Review Reports

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript addresses a relevant topic and includes potentially interesting pharmacological and electrophysiological information regarding ion-channel modulation in glioma. However, in its current form, the scope and type of the article remain unclear, and the novelty relative to previous reviews is not sufficiently established. Several aspects should be clarified before the manuscript can be adequately evaluated.

The rationale for selecting the ion channels and compounds included in the manuscript should be clearly established. In its current form, the selection appears somewhat arbitrary and does not fully reflect the broad scope suggested by the title. If the manuscript is intended to focus on selected ion channels and representative pharmacological modulators, this should be explicitly stated. More importantly, the criteria used to select both the channels and the compounds should be clearly defined.

The manuscript combines features of a narrative review with original experimental results, making the article type unclear. In particular, the inclusion of unpublished electrophysiological data and new docking analyses goes beyond the usual scope of a review. The authors should clearly define whether this work is intended as a Review, a Perspective, or an original research article, and the structure and presentation of the manuscript should be adjusted accordingly.

The concentrations reported in Table 1 should be carefully revised. As currently presented, several values appear to be expressed in M, which would imply concentrations in the range of tens or even hundreds of moles and is clearly inconsistent with the experimental context. The authors should verify all units and ensure that µM, mM, or other appropriate units are correctly and consistently reported.

The specific contribution of this manuscript relative to previous reviews on ion channels in glioma should be more clearly established. Several of the channel families, pharmacological strategies, and drug-repurposing concepts discussed here have already been extensively reviewed, including in Takayasu et al., Ion Channels and Their Role in the Pathophysiology of Gliomas (Mol Cancer Ther. 2020; doi: 10.1158/1535-7163.MCT-19-0929) and Elias et al., Ion Channels in Gliomas—From Molecular Basis to Treatment (Int J Mol Sci. 2023; doi: 10.3390/ijms24032530). The potentially distinctive aspect of the present manuscript appears to be its emphasis on direct electrophysiological modulation and channel-gating properties. If this is the intended contribution, this focus should be explicitly defined and consistently developed throughout the manuscript.

A substantial proportion of the experimental content presented in the manuscript is not original. Figures 1, 8, and 9 are explicitly indicated as adapted from previously published studies, and the Appendix further states that most of the experimental results were adapted from earlier work, with Figure 4 being specifically identified as original and unpublished. This considerably limits the novelty of the manuscript, particularly because several of the pharmacological effects discussed have already been reported elsewhere. In addition, the origin of Figure 5 is not clearly stated. The authors should explicitly indicate for each experimental figure whether the data are original, reproduced, or adapted, and should better define what genuinely new contribution is provided by the present work.

Several figures overlap with the main text, making parts of the manuscript difficult or impossible to read. This occurs in multiple sections and substantially affects readability. The layout should be carefully revised to ensure that figures, legends, and text do not overlap.

Figure 4 is presented as original experimental data within a manuscript that is otherwise framed as a review. This further contributes to the ambiguity regarding the nature of the article. If these data are intended to support an original experimental conclusion, substantially more methodological information would be required, including experimental design, biological replicates, statistical analysis, cell culture conditions, recording criteria, and appropriate controls. However, even with this information, a single preliminary dataset would not be sufficient to support publication as an original research article. Conversely, its inclusion within a review is difficult to justify, since the main purpose of a review is to critically synthesize the available literature rather than introduce isolated preliminary findings. The authors should reconsider whether Figure 4 belongs in the manuscript or substantially redefine the article type and scope.

The manuscript relies substantially on experimental figures and pharmacological observations originating from previous publications involving one of the authors of the present review, particularly S.-N. Wu. For example, Figure 1 is adapted from Lai et al. [10], which includes S.-N. Wu as an author, and Figures 8 and 9 are adapted from Yeh et al. [18], which also includes S.-N. Wu. This is not inappropriate by itself; however, it raises an important concern regarding the representativeness and balance of the literature selected for discussion. The authors should clarify whether the chosen channels and compounds were selected using predefined criteria independent of their own previous work. Otherwise, the manuscript risks appearing as a synthesis centered largely on the authors’ prior research rather than a balanced critical review of the field.

