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.

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 α1ß1 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 α1ß1, α2ß1 and α3ß1 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 α2ß1 and α3ß1 than for the α1ß1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1ß1 than for the α2ß1 and α3ß1 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 α2ß1 and α3ß1 than for the α1ß1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.

Structure-activity relationship analysis of bufadienolide-induced in vitro growth inhibitory effects on mouse and human cancer cells.

The in vitro growth inhibitory effects of 27 bufadienolides and eight degradation products, with two cardenolides (ouabain and digoxin) chosen as reference compounds, were analyzed by means of an MTT colorimetric assay in six human and two mouse cancer cell lines. A structure-activity analysis was then performed to highlight the most important substituents relating to the in vitro growth inhibitory activity of bufadienolides in cancer cells. Thus, the current study revealed that various bufadienolides, including gamabufotalin rhamnoside (1a), bufotalin (2a), and hellebrin (3a), displayed higher growth inhibitory activities for various human cancer cell lines when compared to ouabain and digoxin. Gamabufotalin rhamnoside (1a) was the only compound that displayed growth inhibitory effects of <1 µM in mouse cancer cells that expressed mutated forms of the Na(+),K(+)-ATPase α-1 subunit. In addition, all genins and degradation products displayed weaker (if any) in vitro growth inhibitory effects on cancer cells when compared to their respective glycosylated homologue, with the exception of hellebrigenin (3b), which was as active as hellebrin (3a).

In vitro pharmacological and toxicological effects of norterpene peroxides isolated from the Red Sea sponge Diacarnus erythraeanus on normal and cancer cells.

Eight cyclic peroxide norterpenoids, compounds 1-8, have been isolated and characterized from the Red Sea sponge Diacarnus erythraeanus, including two new norsesterterpene derivatives (3, 4). Among these metabolites, (-)-muqubilin A (5) (nine cell lines analyzed) and the new compounds 3 and 4 (seven cell lines analyzed) displayed mean IC50 growth inhibitory concentrations in vitro of <10 µM, while the remaining compounds (1, 6-8) were inactive in these cancer cell lines. Compound 5 displayed no selectivity between normal and cancer cells in terms of in vitro growth inhibition. Quantitative video microscopy analysis carried out on (-)-muqubilin A-treated cells validated the data obtained by means of the MTT colorimetric assay, while flow cytometry analysis revealed ROS production but no induction of apoptosis in cancer cells.

Rapid structural identification of cytotoxic bufadienolide sulfates in toad venom from Bufo melanosticus by LC-DAD-MS(n) and LC-SPE-NMR.

Toad venom, namely, "Chansu" in China, has been widely used for the treatment of heart failure, sores, pains, and various cancers. Upon LC-MS analysis of the venom from Bufo melanosticus collected in Indonesia, new bufadienolide sulfates were identified. For a complete characterization, the MeOH extract of the toad venom from B. melanosticus was fractionated by preparative HPLC, and the structures of five new buadienolide sulfates (1-5) along with one new bufogenin (6) were rapidly elucidated on the basis of LC-DAD-MS(n) and LC-SPE-NMR data. The in vitro growth inhibitory activity of these six compounds along with hellebrin (positive control) has been assayed by means of the MTT colorimetric assay in four human and two mouse cancer cell lines. Compound 3 and hellebrin displayed similar and marked in vitro cytotoxicity.

Glanduliferins A and B, two new glucosylated steroids from Impatiens glandulifera, with in vitro growth inhibitory activity in human cancer cells.

Impatiens glandulifera has been imported from Himalaya in Europe and is considered as an invasive alien plant whose spreading arouses increasing interest among scientific literature. Via anti-cancer bioguiding, two new glucosylated steroids, named glanduliferins A and B, were isolated from the dried stem of I. glandulifera plants, together with the well-known α-spinasterol and 2-methoxy-1,4-naphthoquinone, which are also isolated from roots and leaves. They were characterized as 17-(2-hydroxy-2-pentamethylcyclopropyl-ethyl)-10,13-dimethyl-2,3,4,5,6,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopents[a]phenathren-3-O-(4-O-acetyl)-α-D-glucopyranoside and 17-(4-ethyl-1,5-dimethyl-hex-2-enyl)-10,13-dimethyl-2,3,4,5,6,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopents[a]phenathren-3-O-(6-O-acetyl)-ß-D-glucopyranoside using various NMR and HRESIMS techniques and chemical methods. In vitro determination of the growth inhibitory activity of the four isolated compounds using the MTT colorimetric assay revealed mean IC50 growth inhibitory value of ~30 µM for glanduliferin A while glanduliferin B and α-spinasterol were poorly active till 100 µM. 2-methoxy-1,4-naphthoquinone revealed to be active in the single micromolar digit range as previously described. Quantitative videomicroscopy analyses of the effects of glanduliferins A and B suggested cytostatic rather than cytotoxic activity in U373 glioblastoma (GBM) cells.