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.

<p>(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.