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

Fujii, Takuto; Shimizu, Takahiro; Shimizu, Yasuharu; Katoh, Mizuki; Okumura, Tomoyuki; Fujii, Tsutomu; Sakai, Hideki

doi:10.3390/ijms27177969

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

by

Takuto Fujii

^1,*

,

Takahiro Shimizu

¹

,

Yasuharu Shimizu

²,

Mizuki Katoh

¹,

Tomoyuki Okumura

³,

Tsutomu Fujii

³

and

Hideki Sakai

^1,*

¹

Department of Pharmaceutical Physiology, Faculty of Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan

²

Tokyo Research Center, Kyushin Pharmaceutical Co, Ltd., Tokyo 166-0012, Japan.

³

Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan.

^*

Authors to whom correspondence should be addressed.

Int. J. Mol. Sci. 2026, 27(17), 7969; https://doi.org/10.3390/ijms27177969

Submission received: 21 May 2026 / Revised: 31 August 2026 / Accepted: 5 September 2026 / Published: 7 September 2026

(This article belongs to the Special Issue Ion Channels and Transporters: Regulation and Roles in Human Diseases)

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Abstract

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.

Keywords: bufadienolide; bufalin; cancer; Na⁺,K⁺-ATPase; membrane microdomain; volume-regulated anion channel

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MDPI and ACS Style

Fujii, T.; Shimizu, T.; Shimizu, Y.; Katoh, M.; Okumura, T.; Fujii, T.; Sakai, H. Cardiac Glycoside 3β-Bufalin Suppresses Cancer Cell Proliferation by Coupling with Na⁺,K⁺-ATPase and Volume-Regulated Anion Channel Within Membrane Microdomains. Int. J. Mol. Sci. 2026, 27, 7969. https://doi.org/10.3390/ijms27177969

AMA Style

Fujii T, Shimizu T, Shimizu Y, Katoh M, Okumura T, Fujii T, Sakai H. Cardiac Glycoside 3β-Bufalin Suppresses Cancer Cell Proliferation by Coupling with Na⁺,K⁺-ATPase and Volume-Regulated Anion Channel Within Membrane Microdomains. International Journal of Molecular Sciences. 2026; 27(17):7969. https://doi.org/10.3390/ijms27177969

Chicago/Turabian Style

Fujii, Takuto, Takahiro Shimizu, Yasuharu Shimizu, Mizuki Katoh, Tomoyuki Okumura, Tsutomu Fujii, and Hideki Sakai. 2026. "Cardiac Glycoside 3β-Bufalin Suppresses Cancer Cell Proliferation by Coupling with Na⁺,K⁺-ATPase and Volume-Regulated Anion Channel Within Membrane Microdomains" International Journal of Molecular Sciences 27, no. 17: 7969. https://doi.org/10.3390/ijms27177969

APA Style

Fujii, T., Shimizu, T., Shimizu, Y., Katoh, M., Okumura, T., Fujii, T., & Sakai, H. (2026). Cardiac Glycoside 3β-Bufalin Suppresses Cancer Cell Proliferation by Coupling with Na⁺,K⁺-ATPase and Volume-Regulated Anion Channel Within Membrane Microdomains. International Journal of Molecular Sciences, 27(17), 7969. https://doi.org/10.3390/ijms27177969

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

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