Cardiac Glycoside 3β-Bufalin Suppresses Cancer Cell Proliferation by Coupling with Na⁺,K⁺-ATPase and Volume-Regulated...

Bufadienolides are toad-derived cardiotonic steroids with anti-cancer activity; however, their Na⁺,K⁺-ATPase-mediated anti-cancer mechanisms remain incompletely understood. Here, we compared the anti-proliferative effects of six bufadienolides, bufalin (3β-bufalin), resibufogenin, cinobufagin, telocinobufagin, cinobufotalin, and desacetylcinobufagin, on human colorectal cancer HT-29 cells. Among them, only 3β-bufalin significantly suppressed cell proliferation in a concentration-dependent manner, with an IC₅₀ of 1.8 nM, whereas its hepatic metabolite, 3α-bufalin, showed markedly weaker activity, with an IC₅₀ of 488 nM. 3β-Bufalin inhibited Na⁺,K⁺-ATPase activity with an IC₅₀ of approximately 37 nM, indicating that it suppressed growth at concentrations lower than those required for pump inhibition. The Na⁺,K⁺-ATPase α1-isoform can function as a receptor-type (non-pumping) signaling platform linked to volume-regulated anion channel (VRAC) activation in membrane microdomains of cancer cells. 3β-Bufalin activated the VRAC with an EC₅₀ of 4.6 nM, whereas 3α-bufalin and the other bufadienolides showed no detectable activation. The pharmacological inhibition of the VRAC and the disruption of cholesterol-rich membrane microdomains significantly attenuated the anti-proliferative effect of 3β-bufalin. Furthermore, 3β-bufalin induced G2/M cell cycle arrest, which was attenuated by VRAC inhibition. These findings indicate that 3β-bufalin selectively suppresses cancer cell proliferation by coupling with receptor-type Na⁺,K⁺-ATPase to activate the VRAC in membrane microdomains rather than inhibiting Na⁺,K⁺-ATPase pump activity.