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

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study aimed to compare the anticancer effects of bufadienolide-derived compounds from toad venom and, in particular, to propose a novel mechanism by which 3β-bufalin inhibits cancer cell proliferation through receptor-type signaling via Na⁺/K⁺-ATPase α1 and VRAC activation. The comparison of structure-activity relationships among various bufadienolides is interesting, and the evaluation of VRAC activation through electrophysiological analysis is also considered a strength.

However, the data currently presented are insufficient to fully support the paper’s key conclusions. In particular, while the authors conclude that “3β-bufalin inhibits cancer cell proliferation through receptor-type Na⁺/K⁺-ATPase α1-mediated VRAC activation,” there is a lack of key mechanistic studies to substantiate this claim.

1. It has not been proven that VRAC activation is the cause of the antiproliferative effect.

The most important conclusion of this study is that the antiproliferative effect of 3β-bufalin is mediated through VRAC activation. However, the current results merely show that VRAC activation and cell proliferation inhibition are observed within similar concentration ranges; they do not establish a causal relationship between the two phenomena.

The current data merely suggest a correlation, and it has not been confirmed whether VRAC activity actually mediates the antiproliferative effect.

To prove this, the following experiments are necessary:

• Blockade experiments using VRAC inhibitors (e.g., DCPIB)
• Knockdown or knockout experiments targeting VRAC components such as LRRC8A
• Rescue experiments to determine whether the antiproliferative effect of 3β-bufalin is reduced or abolished upon inhibition of VRAC function

Without such loss-of-function experiments, it is difficult to conclude that VRAC activity is the direct cause of the antiproliferative effect.

2. The involvement of receptor-type Na⁺/K⁺-ATPase α1 has not been directly demonstrated.

Throughout the paper, the authors argue that receptor-type Na⁺/K⁺-ATPase α1 signaling is the key mechanism underlying this phenomenon. However, no experiments were actually conducted to verify the necessity of Na⁺/K⁺-ATPase α1.

The current conclusions are largely based on the authors’ previous studies and indirect inferences, and the paper does not demonstrate whether receptor-type Na⁺/K⁺-ATPase α1 is actually essential.

Therefore, the following additional experiments are required.

• ATP1A1 (Na⁺/K⁺-ATPase α1) knockdown or knockout
• Evaluate whether VRAC activation is inhibited upon reduced α1 expression
• Evaluate whether the antiproliferative effect of 3β-bufalin is reduced upon reduced α1 expression
• Verification of receptor-type signaling through experiments involving the disruption of membrane microdomains or caveolae

If these experiments are not performed, the phrase “Na⁺/K⁺-ATPase α1-mediated” is considered an overinterpretation.

3. The analysis of the biological mechanisms underlying the antiproliferative effects is overly limited.

This study relies primarily on cell proliferation assays, ATPase activity measurements, and patch-clamp analysis. However, almost no biological explanation is provided as to why cancer cell proliferation is reduced.

Further validation is needed to determine what intracellular changes VRAC activation actually induces to cause proliferation inhibition.

For example, the following analyses should be performed:

• Cell cycle analysis
• Apoptosis analysis (Annexin V/PI, caspase activation, PARP cleavage, etc.)
• Colony formation assay
• ROS production analysis
• Evaluation of previously reported bufalin-related signaling pathways (JNK, ROS, mitochondrial signaling, etc.)

At the current stage, only a reduction in cell numbers has been confirmed; the detailed biological mechanisms underlying the anticancer effects have not been sufficiently elucidated.

Comments on the Quality of English Language

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Author Response

Comments and Suggestions for Authors #1

This study aimed to compare the anticancer effects of bufadienolide-derived compounds from toad venom and, in particular, to propose a novel mechanism by which 3β-bufalin inhibits cancer cell proliferation through receptor-type signaling via Na⁺/K⁺-ATPase α1 and VRAC activation. The comparison of structure-activity relationships among various bufadienolides is interesting, and the evaluation of VRAC activation through electrophysiological analysis is also considered a strength.

However, the data currently presented are insufficient to fully support the paper’s key conclusions. In particular, while the authors conclude that “3β-bufalin inhibits cancer cell proliferation through receptor-type Na⁺/K⁺-ATPase α1-mediated VRAC activation,” there is a lack of key mechanistic studies to substantiate this claim.