Author Response

Please see attached

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The manuscript is improved and of interest for a larger audience now. 

I do reccomend it for publication. 

 

Best. 

Author Response

Please see attached

Author Response File: Author Response.pdf

Reviewer 3 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Review report

General assessment

The manuscript presents a Perspective on ion-channel-targeting modulators in glioma, integrating published electrophysiological evidence with exploratory molecular docking analyses. The topic is relevant for glioma pharmacology, tumor-associated epilepsy, and drug-repurposing strategies. The manuscript is generally well aligned with the format of a Perspective, and the abstract appropriately acknowledges the hypothesis-generating nature of the docking component and the translational limitations of several compounds.

The article is potentially suitable for publication after minor revision. The main remaining issues concern methodological clarity in the docking section, cautious interpretation of docking-derived claims, and final editorial polishing.

Strengths

The manuscript addresses a timely and relevant topic. The focus on functional ion-channel modulation is valuable because it goes beyond expression-level discussion. The inclusion of clinically used drugs and bioactive natural products gives the Perspective translational interest. The authors also acknowledge important limitations related to docking, toxicity, and the need for experimental validation.

Minor comments

1. In Appendix A.2, the statement that the corresponding PDB IDs are “listed as XXXX” appears to be an unresolved placeholder. This should be corrected and replaced with the exact PDB IDs used for each docking analysis. Since the docking component is one of the original elements of the Perspective, the methods should be internally complete and free of placeholders.

2. The docking methods should be made easier to reproduce. The manuscript reports the general workflow and indicates that the lowest-energy pose was selected, while several docking coordinates are provided in figure legends. The Methods would benefit from a compact summary of the operational parameters across all docking cases, including docking box definition, pose-selection criteria, and treatment of retained water molecules where applicable.

3. The perampanel–AMPA section still contains strong translational wording. In particular, the phrase “may represent a paradigm shift” should be softened, since the docking result is exploratory and has not been experimentally validated in the present study.

4. Figure 11 and its legend should be moderated. The current wording implies that the discussed compounds suppress glioma invasiveness through ion-channel modulation. A more cautious formulation would better reflect the manuscript’s own statement that ion-channel modulation alone does not establish a causal anti-glioma mechanism, especially for pleiotropic compounds.

5. The manuscript requires a final language and formatting check. Examples include wording such as “The effects of perampanel effects,” residual grammar issues in the table notes, and several awkward expressions in the docking and conclusion sections.

Recommendation

Minor revision.

The manuscript is close to publication. The remaining issues should be addressable through correction of the docking-methods placeholder, clearer reporting of docking parameters, modest softening of residual overstatements, moderation of Figure 11, and final editorial polishing.

AI disclosure

I confirm that the review reflects my own scientific assessment. I used ChatGPT-5.5 only for wording, organization, and language clarity. The manuscript evaluation, main criticisms, recommendation, and final approval were mine, and I take full responsibility for the report.

Author Response

Please see attached.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Overall, the revised version has improved substantially, and several of the concerns raised in the first round have been addressed. The scope is clearer, the presentation is more consistent, the concentration units have been corrected, and the removal of the unpublished electrophysiological dataset helps reduce some of the ambiguity regarding the nature of the manuscript. The discussion of the limitations of the docking analyses is also more cautious and appropriate.

I still have, however, a few reservations that I believe should be considered in the final evaluation of the manuscript. One of them is the definition of the article type. In the response letter, the manuscript is at one point described as an original research article, while later it is again defined as a review article. In the manuscript itself it is labeled as a Review, although the text also refers to it as a “perspective” and original docking analyses are still included. I think this point could still benefit from greater consistency.

