Cardiac Glycoside 3β-Bufalin Suppresses Cancer Cell Proliferation by Coupling with Na⁺,K⁺-ATPase and Volume-Regulated Anion Channel Within Membrane Microdomains

Figures

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  Figure 1. Effects of toad-derived bufadienolides on cell proliferation in HT-29 cells. Cells were treated with bufalin (3β-bufalin), resibufogenin, cinobufagin, telocinobufagin, cinobufotalin, or desacetylcinobufagin at 30 nM for 24 h. Cell proliferation was evaluated by quantifying the increase in cell number. Data are presented as means ± SEM; n = 4. ** p < 0.01 versus control (cont.).

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  Figure 2. Concentration-dependent anti-proliferative effects of 3β-bufalin and its hepatic metabolite 3α-bufalin on HT-29 cells. Cells were treated with increasing concentrations of each compound for 24 h. Data are presented as means ± SEM; n = 4–7.

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  Figure 3. Effects of bufadienolides on Na+,K+-ATPase activity in membrane fractions of HT-29 cells. (A) Concentration-dependent effects of 3β-bufalin (n = 4) and 3α-bufalin (n = 5) on Na+,K+-ATPase activity in the membrane fractions. (B) Concentration-dependent effects of other bufadienolides, cinobufagin, resibufogenin, telocinobufagin, desacetylcinobufagin, and cinobufotalin, on Na+,K+-ATPase activity (n = 3). Data are presented as mean ± SEM.

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  Figure 4. Effects of 3β-bufalin and 3α-bufalin on H+,K+-ATPase and Ca2+-ATPase activities. (A) Effects of 3β-bufalin and 3α-bufalin on hog gastric H+,K+-ATPase activity. (B) Effects of 3β-bufalin and 3α-bufalin on rabbit Ca2+-ATPase activity. Data are presented as means ± SEM; n = 3.

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  Figure 5. 3β-Bufalin activates VRAC currents in HT-29 cells. (A,B) Upper panels: Representative whole-cell current traces at a holding potential of 0 mV in response to voltage steps to ±40 mV. 3β-Bufalin or 3α-bufalin (100 nM) was applied during the periods indicated by the blue or red lines, respectively. Step-pulse protocols were applied at the time points indicated as (i) and (ii). Lower panels: Current traces obtained in response to step pulses from −100 mV to +100 mV at (i) and (ii). (C) Concentration-dependent effects of 3β-bufalin and the effect of 3α-bufalin (100 nM) on the mean current density induced by a voltage pulse of +100 mV. Data are presented as means ± SEM; n = 3–6.

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  Figure 6. Contribution of VRAC in membrane microdomains to anti-proliferative effect of 3β-bufalin on HT-29 cells. (A) Effect of DCPIB (10 μM) and/or 3β-bufalin (BF; 30 nM) on cell proliferation. n = 5. (B) Effect of MβCD (10 μM) and/or 3β-bufalin (BF; 30 nM) on cell proliferation. n = 6. ** p < 0.01. (C) Proposed model for 3β-bufalin-induced VRAC activation in membrane microdomains. Cholesterol within membrane microdomains is indicated by green symbols.

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  Figure 7. Elucidation of 3β-bufalin-induced cellular responses mediated by receptor-type Na+,K+-ATPase and VRAC in HT-29 cells. (A) Effects of 3β-bufalin (BF; 30 nM) and/or DCPIB (10 μM) on caspase-3/7 activity in cells. n = 4. (B) Effects of 3β-bufalin (BF; 30 nM) and/or DCPIB (10 μM) on cellular dehydrogenase activity in cells. n = 4. (C) Effects of 3β-bufalin (BF; 30 nM) and/or DCPIB (10 μM) on cell cycle. Representative flow cytometric profiles of cell cycle distribution (left) and quantitative analysis of proportion of cells in G2/M phase (right). The G2/M region is indicated by the blue bracket. n = 3. Data are presented as means ± SEM. ** p < 0.01; NS, not significant.

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  Figure 8. Proposed mechanism of 3β-bufalin-induced anti-proliferative action through receptor-type Na+,K+-ATPase-mediated VRAC activation. At relatively higher concentrations, 3β-bufalin inhibits the ion-pumping activity of Na+,K+-ATPase (blue arrow). At low nanomolar concentrations, 3β-bufalin is proposed to act on receptor-type, non-pumping Na+,K+-ATPase in membrane microdomains (red arrow), activating VRAC. VRAC activation induces G2/M cell cycle arrest, thereby suppressing cancer cell proliferation. Cholesterol within membrane microdomains is indicated by green symbols.