Response

We appreciate your insightful and helpful comments and suggestions. In response, we revised the manuscript and performed additional experiments and analyses to further investigate the involvement of VRAC and membrane microdomains in the anti-proliferative effect of 3β-bufalin and to characterize the underlying cellular mechanisms. Based on these additional findings and the reviewer’s comments, we have also revised the title to “Cardiac Glycoside 3β-Bufalin Suppresses Cancer Cell Proliferation by Coupling with Na⁺,K⁺-ATPase and Volume-Regulated Anion Channel within Membrane Microdomains.” We have addressed each point in detail below. In addition, we have provided a marked version of the manuscript in which all revisions are highlighted in red for clarity.

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1. It has not been proven that VRAC activation is the cause of the antiproliferative effect.

The most important conclusion of this study is that the antiproliferative effect of 3β-bufalin is mediated through VRAC activation. However, the current results merely show that VRAC activation and cell proliferation inhibition are observed within similar concentration ranges; they do not establish a causal relationship between the two phenomena. The current data merely suggest a correlation, and it has not been confirmed whether VRAC activity actually mediates the antiproliferative effect. To prove this, the following experiments are necessary:

Blockade experiments using VRAC inhibitors (e.g., DCPIB)

Knockdown or knockout experiments targeting VRAC components such as LRRC8A

Rescue experiments to determine whether the antiproliferative effect of 3β-bufalin is reduced or abolished upon inhibition of VRAC function

Without such loss-of-function experiments, it is difficult to conclude that VRAC activity is the direct cause of the antiproliferative effect.

Response

We thank the reviewer for this important comment. To examine the functional involvement of VRAC in the anti-proliferative effect of 3β-bufalin, we performed additional experiments using the VRAC inhibitor DCPIB. Treatment with 3β-bufalin (30 nM) significantly suppressed the increase in HT-29 cell number, whereas DCPIB (10 μM) significantly attenuated this effect. DCPIB alone did not significantly affect cell number under the same experimental conditions. These results provide pharmacological evidence that VRAC activation contributes to the anti-proliferative effect of 3β-bufalin. Please refer to the Abstract (page 1, lines 29–30), the Results section (pages 6 and 7, lines 191–205), Figure 6A,C and the legend (page 7, lines 207–211), the Discussion section (pages 8 and 9, lines 245–251, lines 261–262), and the Materials and Methods section (page 11, lines 356).

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2. The involvement of receptor-type Na⁺/K⁺-ATPase α1 has not been directly demonstrated.

Throughout the paper, the authors argue that receptor-type Na⁺/K⁺-ATPase α1 signaling is the key mechanism underlying this phenomenon. However, no experiments were actually conducted to verify the necessity of Na⁺/K⁺-ATPase α1. The current conclusions are largely based on the authors’ previous studies and indirect inferences, and the paper does not demonstrate whether receptor-type Na⁺/K⁺-ATPase α1 is actually essential. Therefore, the following additional experiments are required.

ATP1A1 (Na⁺/K⁺-ATPase α1) knockdown or knockout

Evaluate whether VRAC activation is inhibited upon reduced α1 expression

Evaluate whether the antiproliferative effect of 3β-bufalin is reduced upon reduced α1 expression

Verification of receptor-type signaling through experiments involving the disruption of membrane microdomains or caveolae

If these experiments are not performed, the phrase “Na⁺/K⁺-ATPase α1-mediated” is considered an overinterpretation.

Response

We thank the reviewer for this insightful comment. Following this suggestion, we performed an additional experiment to examine whether membrane microdomains are involved in the anti-proliferative effect of 3β-bufalin. Disruption of cholesterol-rich membrane microdomains by methyl-β-cyclodextrin (MβCD; 10 μM) significantly attenuated the suppression of HT-29 cell proliferation induced by 3β-bufalin (Figure 6B). Together with the inhibitory effect of DCPIB described above (Figure 6A), these results support the involvement of VRAC activation within membrane microdomains in the anti-proliferative effect of 3β-bufalin. Furthermore, our previous study demonstrated functional coupling between receptor-type Na⁺,K⁺-ATPase α1-isoform and VRAC in membrane microdomains of HT-29 cells (Reference 18; Fujii et al., BBA, 2018). These findings support a model in which 3β-bufalin activates VRAC functionally coupled with receptor-type Na⁺,K⁺-ATPase in membrane microdomains. We agree, however, that the present study does not genetically establish the requirement for ATP1A1. Therefore, we have revised the relevant statements to avoid the use of the term “Na⁺,K⁺-ATPase α1-mediated” where it could imply a directly established causal relationship. Please refer to the Abstract (page 1, lines 29–30, 30–31), the Results section (pages 6 and 7, lines 191–205), Figure 6B,C and the legend (page 7, lines 207–211), the Discussion section (pages 8 and 9, lines 245–251, lines 261–262), and the Materials and Methods section (page 11, lines 356).

