Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase

Abstract

BackgroundSurface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK.MethodsThe in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides.ResultsAlthough cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation.ConclusionsAltogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.

Cardiac glycosides induced toxicity in human cells expressing α1-, α2-, or α3-isoforms of Na-K-ATPase

“…Upon inhibition of Na ϩ -K ϩ -ATPase with CGs, one of the immediate consequences is inhibition of its ion-pumping activity and as a result dissipation of ion gradients, most likely a scenario of triggering of apoptotic cell death (18,31,34). This conclusion also fits well with a recent finding that the K 0.5 for inhibition of growth of various cell lines by different CGs is linearly related to the K i for inhibition of Na ϩ -K ϩ -ATPase activity of the purified human ␣1␤1-isoform (28). In parallel, internalization of the pump, which is the cellular method of removing inhibited pumps from the plasma membrane, takes place.…”

Effects of active bufadienolide compounds on human cancer cells and CD4+CD25+Foxp3+ regulatory T cells in mitogen-activated human peripheral blood mononuclear cells

“…Indeed, bufalin is one of the well-studied active compounds with respect to its in vitro activity against a wide spectrum of cancer cell lines including leukemia, breast, prostate, gastric and liver cancer (6), and has been demonstrated to induce apoptosis of these cancer cells via modulation of several signaling pathways such as activation of the intrinsic mitochondrial apoptosis pathway and Fas, downregulation of the PI3K/Akt or MEK/ERK pathway and activation of the MAPK cascade including JNK (6)(7)(8). Despite being key active bufadienolide compounds (33,34), the antitumor activity of gamabufotalin and arenobufagin, and their underlying molecular mechanisms have not yet been well determined. Until quite recently, arenobufagin has been demonstrated to induce apoptosis and autophagy through inhibition of the PI3K/Akt/mTOR pathway, and intercalate with DNA leading to G 2 cell cycle arrest in human hepatocellular carcinoma cells (35,36).…”

Bufadienolides of Kalanchoe species: an overview of chemical structure, biological activity and prospects for pharmacological use

“…Studies on human liver microsomes (HLMs) indicated that hydroxylation and dehydrogenation might be the major metabolic pathways of bufadienolides (Han et al 2016 ). Molecular mechanisms of anticancer activities of hellebrin and its aglycone hellebrigenin (compounds that were also found in Kalanchoe plants) were described by Moreno et al ( 2013 ). According to those authors, both compounds are able to bind to the alpha subunits of the Na + /K + -ATPase and display similar growth inhibitory effects in different cancer cell lines, i.e.…”