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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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NaK alpha1-subunit and in vitro growth inhibition of cancer cells. A: Correlation of Ki for inhibition of human α1β1 by cardiac glycosides and the growth inhibition effects (IC50) of human cancer cells; 1: digoxin; 2: hellebrin; 3: ouabain; 4: oleandrin; 5: hellebrigenin; 6: gamabufotalin-rhamnoside. IC50 values are the averages ± SEM of 4 experiments with 10 different human cancer cell lines from the NCI60 library: ACHN (renal cell carcinoma); SF-268 and SNB-75 (glioma); MCF-7 (breast cancer); SKMEL-5 (melanoma); HCT-116 and HT-29 (colon cancer); A549 (NSCLC); TK-10 (kidney cancer); and Ovcar-3 and Ovcar-4 (ovarian cancer). In this experiment, the IC50 values were determined after two days of culture using the crystal violet assay [54]. B: Illustration of the IC50in vitro growth inhibitory concentration (MTT colorimetric assay; Y axis) as opposed to the mRNA levels (by means of quantitative RT-PCR as detailed in [10]) of the NaK α1 subunit in five human cancer cell lines, including Hs683 oligodendroglioma (207 mRNA copies / μg cDNA); T98G GBM (911 mRNA copies / μg cDNA); A549 NSCLC (1,450 mRNA copies / μg cDNA); U373 GBM (2,091 mRNA copies / μg cDNA); and PC-3 prostate adenocarcinoma (5,337 mRNA copies / μg cDNA) cells. The IC50 growth inhibitory concentrations that are reported in Figure 2B are from Table 1.
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Figure 2: NaK alpha1-subunit and in vitro growth inhibition of cancer cells. A: Correlation of Ki for inhibition of human α1β1 by cardiac glycosides and the growth inhibition effects (IC50) of human cancer cells; 1: digoxin; 2: hellebrin; 3: ouabain; 4: oleandrin; 5: hellebrigenin; 6: gamabufotalin-rhamnoside. IC50 values are the averages ± SEM of 4 experiments with 10 different human cancer cell lines from the NCI60 library: ACHN (renal cell carcinoma); SF-268 and SNB-75 (glioma); MCF-7 (breast cancer); SKMEL-5 (melanoma); HCT-116 and HT-29 (colon cancer); A549 (NSCLC); TK-10 (kidney cancer); and Ovcar-3 and Ovcar-4 (ovarian cancer). In this experiment, the IC50 values were determined after two days of culture using the crystal violet assay [54]. B: Illustration of the IC50in vitro growth inhibitory concentration (MTT colorimetric assay; Y axis) as opposed to the mRNA levels (by means of quantitative RT-PCR as detailed in [10]) of the NaK α1 subunit in five human cancer cell lines, including Hs683 oligodendroglioma (207 mRNA copies / μg cDNA); T98G GBM (911 mRNA copies / μg cDNA); A549 NSCLC (1,450 mRNA copies / μg cDNA); U373 GBM (2,091 mRNA copies / μg cDNA); and PC-3 prostate adenocarcinoma (5,337 mRNA copies / μg cDNA) cells. The IC50 growth inhibitory concentrations that are reported in Figure 2B are from Table 1.

Mentions: The histological types and origins of the eight human cancer cell lines that were used for the MTT colorimetric assay are detailed in the legend of Table 1. Two mouse cancer cell lines were also used, the CT26.WT colon cancer cell line (ATCC code CRL-2638) and the B16F10 melanoma cell line (ATCC code CRL-6475). Both cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). A control cell line (human NHDF fibroblasts) was obtained from PromoCell (code c-12300; Heidelberg, Germany). The cell lines detailed in Figure 2A are a generous gift from the National Cancer Institute (NCI, Bethesda, USA) to Steven Karlish’s lab.


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)

NaK alpha1-subunit and in vitro growth inhibition of cancer cells. A: Correlation of Ki for inhibition of human α1β1 by cardiac glycosides and the growth inhibition effects (IC50) of human cancer cells; 1: digoxin; 2: hellebrin; 3: ouabain; 4: oleandrin; 5: hellebrigenin; 6: gamabufotalin-rhamnoside. IC50 values are the averages ± SEM of 4 experiments with 10 different human cancer cell lines from the NCI60 library: ACHN (renal cell carcinoma); SF-268 and SNB-75 (glioma); MCF-7 (breast cancer); SKMEL-5 (melanoma); HCT-116 and HT-29 (colon cancer); A549 (NSCLC); TK-10 (kidney cancer); and Ovcar-3 and Ovcar-4 (ovarian cancer). In this experiment, the IC50 values were determined after two days of culture using the crystal violet assay [54]. B: Illustration of the IC50in vitro growth inhibitory concentration (MTT colorimetric assay; Y axis) as opposed to the mRNA levels (by means of quantitative RT-PCR as detailed in [10]) of the NaK α1 subunit in five human cancer cell lines, including Hs683 oligodendroglioma (207 mRNA copies / μg cDNA); T98G GBM (911 mRNA copies / μg cDNA); A549 NSCLC (1,450 mRNA copies / μg cDNA); U373 GBM (2,091 mRNA copies / μg cDNA); and PC-3 prostate adenocarcinoma (5,337 mRNA copies / μg cDNA) cells. The IC50 growth inhibitory concentrations that are reported in Figure 2B are from Table 1.
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Related In: Results  -  Collection

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Figure 2: NaK alpha1-subunit and in vitro growth inhibition of cancer cells. A: Correlation of Ki for inhibition of human α1β1 by cardiac glycosides and the growth inhibition effects (IC50) of human cancer cells; 1: digoxin; 2: hellebrin; 3: ouabain; 4: oleandrin; 5: hellebrigenin; 6: gamabufotalin-rhamnoside. IC50 values are the averages ± SEM of 4 experiments with 10 different human cancer cell lines from the NCI60 library: ACHN (renal cell carcinoma); SF-268 and SNB-75 (glioma); MCF-7 (breast cancer); SKMEL-5 (melanoma); HCT-116 and HT-29 (colon cancer); A549 (NSCLC); TK-10 (kidney cancer); and Ovcar-3 and Ovcar-4 (ovarian cancer). In this experiment, the IC50 values were determined after two days of culture using the crystal violet assay [54]. B: Illustration of the IC50in vitro growth inhibitory concentration (MTT colorimetric assay; Y axis) as opposed to the mRNA levels (by means of quantitative RT-PCR as detailed in [10]) of the NaK α1 subunit in five human cancer cell lines, including Hs683 oligodendroglioma (207 mRNA copies / μg cDNA); T98G GBM (911 mRNA copies / μg cDNA); A549 NSCLC (1,450 mRNA copies / μg cDNA); U373 GBM (2,091 mRNA copies / μg cDNA); and PC-3 prostate adenocarcinoma (5,337 mRNA copies / μg cDNA) cells. The IC50 growth inhibitory concentrations that are reported in Figure 2B are from Table 1.
Mentions: The histological types and origins of the eight human cancer cell lines that were used for the MTT colorimetric assay are detailed in the legend of Table 1. Two mouse cancer cell lines were also used, the CT26.WT colon cancer cell line (ATCC code CRL-2638) and the B16F10 melanoma cell line (ATCC code CRL-6475). Both cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). A control cell line (human NHDF fibroblasts) was obtained from PromoCell (code c-12300; Heidelberg, Germany). The cell lines detailed in Figure 2A are a generous gift from the National Cancer Institute (NCI, Bethesda, USA) to Steven Karlish’s lab.

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