Abstract
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.
Highlights
The Na+/K+-ATPase is an enzyme that actively transports ions Na+ and K+ across cell membranes and has cardiotonic steroids (CTS—cardenolides and bufadienolides) as specific inhibitors of its pumping function
In this work we compared the effect of two chemically similar bufadienolides, telocinobufagin, and marinobufagin, on the inhibition of Na+/K+-ATPase with their cellular effects and the molecular mechanisms involved, and our results suggest that they have functional selectivity
We showed that 100 nM telocinobufagin inhibited pig kidney Na+/K+-ATPase activity by 25%, probably insufficient for a significant change in the global intracellular Na+ concentration, as shown by Cai et al for 100 nM ouabain in LLC-PK1 cells [67]
Summary
The Na+/K+-ATPase is an enzyme that actively transports ions Na+ and K+ across cell membranes and has cardiotonic steroids (CTS—cardenolides and bufadienolides) as specific inhibitors of its pumping function. Xie and coworkers have established that, besides performing the active ion transport, Na+/K+-ATPase functions as a signal transducer through protein–protein interactions, relaying extracellular signals to intracellular compartments via the activation of cascades of different protein kinases, as well as production of second messengers [1,2]. This intracellular signaling is activated by binding of CTS to Na+/K+-ATPase using the Src tyrosine kinase as a transducer. Studies show that the Na+/K+-ATPase may interact with protein kinases, phosphatases, membrane transporters, and other cellular proteins, and these interactions make this enzyme an important signal transducer [3]
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