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3. The analysis of the biological mechanisms underlying the antiproliferative effects is overly limited.

This study relies primarily on cell proliferation assays, ATPase activity measurements, and patch-clamp analysis. However, almost no biological explanation is provided as to why cancer cell proliferation is reduced. Further validation is needed to determine what intracellular changes VRAC activation actually induces to cause proliferation inhibition.

For example, the following analyses should be performed:

Cell cycle analysis, Apoptosis analysis (Annexin V/PI, caspase activation, PARP cleavage, etc.)

Colony formation assay, ROS production analysis, Evaluation of previously reported bufalin-related signaling pathways (JNK, ROS, mitochondrial signaling, etc.)

At the current stage, only a reduction in cell numbers has been confirmed; the detailed biological mechanisms underlying the anticancer effects have not been sufficiently elucidated.

Response

We thank the reviewer for this valuable suggestion. To further characterize the cellular mechanisms underlying the anti-proliferative effect of 3β-bufalin, we performed additional analyses of caspase-3/7 activity, cellular dehydrogenase activity, and cell-cycle distribution. Treatment with 3β-bufalin (30 nM) did not significantly increase caspase-3/7 activity in HT-29 cells under our experimental conditions (Figure 7A). In contrast, 3β-bufalin significantly reduced cellular dehydrogenase activity, and this reduction was significantly attenuated by DCPIB (Figure 7B). Furthermore, 3β-bufalin significantly increased the proportion of cells in the G2/M phase, and this increase was also significantly attenuated by DCPIB (Figure 7C). These results suggest that VRAC activation contributes to both the reduction in cellular dehydrogenase activity and G2/M cell-cycle arrest induced by 3β-bufalin, rather than substantial induction of caspase-dependent apoptosis under the present experimental conditions. Please refer to the Abstract (page 1, lines 31–32), the Results section (page 7, lines 212–223), Figure 7 and the legend (page 8, lines 225–231), the Discussion section (page 9, lines 299–308), and the Materials and Methods section (page 13, lines 445–468).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents an interesting study investigating the anti-cancer mechanism of selected bufadienolides, with particular emphasis on the role of receptor-type Na⁺,K⁺-ATPase α1-mediated VRAC activation. The work is well designed and provides valuable mechanistic insights into the structure–activity relationship of bufalin derivatives.

However, several issues should be addressed before the manuscript can be considered for publication.

1. Conclusions. The manuscript would benefit from a clearly separated Conclusions section. At present, the concluding remarks are embedded within the Discussion, making it difficult for readers to identify the main findings and their broader implications. A distinct Conclusions subsection should succinctly summarize the key outcomes and emphasize the novelty and significance of the study.
2. Figure preparation. The authors should specify in the figure legends or in the Materials and Methods section which software was used to prepare all graphical figures and schemes. This information is important for transparency and reproducibility.
3. Characterization of tested compounds. Since the biological conclusions rely on commercially obtained bufadienolides, the authors should provide evidence of their chemical identity and purity. At minimum, the purity of all investigated compounds should be reported. In addition, supporting analytical data such as ¹H NMR spectra together with HRMS data, or alternatively HPLC chromatograms demonstrating compound purity, should be included as Supplementary Information. This recommendation applies even if the compounds were purchased from a commercial supplier, as confirmation of compound quality is essential for ensuring the reliability and reproducibility of the biological results.
4. Preliminary pharmacokinetic assessment. To strengthen the translational value of the study, the authors are encouraged to include a preliminary in silico evaluation of the pharmacokinetic (ADME) properties of the investigated bufadienolides. Prediction of parameters such as gastrointestinal absorption, blood–brain barrier permeability, P-glycoprotein interaction, cytochrome P450 liabilities, and drug-likeness would provide valuable information regarding the therapeutic potential of these compounds and complement the biological findings.
5. Section 4.2 (Cell culture). Section 4.2 should be carefully revised, as part of the text is displayed in red font, making it unclear whether these passages represent tracked changes, editorial comments, or content intended to remain in the final version of the manuscript. The authors should ensure that all tracked changes and formatting issues are resolved before submission, and that the final text is presented in a clear and consistent format.

Author Response

Comments and Suggestions for Authors #2

The manuscript presents an interesting study investigating the anti-cancer mechanism of selected bufadienolides, with particular emphasis on the role of receptor-type Na⁺,K⁺-ATPase α1-mediated VRAC activation. The work is well designed and provides valuable mechanistic insights into the structure–activity relationship of bufalin derivatives.

