Time-Resolved Oxygen Dynamics Reveals Redox-Selective Apoptosis Induced by Cold Atmospheric Plasma in HT-29 Colorectal Cancer Cells

Round 1

Reviewer 1 Report

 

the research topic is very interesting and could bring benefit to the scientific community. the authors propose and analyze novel treatment for colorectal cancer by the use of the cold atmospheric plasma. the whole research is focused on the reactive oxidative species, and their influence on cancers and treatment of it.

the introduction is very well organized authors describe principle of cold atmospheric plasma, its influence and the way of action. after that the Bauer theory was explained and given as starting point for mechanism of reaction, cytotoxicity was also described and pointed out the most important factors for the research.

the experimental part thoroughly describe conditions, design of the machine for producing cold atmospheric plasma, and its regimes used in the research. the preparation of cell culture and determination of cell viability has been described very precisely and reader can easily repeat experiments by itself.

the results are presented in logical manner for simple to the more complex one. the authors take opportunity and used figures wherever its possible by that approach in my opinion they improve article quality visualize very complex topic and give opportunity to researcher to make conclusion by itself.

the discussion is very deep covering all key topics and led to the adequate conclusion

the conclusion summarize the finding underline the most important one but also gives potential flaws of the method.

but i have few suggestions to authors:

the abstract shouldn't start with abbreviations please put full name and abbreviations in bracket!

in the line 48and 49 what RONS means?

in the line 492 Figure 7 is highlighted  why?

please increase the font on figure 10.

the authors mentioned importance of the pH values but i don't find the table of graphs with that values?

 

the research topic is very interesting and could bring benefit to the scientific community. the authors propose and analyze novel treatment for colorectal cancer by the use of the cold atmospheric plasma. the whole research is focused on the reactive oxidative species, and their influence on cancers and treatment of it.

the introduction is very well organized authors describe principle of cold atmospheric plasma, its influence and the way of action. after that the Bauer theory was explained and given as starting point for mechanism of reaction, cytotoxicity was also described and pointed out the most important factors for the research.

the experimental part thoroughly describe conditions, design of the machine for producing cold atmospheric plasma, and its regimes used in the research. the preparation of cell culture and determination of cell viability has been described very precisely and reader can easily repeat experiments by itself.

the results are presented in logical manner for simple to the more complex one. the authors take opportunity and used figures wherever its possible by that approach in my opinion they improve article quality visualize very complex topic and give opportunity to researcher to make conclusion by itself.

the discussion is very deep covering all key topics and led to the adequate conclusion

the conclusion summarize the finding underline the most important one but also gives potential flaws of the method.

but i have few suggestions to authors:

the abstract shouldn't start with abbreviations please put full name and abbreviations in bracket!

in the line 48and 49 what RONS means?

in the line 492 Figure 7 is highlighted  why?

please increase the font on figure 10.

the authors mentioned importance of the pH values but i don't find the table of graphs with that values?

Author Response

We sincerely thank the reviewer for the very positive evaluation of our manuscript and for the encouraging comments regarding its scientific relevance, organization, and experimental rigor. We greatly appreciate the reviewer’s careful reading and the constructive suggestions, all of which have been addressed in the revised manuscript as detailed below.

Specific comments

Comment:
The abstract should not start with abbreviations; please put the full name and abbreviations in brackets.

Response:
This suggestion has been fully implemented. In the revised manuscript, all abbreviations appearing at the beginning of the abstract have been replaced by their full terms, followed by the corresponding abbreviations in parentheses.

Comment:
In lines 48–49, what does RONS mean?

Response:
The abbreviation RONS has now been explicitly defined as “reactive oxygen and nitrogen species” in the revised manuscript. Similar abbreviations have also been checked and clarified throughout the text to ensure consistency and clarity.

Comment:
In line 492, Figure 7 is highlighted. Why?

Response:
This was a typographical error. It has been corrected in the revised manuscript. The entire manuscript has been carefully rechecked to ensure that no similar typographical or formatting issues remain.

Comment:
Please increase the font size in Figure 10.

Response:
The font size in Figure 10, as well as in other figures with similar formatting, has been increased to improve readability and visual clarity.

Comment:
The authors mentioned the importance of pH values, but no table or graph is provided.

Response:
As stated in the manuscript, pH measurements were performed to validate the reliability of the DCFH-DA probe. In the revised version, the pH-related data, corresponding graphs, and statistical analyses have been transferred to the Supplementary Materials to avoid interrupting the main mechanistic narrative. The pH behavior in the presence of DCFH-DA is now clearly documented in the Supplementary Materials.
Furthermore, since pH variations were not directly involved in the core mechanistic analysis presented in the manuscript, relocating these results helps prevent potential confusion while preserving methodological transparency.

Author Response File: Author Response.docx

Reviewer 2 Report

In my point of view, after some revisions, this interesting manuscript can be considered for publication in the Antioxidants journal. These are my suggestions:

Keywords are missing.

The meaning of “CAP” has to be explained (Lines 18 and 42).

The study’s novelty has to be better explained in the Introduction. The objectives of the current study also have to be clarified at the end of the introductory section.

The Materials and Methods are adequate.

Results and Discussion: I miss further discussion with other similar investigations to compare the obtained results. A subsection to address the study’s strengths and limitations could be included as a subsection.

Section 4 should be incorporated in section 3. Currently you have two sections entitled “Discussion”. The Conclusions should be more concise and provide more data for further research to be carried out.

In my point of view, after some revisions, this interesting manuscript can be considered for publication in the Antioxidants journal. These are my suggestions:

Keywords are missing.

The meaning of “CAP” has to be explained (Lines 18 and 42).

