Telocinobufagin and Marinobufagin Produce Different Effects in LLC-PK1 Cells: A Case of Functional Selectivity of Bufadienolides

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.

Marinobufagenin Inhibits Neutrophil Migration and Proinflammatory Cytokines

“…This isoform is possibly the only one expressed in peritoneal macrophages [39]. Additionally, we have shown here and elsewhere [25, 40] that marinobufagenin is 20-80 times less potent than ouabain. Indeed, 1 mM marinobufagenin did not inhibit the murine Na + /K + -ATPase α 1 isoform, and the inhibition of the other isoforms, α 2 and α 3, only begins at around 1 μ M (IC 50 = 3.8 μ M).…”

“…Differences of the affected cytokines may be associated with the pharmacological profile of these steroids. We have recently shown that structurally related bufadienolides generate distinct cellular effects probably by a particular selection of intracellular signaling pathways, which is called functional selectivity [25]. At last, the reduction in cytokine levels and polymorphonuclear migration caused by marinobufagenin suggests an anti-inflammatory role in vivo.…”

“…Marinobufagenin has been considered a unique cardiotonic steroid regarding its selectivity to the ouabain-resistant rodent α 1 isoform; namely, in contrast to any other bufadienolide or cardenolide, it was shown to be more potent to inhibit the rat Na + /K + -ATPase α 1 isoform than α 2/ α 3 isoforms [41], and it has been claimed as an endogenous Na + /K + -ATPase α 1 isoform ligand [42]. Nevertheless, we [25, 43] and others [44] have found that marinobufagenin has a selectivity profile against rodent Na + /K + -ATPase α isoforms similar to all known cardiotonic steroids ( α 2/ α 3 > α 1) and is a poor α 1 ligand. The reason of these discrepancies is unknown and is now being better investigated.…”

“…Marinobufagenin was purified from the Rhinella schneideri toad venom at the Institute of Biomedical Sciences (ICB) of the Federal University of Rio de Janeiro (UFRJ) as reported elsewhere [25]. A marinobufagenin sample was solubilized in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/mL and stored at -20°C.…”

Bufadienolides induce apoptosis and autophagy by inhibiting the AKT signaling pathway in melanoma A‑375 cells

“…hepatoma, gallbladder carcinoma and lung cancer (16). in previously published studies, bufadienolide compounds have been demonstrated to induce arrest of the tumor cell cycle and apoptosis (17,18). in addition, bufadienolides were revealed to markedly inhibit proliferation and induce autophagy in liver cancer cells (19), and alterations in the expression levels of microtubule-associated proteins 1a/1B light chain 3-ii (lc3-ii), p62, Bax, Bcl-2, cyclin d1 and caspase-3 are involved in the underlying mechanism (20).…”