I also remain somewhat concerned about the novelty of the manuscript relative to previous reviews on ion channels in glioma. The relevant previous reviews are now cited, but the distinctive contribution of the present manuscript is still not completely evident in the main text. The emphasis on electrophysiological modulation and channel-gating properties is potentially interesting, but in my view this aspect could have been developed more clearly as the central differentiating element of the review.

Another point is the balance of the literature. Although the authors indicate that additional studies from independent groups were incorporated, several of the key pharmacological examples and experimental references remain closely related to previous work from one of the authors. This is not problematic in itself, but it may still give the impression that the review is somewhat centered on the authors’ own research line rather than providing a fully balanced overview of the field.

Finally, after removal of the unpublished valproic-acid electrophysiological figure, some statements regarding inhibition of INa in U87 glioma cells appear to remain in the text. I think it is important that these statements are clearly supported by previously published evidence if they are to remain in a review article.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting and generally well-written manuscript on pharmacological modulation of ion channels in glioma. The topic is relevant and potentially useful for readers interested in ion-channel-targeted therapeutic strategies. However, I have substantial concerns about the current form of the manuscript.

My main concern is that the manuscript is submitted as a review, but it appears to include unpublished original data, including electrophysiological recordings (Figures 4 and 5) and molecular docking analyses (Figures 2, 3, 6, and 10). These data are presented and interpreted as primary results, but in review no Materials and Methods are provided. Therefore, the experimental and computational results cannot be properly peer-reviewed. The authors should either remove these data or convert the manuscript into a format that includes full methodological details, etc.

A specific problem concerns cilostazol and BK_Ca channels. In Figure 5, the authors show increased BK_Ca-channel activity in U87 glioma cells in cell-attached patches. However, the statement that this effect was accompanied by prolongation of mean open time is not supported by the data shown, because no open-time or dwell-time analysis is presented. This conclusion should either be removed or clearly attributed to Ref. [13], where inside-out patch recordings and kinetic analysis were performed.

The authors should also be more cautious about the mechanism of cilostazol action. Ref. [13] provides stronger evidence for a phosphodiesterase-independent and possibly direct effect on BK_Ca channels, because the experiments were performed in inside-out patches. In contrast, the present Figure 5 uses the cell-attached configuration, which preserves intracellular signalling pathways. Thus, indirect mechanisms involving PDE inhibition, cAMP/PKA signalling, adenosine-related pathways, or other cytosolic processes cannot be excluded. And yet, in Figure 6, cilostazol is docked to a cytoplasmic region of the BK_Ca channel, and specific amino-acid contacts are discussed. Moeover, the rationale for selecting this site and pose are not clear . The Authors should state whether this is the most energetically favorable pose, whether alternative poses were obtained, and how the docking search space was defined. Similar concerns apply to the other docking figures. In the absence of methodological detail and experimental validation, these docking results remain speculative and should not be discussed at the level of individual amino-acid interactions.

In summary, the manuscript has potential value as a review, but the inclusion of unpublished electrophysiological and docking data without adequate methods and validation substantially weakens it. I recommend major revision. The authors should clearly separate literature review from original results.

Author Response

Please see attached.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. General assessment

The manuscript discusses ion-channel-targeting modulators in glioma, with emphasis on NaV, BKCa, IKCa, and Kir channels. The topic is relevant, particularly in relation to glioma-associated epilepsy, invasion, and pharmacological repurposing. The manuscript also includes molecular docking analyses for selected ligand–channel pairs, which could provide a useful hypothesis-generating layer if properly integrated.

However, the current version requires major revision. The main issue is the unclear identity of the article. The manuscript reads partly as a broad narrative review, partly as a Perspective, and partly as an article containing original docking analyses. This hybrid structure is not problematic in itself, but it must be explicitly defined. A Perspective integrating selected docking hypotheses could be acceptable, provided that the scope, methods, and evidentiary limits are clearly stated.

