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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

Laetitia Moreno Y Banuls1, Adriana Katz2, Walter Miklos3, Alessio Cimmino4, Daniel M Tal2, Elena Ainbinder2, Martin Zehl5, Ernst Urban6, Antonio Evidente4, Brigitte Kopp5, Walter Berger3, Olivier Feron7, Steven Karlish2 and Robert Kiss1*

Author Affiliations

1 Laboratoire de Toxicologie; Faculté de Pharmacie; Université Libre de Bruxelles (ULB), Brussels, 1050, Belgium

2 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel

3 Department of Medicine I, Institute of Cancer Research and Comprehensive Cancer Center, Medical University Vienna, Vienna, Austria

4 Dipartimento di Scienze Chimiche, Complesso Universitario Monte Sant’Angelo, Università di Napoli Federico II, Napoli, 80126, Italy

5 Department of Pharmacognosy, University of Vienna, Vienna, 1090, Austria

6 Department of Medicinal Chemistry, University of Vienna, Vienna, 1090, Austria

7 Pole of Pharmacology & Therapeutics (UCL-FATH), Angiogenesis & Cancer Research Laboratory, Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain (UCL), Brussels, Belgium

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Molecular Cancer 2013, 12:33  doi:10.1186/1476-4598-12-33

Published: 26 April 2013

Abstract

Background

Surface-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.

Methods

The 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.

Results

Although 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.

Conclusions

Altogether, 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.

Keywords:
Cardiotonic steroids; Cardenolides; Bufadienolides; Cancer; Glycoside / aglycone forms; Lactate release - oxygen consumption