Bufadienolides are cardiotonic steroids (CTS) identified in mammals. Besides Na⁺/K⁺-ATPase inhibition, they activate signal transduction via protein⁻protein interactions. Diversity of endogenous bufadienolides and mechanisms of action may indicate the presence of functional selectivity and unique cellular outcomes. We evaluated whether the bufadienolides telocinobufagin and marinobufagin induce changes in proliferation or viability of pig kidney (LLC-PK1) cells and the mechanisms involved in these changes. In some experiments, ouabain was used as a positive control. CTS exhibited an inhibitory IC50 of 0.20 (telocinobufagin), 0.14 (ouabain), and 3.40 μM (marinobufagin) for pig kidney Na⁺/K⁺-ATPase activity and concentrations that barely inhibited it were tested in LLC-PK1 cells. CTS induced rapid ERK1/2 phosphorylation, but corresponding proliferative response was observed for marinobufagin and ouabain instead of telocinobufagin. Telocinobufagin increased Bax:Bcl-2 expression ratio, sub-G0 cell cycle phase and pyknotic nuclei, indicating apoptosis. Src and MEK1/2 inhibitors blunted marinobufagin but not telocinobufagin effect, which was also not mediated by p38, JNK1/2, and PI3K. However, BIO, a GSK-3β inhibitor, reduced proliferation and, as telocinobufagin, phosphorylated GSK-3β at inhibitory Ser9. Combination of both drugs resulted in synergistic antiproliferative effect. Wnt reporter activity assay showed that telocinobufagin impaired Wnt/β-catenin pathway by acting upstream to β-catenin stabilization. Our findings support that mammalian endogenous bufadienolides may exhibit functional selectivity.
Keywords: ERK1/2; GSK-3β; Na+/K+-ATPase; Wnt/β-catenin pathway; bufadienolides; cardiotonic steroids; functional selectivity.