At present, the manuscript is too descriptive, insufficiently critical, and methodologically incomplete with respect to the computational component. The therapeutic conclusions are also stronger than the evidence supports.

2. Strengths

The topic is timely and relevant for glioma biology and pharmacology.

The focus on functional ion-channel modulation is valuable, since it goes beyond expression-based discussions.

The selection of compounds includes clinically used agents and bioactive natural products, which makes the topic potentially useful for drug-repurposing discussions.

The docking analyses could add value if presented as a clearly defined, reproducible, hypothesis-generating component.

3. Major comments

1. The manuscript must define its article identity and scope.
The manuscript should clearly state whether it is intended as a review, a Perspective, or a review/Perspective with original docking analyses. At present, this is ambiguous. The authors discuss previously published electrophysiological findings, include adapted figures, and then introduce docking analyses that appear to be newly performed. This mixture makes it difficult to understand what the manuscript contributes beyond summarizing the literature.

A viable solution would be to reframe the work as a focused Perspective on ion-channel pharmacology in glioma, with docking used as a complementary hypothesis-generating tool. In that case, the title, abstract, introduction, and conclusions should be revised accordingly. The manuscript should not present the docking as definitive mechanistic evidence, and it should not read as an original experimental study unless full methods and validation are provided.

2. The background is excessive and should be substantially shortened.
Large parts of the manuscript provide general descriptions of ion-channel biology and drug pharmacology. This dilutes the main message and makes the article appear more like a textbook chapter than a focused scientific Perspective. The background should be reduced to what is necessary to understand the glioma-relevant channel modulation.

The revised manuscript should be organized around the strength of evidence for each compound–channel pair. This would make the article more useful and would help distinguish established electrophysiological findings from speculative or docking-based hypotheses.

3. The docking analyses require a dedicated Methods section.
If the docking was performed by the authors, the manuscript must include a reproducible computational Methods section. Essential details are currently missing, including protein preparation, ligand preparation, protonation states, docking software version, grid center, grid dimensions, pose-selection criteria, number of docking runs, treatment of water molecules, treatment of membrane context, and any validation procedure.

For ion channels, this issue is especially important because binding-site accessibility, conformational state, membrane environment, and ligand protonation can strongly affect docking results. The authors should also explain why docking was performed for some compounds but not systematically for all compounds discussed in the review.

4. Docking results are overinterpreted.
The docking analyses provide plausible binding poses and contact residues, but they do not demonstrate direct binding, state-dependent modulation, or causal effects on glioma phenotypes. Binding energies should not be treated as quantitative affinity estimates. Statements implying that docking explains the observed electrophysiology should be softened.

The authors should consistently present docking as hypothesis-generating. If they wish to make stronger mechanistic claims, they should provide orthogonal support, such as comparison with known ligands, redocking controls, mutational rationale, or experimental validation from the literature.

5. Therapeutic claims should be aligned with the level of evidence.
The manuscript often moves from ion-current modulation to therapeutic potential too quickly. This is particularly problematic for pleiotropic compounds such as valproic acid, berberine, arecoline, triptolide, and oxaliplatin. Channel modulation does not by itself establish channel-mediated anti-glioma activity.

The authors should clearly separate four levels of evidence: electrophysiological modulation, effects on glioma cell behavior, in vivo or clinical relevance, and docking-based hypotheses. Claims such as “therapeutic benefits,” “drug repurposing candidate,” or “suppression of glioma invasiveness” should be moderated unless directly supported by glioma-relevant data.

6. The manuscript needs a critical evidence table.
The current table lists compounds and channels but does not convey evidence quality. A revised table should include the channel subtype, cell model, concentration range or IC50/EC50, direction of modulation, type of electrophysiological evidence, glioma phenotype tested, in vivo or clinical support, docking availability, and main limitations.

This table would help the reader understand which conclusions are well supported and which remain speculative.

