title: 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: 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.
authors.affiliation: Laboratoire de Toxicologie, Faculté de Pharmacie, Université Libre de Bruxelles, Brussels 1050, Belgium.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Moreno Y Banuls
authors.forename: Adriana
authors.initials: A
authors.lastname: Katz
authors.forename: Walter
authors.initials: W
authors.lastname: Miklos
authors.forename: Alessio
authors.initials: A
authors.lastname: Cimmino
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Tal
authors.forename: Elena
authors.initials: E
authors.lastname: Ainbinder
authors.forename: Martin
authors.initials: M
authors.lastname: Zehl
authors.forename: Ernst
authors.initials: E
authors.lastname: Urban
authors.forename: Antonio
authors.initials: A
authors.lastname: Evidente
authors.forename: Brigitte
authors.initials: B
authors.lastname: Kopp
authors.forename: Walter
authors.initials: W
authors.lastname: Berger
authors.forename: Olivier
authors.initials: O
authors.lastname: Feron
authors.forename: Steven
authors.initials: S
authors.lastname: Karlish
authors.forename: Robert
authors.initials: R
authors.lastname: Kiss
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002018:Bufanolides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D002301:Cardiac Glycosides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D006031:Glycosylation; D019073:HT29 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D007527:Isoenzymes; D019344:Lactic Acid / Q000378:metabolism; D055442:Metabolome; D009369:Neoplasms / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D010101:Oxygen Consumption; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D002018:Bufanolides; D002301:Cardiac Glycosides; D007527:Isoenzymes; D019344:Lactic Acid; C001330:hellebrigenin; C499076:ATP1A1 protein, human; D000254:Sodium-Potassium-Exchanging ATPase; C007913:hellebrin