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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.

Moreno Y Banuls L, Katz A, Miklos W, Cimmino A, Tal DM, Ainbinder E, Zehl M, Urban E, Evidente A, Kopp B, Berger W, Feron O, Karlish S, Kiss R - Mol. Cancer (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Toxicologie, Faculté de Pharmacie, Université Libre de Bruxelles, Brussels 1050, Belgium.

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.

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Metabolic profiling of human HT29 colon cancer cells treated with cardiotonic steroids. A: Extracellular lactate concentrations and B: O2 consumption rates in HT29 cancer cells treated with the indicated compounds at their IC50in vitro growth inhibitory concentrations (see Table 1). The data (mean ± SEM from triplicates) are expressed in mM lactate accumulated in the extracellular medium after 24 hours and arbitrary units derived from time-resolved fluorescent signal.min-1.
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Figure 6: Metabolic profiling of human HT29 colon cancer cells treated with cardiotonic steroids. A: Extracellular lactate concentrations and B: O2 consumption rates in HT29 cancer cells treated with the indicated compounds at their IC50in vitro growth inhibitory concentrations (see Table 1). The data (mean ± SEM from triplicates) are expressed in mM lactate accumulated in the extracellular medium after 24 hours and arbitrary units derived from time-resolved fluorescent signal.min-1.

Mentions: The vacuolization processes reported in the previous section with respect to 19-hydroxy-2″-oxovoruscharin cardenolide in human A549 NSCLC [30] and U373 GBM [10] were paralleled by marked decreases in intracellular ATP concentration ([ATP]i) in these cells [10,12], while much weaker effects were observed in normal cells [10,12]. Therefore, we analyzed the effects induced by four cardenolides and four bufadienolides (including hellebrin and hellebrigenin) on glycolysis and O2 consumption rates in human HT-29 colon cancer cells. The preliminary data (not shown) indicated that the HT-29 cell line exhibited both glycolytic (glucose to lactate) and oxidative (glucose to CO2) metabolism. While cardenolides and bufadienolides did not alter the glycolytic flux as determined by the measurements of lactate release in the extracellular medium (Figure 6A), each compound significantly (P < 0.01) influenced cell respiration (Figure 6B). The O2 consumption rate was significantly reduced following treatment with digoxin, ouabain, hellebrin and gamabufotalin-rhamnoside at the IC50in vitro growth inhibition of each compound (as determined in the MTT assay). A trend to a higher activity of the glycosides versus aglycones was also observed. Of note, these measurements were obtained after 24 h (Figure 6), a delay for which the confounding effects of cell death could be excluded (Figure 4).


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.

Moreno Y Banuls L, Katz A, Miklos W, Cimmino A, Tal DM, Ainbinder E, Zehl M, Urban E, Evidente A, Kopp B, Berger W, Feron O, Karlish S, Kiss R - Mol. Cancer (2013)

Metabolic profiling of human HT29 colon cancer cells treated with cardiotonic steroids. A: Extracellular lactate concentrations and B: O2 consumption rates in HT29 cancer cells treated with the indicated compounds at their IC50in vitro growth inhibitory concentrations (see Table 1). The data (mean ± SEM from triplicates) are expressed in mM lactate accumulated in the extracellular medium after 24 hours and arbitrary units derived from time-resolved fluorescent signal.min-1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3722118&req=5

Figure 6: Metabolic profiling of human HT29 colon cancer cells treated with cardiotonic steroids. A: Extracellular lactate concentrations and B: O2 consumption rates in HT29 cancer cells treated with the indicated compounds at their IC50in vitro growth inhibitory concentrations (see Table 1). The data (mean ± SEM from triplicates) are expressed in mM lactate accumulated in the extracellular medium after 24 hours and arbitrary units derived from time-resolved fluorescent signal.min-1.
Mentions: The vacuolization processes reported in the previous section with respect to 19-hydroxy-2″-oxovoruscharin cardenolide in human A549 NSCLC [30] and U373 GBM [10] were paralleled by marked decreases in intracellular ATP concentration ([ATP]i) in these cells [10,12], while much weaker effects were observed in normal cells [10,12]. Therefore, we analyzed the effects induced by four cardenolides and four bufadienolides (including hellebrin and hellebrigenin) on glycolysis and O2 consumption rates in human HT-29 colon cancer cells. The preliminary data (not shown) indicated that the HT-29 cell line exhibited both glycolytic (glucose to lactate) and oxidative (glucose to CO2) metabolism. While cardenolides and bufadienolides did not alter the glycolytic flux as determined by the measurements of lactate release in the extracellular medium (Figure 6A), each compound significantly (P < 0.01) influenced cell respiration (Figure 6B). The O2 consumption rate was significantly reduced following treatment with digoxin, ouabain, hellebrin and gamabufotalin-rhamnoside at the IC50in vitro growth inhibition of each compound (as determined in the MTT assay). A trend to a higher activity of the glycosides versus aglycones was also observed. Of note, these measurements were obtained after 24 h (Figure 6), a delay for which the confounding effects of cell death could be excluded (Figure 4).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Toxicologie, Faculté de Pharmacie, Université Libre de Bruxelles, Brussels 1050, Belgium.

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.

Show MeSH
Related in: MedlinePlus