4. Minor comments

The abstract should be shortened and rewritten to reflect the final article identity.

The term “metastatic spread” is potentially misleading for glioma and should be replaced by “diffuse infiltration” or “local invasion,” unless extracranial metastasis is specifically intended.

Safety and translational limitations should be discussed more explicitly. Arecoline, triptolide, berberine, and oxaliplatin each raise distinct issues that should temper the therapeutic perspective.

Docking figures should be improved with clearer labels, binding-site context, and a statement that the poses are computational predictions.

The manuscript should avoid presenting adapted experimental figures in a way that could be confused with newly generated data.

Several language and nomenclature issues require correction, including “KCNNA1,” “illusrates,” “subseequent,” “unliked,” “sole as a dietary supplement,” and “Chinese Thunder Good Vine.”

The triptolide subsection should be numbered consistently.

5. Recommendation

Major revision.

The manuscript addresses a relevant topic and could be suitable as a focused Perspective integrating literature evidence with exploratory docking. However, the current version does not yet define its identity clearly enough. The authors should explicitly frame the article type, shorten the broad background, provide full computational methods for the docking analyses, and moderate therapeutic claims. The work may become publishable if it is restructured as a critical, evidence-based Perspective rather than as a broad compilation of literature plus underdescribed docking observations.

Author Response

Please see attached!

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, Wu et al. provide a comprehensive review of pharmacological modulators targeting ion channels in glioma, with a particular focus on voltage-gated sodium channels, BK, IK, and Kir channels. The authors summarize the available evidence supporting the role of these channels in glioma biology and discuss several clinically approved drugs and natural compounds that may modulate their activity, highlighting their potential therapeutic implications and drug-repurposing opportunities. The review also incorporates molecular docking analyses to propose putative interactions between selected compounds and their ion channel targets. Overall, the manuscript addresses an interesting and timely topic, as ion channels are increasingly recognized as promising therapeutic targets in glioma.

Overall, the manuscript addresses an interesting and timely topic with clear translational relevance. The growing interest in ion channel-targeting strategies and drug repurposing for glioma therapy makes this review potentially valuable for readers in both the basic and translational neuroscience and oncology fields.

 

The manuscript is generally well written and addresses a timely topic with clear translational relevance. However, I have a few comments that, in my opinion, would further improve the quality and impact of the review:

1. The authors should better justify the rationale underlying the selection of the compounds discussed in this review. It is currently unclear why these specific molecules were included over other ion channel modulators. Defining the selection criteria (e.g., FDA/EMA approval, clinical use, translational potential, availability of electrophysiological evidence, drug repurposing potential, etc.) would strengthen the structure and rationale of the manuscript.
2. Throughout the sections describing the different ion channel families, it would be preferable to cite original experimental studies rather than relying predominantly on review articles. For example, in the section dedicated to voltage-gated sodium channels, the authors should consider including recent experimental studies such as Alameri et al. (2025), Giammello et al. (2024)—which also compares the effects of multiple sodium channel blockers—and Ai et al. (2023). These references would provide a more up-to-date and comprehensive overview of the field.
3. The statement indicating that temozolomide may also be effective in MGMT-positive patients should be reconsidered or rephrased. The reference provided is based on pediatric glioma models and does not report the MGMT methylation status of the cell lines analyzed. Therefore, the current statement appears stronger than the available evidence supports and should be appropriately qualified.

Author Response

Please see attached.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Review report

General assessment

The revised manuscript is improved in scope and positioning. It is now more clearly framed as a Perspective on ion-channel-targeting modulators in glioma, integrating published electrophysiological evidence with original molecular docking analyses. This is a more appropriate format for the article.

The topic is relevant, and the focus on functional ion-channel modulation is useful. However, the manuscript still requires major revision. The main remaining issues concern docking reproducibility, interpretation of static ion-channel structures, consistency of the cautionary language, and clearer separation between published experimental evidence and original computational hypotheses.

