Fig 1.
Digoxin inhibits the viability of various types of lung cancer cells.
Five lung cancer cell lines, (a) A549, (b) H3255, (c) H1975, (d) PC9 and (e) PC9/gef, were treated with 6 different doses (0, 0.01, 0.05, 0.1, 1 and 5 μM) of digoxin at different time points (0, 24, 48, 72 and 96 h), and cell viability was measured using the PrestoBlue Cell Viability reagent after treatment. Quantitative data are presented as the mean±SD (n = 3); *p<0.05 compared with solvent control (0.1% DMSO) at the corresponding time point.
Fig 2.
Suppression of in vitro colony formation, migration and invasion capabilities of A549 cells by digoxin treatment.
A549 cells grown in a culture dish with (b) or without (a) soft agar were treated with the desired concentrations of digoxin and then subjected to colony formation analyses. (c) Digoxin decreases A549 cell migration ability, as assessed by the transwell migration assay. (d) Invasiveness of A549 cells treated with digoxin was evaluated using a Matrigel-based transwell invasion assay. *p<0.05 compared with vehicle-treated control (0.1% DMSO). Each treatment was independently performed in triplicate.
Fig 3.
Digoxin inhibits phosphorylation of Src and the related EGFR/STAT3 pathway in various types of lung cancer cells in a dose- and time-dependent manner.
(a) A549 cell viability was measured using the Prestoblue Cell Viability Reagent at different time points (0, 24, 48, 72 and 96 h) with or without 100 nM digoxin or dasatinib. (b) Analysis of phosphorylated and non-phosphorylated Src, EGFR and STAT3 in A549 cells in a dose-dependent manner. The cancer cells treated with or without various concentrations of digoxin (50, 100, 250 and 500 nM) or 100 nM dasatinib for 24 h were analyzed by Western blot. (c) Analysis of phosphorylated and non-phosphorylated Src, EGFR and STAT3 in A549 cells in a time-dependent manner. After digoxin treatment (250 nM) for 2, 4, 8, 12 or 24 h, the cancer cells were harvested and analyzed by Western blot. The effect of digoxin on Src, EGFR, and STAT3 in (d) H3255 cells and (e) H1975 cells. GAPDH was used as an internal control. Each treatment was independently performed in triplicate. (f) Effect of Src Y527F on the phosphorylation of STAT3 and FAK after digoxin treatment in A549 cells. Mock and EGFP-Src Y527F transfectants were treated with 100 or 250 nM of digoxin for 24 h. The phosphorylation and total protein levels of Src, STAT3 and FAK were detected by Western blotting. GAPDH was used as an internal control. Each treatment was independently performed in triplicate. (g) Digoxin inhibits the viability of cells transfected with the Src Y527F mutant. After transfection with Src Y527F and treatment with 100 or 250 nM of digoxin for 24 h, A549 cell viability was determined by PrestoBlue Cell Viability reagent. Quantitative data are presented as the mean±SD (n = 3); *p<0.05 compared with the vehicle control (0.1% DMSO) of EGFP-Src Y527F transfectant.
Fig 4.
The effect of digoxin on cell signaling.
Western blotting analyses of phosphorylated and non-phosphorylated PI3K, AKT, MEK1/2, ERK, FAK, SAPK/JNK, paxillin and p130Cas in (a) A549, (b) H3255, and (c) H1975 cell lines after a 24-h treatment with 100, 250 or 500 nM digoxin. The solvent control was 0.1% DMSO; GAPDH was used as an internal control. (d) Western blotting analyses of phosphorylated and total ERK, MEK1/2 and AKT in A549 cells after a 24-h treatment with 10 μM of U0126 and 100 or 250 nM of digoxin. GAPDH was used as an internal control. (e) ERK inhibitor has no effect on digoxin-induced cell death. A549 cells were treated with the designated concentrations of U0126 and digoxin. The cell viability was measured by PrestoBlue Cell Viability reagent. Data are presented as means ± S.D. (n = 3).
Fig 5.
Inhibition of mRNA expression of Src, EGFR, STAT3 and FAK in digoxin-treated cells.
(a) A549 cells were pretreated with or without MG132 (10 μM) for 2 h, then treated with digoxin (250 nM) or 0.1% DMSO (solvent control) and collected at 24 h. Protein expression was evaluated by Western blot analysis. GAPDH was a control for protein loading and transfer. (b) Cells were treated with digoxin (100 and 250 nM) for 24 h, and the detection of Src, EGFR, STAT3 and FAK mRNA expression was determined by real-time RT-PCR. *p<0.05, significantly different from the vehicle-treated control (0.1% DMSO). Each experiment was independently performed triplicate.
Fig 6.
A proposed model of digoxin-mediated anticancer function.
In digoxin-treated cells, the phosphorylation of Src and its related proteins was inhibited, which may lead to the inhibition of lung cancer cell proliferation, migration, and invasion.