Figure 1.
Structure and pharmacophoric conformation of 21-BD.
(A) Chemical structure of ouabain, digoxin, and 21-BD. (B) Left: whole structure of Na,K-ATPase (PDB: 4HYT) showed in solid ribbon representation, where the alpha-helix, beta-sheets and turns are in red, blue and gray, respectively; right: highlight of the binding site with ouabain shown in tube representation. Dashed red lines indicate hydrogen bonds. Only polar hydrogen atoms were showed for a better visualization. Pharmacophoric conformation of, (C) digoxin and (D) 21-BD; polar and nonpolar interactions are depicted by magenta and green colors, respectively. Dashed lines indicate hydrogen bonds. Residue interactions are color coded as indicated in the inserted scale.
Figure 2.
21-BD effect on Na,K-ATPase and Pdr5p activity.
(A) 21-BD competition of 3H-ouabain binding on HeLa cells; the control for maximal binding is represented with a white circle and a long dashed line, competition of ouabain and 21-BD is shown with blue and red circles respectively. (B) Inhibition of rat´s brain hemisphere Na,K-ATPase after 2 h incubation with digoxin (green circles) or 21-BD (red circles). (C) Effect of 21-BD on the Na,K-ATPase activity on proteins expressed in Sf9 insect cells, Na,K-ATPase activity was measured on Sf9 cells expressing the rat α1 β1 (orange circles) or β1 (red circles) after 15 min treatment with the indicated concentrations of 21-BD. (D) Dose-response curve for the effects of 21-DB on Na,K-ATPase activity of mouse kidney membrane preparations. E) Effect of 21-BD (red circles) or digoxin (green circles) on the activity of the Pdr5p transporter.
Figure 3.
21-BD increases Na,K-ATPase expression in cancer cells.
(A) Na,K-ATPase activity after incubation of HeLa cells with 21-BD or digoxin for 48 h with different concentrations of 21-BD. B) mRNA content of the Na,K-ATPase α1 and β1 subunits of HeLa cells after 48 h incubation with various concentrations of 21-BD.
Figure 4.
High concentrations of 21-BD reduce cell viability of HeLa and RKO.
HeLa (A) or RKO (B) cells were treated with digoxin (green symbols) or 21-BD (red symbols) for 24 (circles) or 48 (squares) h. Viability was measured by MTT reduction assay. 100 and 25 µM 21-BD induced the statistically significant reduction of HeLa and RKO viability, respectively (p<0.0084). Digoxin reduces HeLa viability starting with 150 µM for 24 h and 50 µM for 48 h (p<0.001). RKO cells have a higher sensitivity to digoxin that induces statistically significant differences starting from 1.6 µM for 48 h (p<0.0001).
Figure 5.
21-BD induces apoptosis in HeLa and CHO-K1 cells.
(A) Score value obtained from the comet assay of CHO-K1 cells incubated 24 h with 21-BD at different concentrations (red circles). (B) Micronucleated cells percentage of CHO-K1 cultures incubated with 21-BD at different concentrations for 24 h. A 24 h incubation with 0.4 mM Methyl methanesulfonate (MMS) was used as a control (A, B, blue circles). (C) Apoptotic and necrotic HeLa cells after 24 h of incubation in control media (white bars), media with 50 µM 21-BD (red bars) or 2 µM digoxin (green bars) for 24 h. Apoptosis and necrosis were detected by flow cytometry ussing an annexin-V translocation assay and the incorporation of propidium iodide in to the nucleus, respectively. P<0.01.
Figure 6.
21-BD regulates tight junctions.
MDCK cells were cultured in transwell permeable supports and treated with 5, 10 and 50 µM 21-BD. (A) TER was measured as a function of time. The control TER data (white circles, dotted line) averaged 183±8 Ω.cm2 (n = 13) and were normalized to 100%. 5 and 10 µM 21-BD provoke transient small increases of TER, while 50 µM 21-BD causes a stronger and a sustained TER increase (red circles). (B) MDCK cells were incubated 48 h with different concentrations of 21-BD (red symbols) or digoxin (green symbols). mRNA cell content of claudins -4 (circles) and -2 (triangles) were measured by quantitative real time PCR. (C) Protein cell content of the tight junction integral membrane proteins claudins -4 and -2 and the membrane-associated protein ZO-1 as a function of 21-BD concentration in the media for 48 h. Images from the left part of the figure C are representative immunoblots and the graph in the right part is the statistical analysis.
Figure 7.
21-BD regulates tight junctiońs proteins localization.
Confluent monolayers of MDCK cells, grown on filters, were maintained in control medium (A, B, C) or treated with 50 µM 21-BD for 48 h (D, F, G) and processed for immunofluorescence, using antibodies against the TJs proteins: the integral membrane proteins claudin-4 (Cldn-4, A, D green) and claudin-2 (Cldcn-2, B, E red) and the peripheral membrane protein ZO-1 (C, E, red). Nuclei were stained with TOPRO (blue). 21-BD increases claudin-4 and ZO-1 expression at the tight junction (arrows) and in the cytoplasma (arrow heads), while simultaneously reduces the expression of claudin-2.
Figure 8.
21-BD increases the expression of Na,K-ATPase in MDCK cells.
(A) Protein cell content of the α1 subunit of the Na,K-ATPase of confluent monolayers of MDCK cells grown on filters in control medium (white bar) or treated with different concentrations of 21-BD (red bars) for 48 h; upper part of the figure A shows representative immunoblots of the α1 subunit of the Na,K-ATPase and actin, the lower part the densitometric analysis. (B and C) Na,K-ATPase α1 subunit stained with a fluoresceinated antibody (B, C, white) or Topro (blue) to detect the nuclei.