Strengths

The manuscript addresses a timely topic in glioma pharmacology and tumor-associated epilepsy. The revised framing as a Perspective is clearer. Table 1 is more informative than before, and the authors now acknowledge that docking is exploratory and requires validation.

Major comments

1. Docking methods remain insufficiently reproducible.

The Appendix describes the general workflow, but it does not provide enough operational detail. For each docking analysis, the authors should report protein and ligand preparation, protonation states, retained or removed waters, ions or cofactors, docking box center and dimensions, exhaustiveness, number of poses, pose-selection criteria, and any control or redocking procedure. The placeholder “PDB ID: XXXX” should also be corrected.

2. Static cryo-EM ion-channel structures need better contextualization.

The docking interpretation relies on residue-level contacts in conformationally dynamic membrane proteins. The authors should explicitly acknowledge that predicted poses can be affected by channel state, local cryo-EM uncertainty, side-chain placement, ligand protonation, membrane context, and binding-site accessibility. Relevant methodological studies may be cited as examples, such as DOI: 10.1021/acs.jcim.1c00744 and DOI: 10.1021/acs.jmedchem.3c01134.

3. Docking is still overinterpreted in some sections.

The manuscript states that docking is hypothesis-generating, but this caution is not applied consistently. For example, the perampanel–NaV1.6 section concludes that the effects on INa are “most likely attributable” to a direct effect on NaV1.6. This is too strong for docking data alone. Similarly, the perampanel–AMPA section links a predicted pose to a possible “paradigm shift” in brain tumor therapy. These claims should be softened. Docking should be presented as a source of testable hypotheses, not as mechanistic confirmation.

4. The evidence hierarchy should be clearer.

Table 1 should distinguish direct electrophysiological modulation, glioma-relevant phenotypic evidence, in vivo or clinical support, causal channel involvement, and docking-based hypotheses. This distinction is important because channel modulation does not automatically establish channel-mediated anti-glioma activity.

5. Adapted figures and previously published data should be clarified earlier.

Several electrophysiological figures are adapted from previous studies, but this is mainly stated in the legends. The authors should clarify earlier, preferably in the Introduction or in a short methodological note, that these figures provide contextual support, whereas the original component is the selected docking analysis. Wording such as “we further investigated” or “recordings were performed in this study” should be revised when referring to previously published data.

6. Therapeutic claims remain somewhat overextended.

The manuscript now acknowledges that ion-channel modulation alone does not establish causal anti-glioma mechanisms. However, the conclusions and Figure 11 still imply that several compounds suppress glioma invasiveness through ion-channel modulation. This should be moderated, especially for pleiotropic compounds such as valproic acid, berberine, arecoline, triptolide, and oxaliplatin.

Minor comments

The abstract should be shortened further. Some channel-specific background sections remain too textbook-like. The phrase “sing-target agents” should be corrected. Several sentences require language editing, including “mediate through,” “producing suppressing,” and “the concentration range of each compound tested for each compound.” The safety and translational limitations of arecoline, triptolide, berberine, and oxaliplatin should be discussed more explicitly.

Recommendation

Major revision.

The revised manuscript has moved in the right direction, but further work is needed before publication. The authors should make the docking workflow reproducible, better contextualize cryo-EM-based docking on ion channels, moderate docking-derived claims, clarify the origin of adapted figures, and present the evidence hierarchy more critically.

 

I confirm that the review reflects my own scientific assessment. I used ChatGPT-5.5 only for wording, organization, and language clarity. The manuscript evaluation, main criticisms, recommendation, and final approval were mine, and I take full responsibility for the report.

Author Response

Please see attached.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has been improved and it is now suitable for IJMS.

 

Best,

Author Response

Please see attached.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

the manuscript is improved and acceptable in the new version presented

Author Response

Thanks for the reviewer's comments regarding the acceptability of our manuscript.