However, several issues should be addressed before the manuscript can be considered for publication.

Response

We sincerely thank the reviewer for the careful evaluation of our manuscript and for the valuable and constructive comments and suggestions. We have addressed each comment and revised the manuscript accordingly. In addition, we have provided a marked version of the manuscript in which all revisions are highlighted in red for clarity.

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Conclusions. The manuscript would benefit from a clearly separated Conclusions section. At present, the concluding remarks are embedded within the Discussion, making it difficult for readers to identify the main findings and their broader implications. A distinct Conclusions subsection should succinctly summarize the key outcomes and emphasize the novelty and significance of the study.

Response

We thank the reviewer for this helpful suggestion. As recommended, we have added a separate Conclusions section to the revised manuscript. The concluding statements previously included in the Discussion have been revised and moved to this section to clearly summarize the main findings of the present study. Please refer to the Conclusions section (pages 10 and 11, lines 337–345).

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Figure preparation. The authors should specify in the figure legends or in the Materials and Methods section which software was used to prepare all graphical figures and schemes. This information is important for transparency and reproducibility.

Response

We thank the reviewer for this comment. We have added information regarding the software used to prepare graphs, figures, and schematic illustrations to the Materials and Methods section. Graphs were generated using Origin 2019 and Microsoft Excel (Microsoft 365), and figures and schematic illustrations were prepared using Microsoft PowerPoint (Microsoft 365). Please refer to the Materials and Methods section (page 13, lines 478–481).

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Characterization of tested compounds. Since the biological conclusions rely on commercially obtained bufadienolides, the authors should provide evidence of their chemical identity and purity. At minimum, the purity of all investigated compounds should be reported. In addition, supporting analytical data such as ¹H NMR spectra together with HRMS data, or alternatively HPLC chromatograms demonstrating compound purity, should be included as Supplementary Information. This recommendation applies even if the compounds were purchased from a commercial supplier, as confirmation of compound quality is essential for ensuring the reliability and reproducibility of the biological results.

Response

We thank the reviewer for this important suggestion. All bufadienolides used in this study were purified by Kyushin Pharmaceutical Co., Ltd., and their purities were determined by HPLC. The purities were 99.5% for 3β-bufalin, 98.8% for 3α-bufalin, 99.7% for resibufogenin, 99.3% for cinobufagin, 94.5% for telocinobufagin, 97.0% for cinobufotalin, and 99.4% for desacetylcinobufagin. We have added this information to the Materials and Methods section. In addition, the corresponding HPLC chromatograms have been included as Supplementary Figure S3 to document the purity of the compounds used in the present study. Please refer to the Materials and Methods section (page 11, lines 350–354) and Supplementary Figure S3.

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Preliminary pharmacokinetic assessment. To strengthen the translational value of the study, the authors are encouraged to include a preliminary in silico evaluation of the pharmacokinetic (ADME) properties of the investigated bufadienolides. Prediction of parameters such as gastrointestinal absorption, blood–brain barrier permeability, P-glycoprotein interaction, cytochrome P450 liabilities, and drug-likeness would provide valuable information regarding the therapeutic potential of these compounds and complement the biological findings.

Response

We thank the reviewer for this valuable suggestion. As recommended, we performed a preliminary in silico ADME analysis of the investigated bufadienolides using SwissADME and have included the results as Supplementary Table S1. The analysis included gastrointestinal absorption, blood–brain barrier permeability, P-glycoprotein substrate prediction, inhibition of major cytochrome P450 isoforms, and drug-likeness parameters. We have also added a discussion of these predictions and their potential pharmacokinetic and therapeutic implications to the revised manuscript. Please refer to the Discussion section (page 10, lines 309–327) and Supplementary Table 1.

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Section 4.2 (Cell culture). Section 4.2 should be carefully revised, as part of the text is displayed in red font, making it unclear whether these passages represent tracked changes, editorial comments, or content intended to remain in the final version of the manuscript. The authors should ensure that all tracked changes and formatting issues are resolved before submission, and that the final text is presented in a clear and consistent format.

Response

We thank the reviewer for pointing this out. The red font in the Cell Culture section was an unintended formatting issue. We have corrected the formatting and carefully checked the entire manuscript to ensure that all text is presented consistently in the revised version. Please refer to the Materials and Methods section (page 11, lines 359–367).

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have revised the manuscript and the supplementary material in accordance with the recommendations, and the work is now suitable for publication in this form.