Transcriptomic analysis of digitoxin: Synergy with doxorubicin in HER2-overexpressing MDA-MB-453 breast cancer cells

The aim of this research is to further elucidate the mechanism of action of digitoxin and explore its potential synergistic effects with doxorubicin. MDA-MB-453 breast cancer cells, characterized by HER2 overexpression and low ER levels, were exposed to digitoxin at three doses (0.1 (0.13 microM), 0.2, and 1.0 microg/ml). RNA was extracted over 6 and 24-h periods to subject to transcriptomic analysis, using IPA software. To validate the findings, cell growth inhibitory, Western blot, and enzymatic assays were performed. In addition, molecular docking was carried out to assess the interaction of digitoxin and doxorubicin with the Na⁺/K⁺-ATPase. IPA analysis indicates that the effects of digitoxin are dose and time-dependent; at the highest dose, digitoxin activates the transcription of cholesterol biosynthetic genes at early times, and the stress response gene ATF3 at later times. Key genes at the central point of the pathways altered by digitoxin include: (activated) TP53, CREB1, and TGFB1 at the highest dose at 6 and 24 h and (repressed) MYCN at the middle dose at 24 h. ATF3 also plays a role in the action of doxorubicin, and digitoxin exhibits synergy with doxorubicin in MDA-MB-453 cells. Molecular docking studies demonstrated binding potential of both digitoxin and doxorubicin to Na⁺/K⁺-ATPase, with doxorubicin showing a stronger binding affinity. Our results highlight the role of bioelectric signaling through ion channel proteins, like Na⁺/K⁺-ATPase, in cancer development. Our findings suggest it is worthwhile to study the use of digitoxin, alone or combined with doxorubicin, for treating estrogen receptor-negative breast cancer, but caution of possible risks to patients who take both drugs in combination.