1
Digoxin 20 nM 98.55 ± 6.12 5.27 72.47 ± 6 3.91
Digoxin 40 nM 37.18 ± 4.75 13.97 39.54 ± 4.61 7.17
Digoxin 80 nM 14.5 ± 2.03 35.83 13.87 ± 2.16 20.43


 

 

 

 


Etoposide (μM) Control 633.19 ± 40.77 1 445.82 ± 30.85 1
Digoxin 20 nM 445.77 ± 22.04 1.42 327.28 ± 20.82 1.36
Digoxin 40 nM 232.98 ± 18.38 2.72 172.46 ± 10.57 2.59
Digoxin 80 nM 110.95 ± 13.63 5.71 119.34 ± 12.85 3.74


 

 

 

 


Paclitaxel (nM) Control 293.07 ± 19.72 1 216.53 ± 16.27 1
Digoxin 20 nM 209.57 ± 17.65 1.4 144.27 ± 11.57 1.5
Digoxin 40 nM 61.04 ± 4.99 4.8 67.82 ± 4.05 3.19
Digoxin 80 nM 35.77 ± 4.31 8.19 37.01 ± 4.99 5.85


 

 

 

 


Cisplatin (μM) Control 186.47 ± 11.41 1 155.53 ± 11.78 1
Digoxin 20 nM 112.87 ± 8.52 1.65 107.17 ± 7.35 1.45
Digoxin 40 nM 86.24 ± 6.39 2.16 68.15 ± 6.24 2.28
Digoxin 80 nM 51.73 ± 6.28 3.6 58.44 ± 6.06 2.66


 

 

 

 


5-FU (μM) Control 185.38 ± 14.67 1 168.39 ± 10.8 1
Digoxin 20 nM 118.23 ± 10.08 1.57 110.88 ± 7.6 1.52
Digoxin 40 nM 86.55 ± 7.17 2.14 73.46 ± 7.73 2.29
Digoxin 80 nM 55.25 ± 7.06 3.36 38.89 ± 4.72 4.33


 

 

 

 


Ara-C (μM) Control 16.88 ± 1.15 1 10.93 ± 0.96 1
Digoxin 20 nM 11.4 ± 1.07 1.48 8.5 ± 0.74 1.29
Digoxin 40 nM 6.65 ± 0.5 2.54 4.66 ± 0.3 2.35
Digoxin 80 nM 2.99 ± 0.32 5.65 1.61 ± 0.33 6.79


 

 

 

 


Doxorubicin (μM) Control 29.24 ± 2.74 1 24.4 ± 1.84 1
Digoxin 20 nM 14.03 ± 1.54 2.08 15.02 ± 1.68 1.62
Digoxin 40 nM 11.06 ± 1.19 2.64 10.82 ± 1.16 2.26
Digoxin 80 nM 6.74 ± 0.47 4.34 7.52 ± 0.92 3.24
Effects of digoxin on the sensitivity of SW1990/Gem and Panc-1/Gem cells to different anticancer drugs were detected by MTT assay. Data were expressed as mean ± SD of three independent experiments.