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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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Computer-assisted phase-contrast microscopy (quantitative videomicroscopy) illustrations of human U373 glioblastoma cells treated with 10 nM hellebrigenin for 72 h. The squares on the left-handed panel of images (0 – 32 – 48 h) highlight the morphological aspects of the U373 GBM cells on the right-handed images at a higher magnification, with the appearance of marked vacuolization processes. Cells were not starved during the experiments. The images provided here are representative images obtained from triplicates in each experimental condition.
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Figure 5: Computer-assisted phase-contrast microscopy (quantitative videomicroscopy) illustrations of human U373 glioblastoma cells treated with 10 nM hellebrigenin for 72 h. The squares on the left-handed panel of images (0 – 32 – 48 h) highlight the morphological aspects of the U373 GBM cells on the right-handed images at a higher magnification, with the appearance of marked vacuolization processes. Cells were not starved during the experiments. The images provided here are representative images obtained from triplicates in each experimental condition.

Mentions: Computer-assisted phase contrast microscopy (quantitative videomicroscopy) analyses were used to morphologically visualize the effects induced by hellebrin (data not shown) and hellebrigenin (Figure 5) in human U373 GBM cells. Hellebrigenin was assayed in U373 GBM cells at the IC50 growth inhibitory concentration of 10 nM, which was determined by the MTT colorimetric assay (Table 1). Numerous cytoplasmic vacuoles were observed after 12 h of U373 cell treatment with 10 nM hellebrigenin, and this effect was sustained up to 32–40 h after treatment (Figure 5). Then, between 45–50 h, U373 GBM cells began to die, and, in accordance with the MTT assay-related data, approximately 50% of the U373 GBM cells died after 72 h of treatment with 10 nM hellebrigenin (Figure 5). These morphological analyses revealed that hellebrigenin is a cytotoxic compound and not a cytostatic one. Similar results were observed with respect to hellebrin (data not shown).


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)

Computer-assisted phase-contrast microscopy (quantitative videomicroscopy) illustrations of human U373 glioblastoma cells treated with 10 nM hellebrigenin for 72 h. The squares on the left-handed panel of images (0 – 32 – 48 h) highlight the morphological aspects of the U373 GBM cells on the right-handed images at a higher magnification, with the appearance of marked vacuolization processes. Cells were not starved during the experiments. The images provided here are representative images obtained from triplicates in each experimental condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Computer-assisted phase-contrast microscopy (quantitative videomicroscopy) illustrations of human U373 glioblastoma cells treated with 10 nM hellebrigenin for 72 h. The squares on the left-handed panel of images (0 – 32 – 48 h) highlight the morphological aspects of the U373 GBM cells on the right-handed images at a higher magnification, with the appearance of marked vacuolization processes. Cells were not starved during the experiments. The images provided here are representative images obtained from triplicates in each experimental condition.
Mentions: Computer-assisted phase contrast microscopy (quantitative videomicroscopy) analyses were used to morphologically visualize the effects induced by hellebrin (data not shown) and hellebrigenin (Figure 5) in human U373 GBM cells. Hellebrigenin was assayed in U373 GBM cells at the IC50 growth inhibitory concentration of 10 nM, which was determined by the MTT colorimetric assay (Table 1). Numerous cytoplasmic vacuoles were observed after 12 h of U373 cell treatment with 10 nM hellebrigenin, and this effect was sustained up to 32–40 h after treatment (Figure 5). Then, between 45–50 h, U373 GBM cells began to die, and, in accordance with the MTT assay-related data, approximately 50% of the U373 GBM cells died after 72 h of treatment with 10 nM hellebrigenin (Figure 5). These morphological analyses revealed that hellebrigenin is a cytotoxic compound and not a cytostatic one. Similar results were observed with respect to hellebrin (data not shown).

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