The study’s novelty has to be better explained in the Introduction. The objectives of the current study also have to be clarified at the end of the introductory section.

The Materials and Methods are adequate.

Results and Discussion: I miss further discussion with other similar investigations to compare the obtained results. A subsection to address the study’s strengths and limitations could be included as a subsection.

Section 4 should be incorporated in section 3. Currently you have two sections entitled “Discussion”. The Conclusions should be more concise and provide more data for further research to be carried out.

Author Response

Reviewer 2

We sincerely thank the reviewer for the positive assessment of our manuscript and for the constructive suggestions provided. We appreciate the reviewer’s valuable feedback, which helped us to further improve the manuscript. All comments have been carefully addressed, as detailed below.

Specific comments

Comment:
Keywords are missing.

Response:
Keywords have been added to the revised manuscript.

Comment:
The meaning of “CAP” has to be explained (Lines 18 and 42).

Response:
The abbreviation CAP has been explicitly defined as “cold atmospheric plasma” at its first occurrence in the revised manuscript. Similar abbreviations have also been reviewed and clarified where necessary.

Comment:
The study’s novelty has to be better explained in the Introduction. The objectives of the current study also have to be clarified at the end of the introductory section.

Response:
The Introduction has been revised accordingly. In particular, the final paragraph of the Introduction has been rewritten to clearly articulate the novelty of the study and to explicitly state the objectives, while avoiding the inclusion of experimental results.

Comment:
The Materials and Methods are adequate.

Response:
We thank the reviewer for this positive evaluation.

Comment:
Results and Discussion: further discussion with similar investigations is missing. A subsection addressing strengths and limitations could be included.

Response:
The Discussion section has been revised to include a more comprehensive comparison with previous studies, particularly mechanistic investigations reported by Bauer and co-workers regarding tumor selectivity and redox-driven apoptosis induced by cold atmospheric plasma.
In addition, a dedicated subsection addressing the strengths, limitations, and future perspectives of the study has been added to the revised manuscript.

Comment:
Section 4 should be incorporated into Section 3. Currently, there are two sections entitled “Discussion.” The Conclusions should be more concise.

Response:
This structural issue has been corrected in the revised manuscript. Section titles and numbering have been standardized, and the Conclusions section has been rewritten as a separate, concise section that clearly summarizes the main findings and outlines directions for future research.

Author Response File: Author Response.docx

Reviewer 3 Report

While the manuscript is interesting and shows a promising approach to cancer treatment, the objective of the manuscript should be emphasized. Furthermore, it is important that the authors perform the corresponding statistical analyses of the variables analyzed.

While the manuscript is interesting, why didn't the authors use a non-tumor cell line, i.e., BEAS cells, to validate that the treatment is specific? In this sense, the selectivity index is important.

Specific points such as suggestions and questions are shown below.

Abstract
L.17. Adjust the abstract to less than 200 words, in accordance with the author guidelines. Puntualizar el objetivo del manuscrito.
L.18. Include the meaning of CAP.

Include the keywords

Introduction
L.41. An introduction typically concludes with the manuscript's objective. Please highlight the manuscript's objective. The introduction does not include results derived from the authors' experiments.
L.42. Include the meaning of CAP.
L.49. Include the meaning of RONS.
L.81. Comprueba el significado de la oración.

Materials and methods
L.168. Include brand, model, state, country.
L.178. Please check. This wavelength corresponds to the visible region, not the UV region.
L.257. Check the meaning of this sentence.
L.263-264. Sentences generally do not begin with numbers; please make the corresponding change.
L.283. Include the meaning of DMSO.
L.302-303. Sentences generally do not begin with numbers; please make the corresponding change.
L.321. Change "complete" to "completely".
L.361-365. This part is not needed in this section; please remove it and place it in the appropriate section.
L.366-368. Did the numerical variables meet the assumption of normality and homogeneity of variance? What tests did the authors perform? Why were analyses of variance (ANOVA) not performed followed by multiple range testing (e.g., Tukey)?

Results
L.369. This is the results section only.
L.379-380. In this case it was not 12KV, 7KHz but 11KV, 7KHz. Why do not the authors explain why some regimes increase viability?
L.394. Figure 4,
L.433. Why was not statistical analysis performed for this variable? Please perform it.
L.441. Important! Perform the statistical analysis of the data in Figure 5c.

Figure 6. Please indicate if this series of images corresponds to 13KV, -6kHz.
Figure 7 is not referenced in the text.

L.504. Standardize the way figures are referenced, since parentheses or periods, etc., are sometimes used.

Discussion and conclusion
L.585. Separate the conclusion from the discussion.
L.590. But this must be verified with non-tumor cell lines (BEAS cells) and the selectivity index calculated to confirm it.

Comments for author File: Comments.pdf

Author Response

Reviewer 3

We sincerely thank the reviewer for the positive evaluation of our manuscript and for the insightful and constructive comments. We appreciate the reviewer’s careful assessment, which helped us to improve the clarity, focus, and methodological rigor of the revised manuscript. All comments have been carefully considered and addressed, as detailed below.

General comments

Comment:
While the manuscript is interesting and shows a promising approach to cancer treatment, the objective of the manuscript should be emphasized. Furthermore, it is important that the authors perform the corresponding statistical analyses of the variables analyzed.

Response:
We thank the reviewer for this valuable comment. In the revised manuscript, the objective of the study has been clearly emphasized at the end of the Introduction. The final paragraph of the Introduction has been rewritten to explicitly define the scope, motivation, and novelty of the present work, while avoiding the inclusion of experimental results.

In addition, comprehensive statistical analyses have now been performed for the relevant variables, including tests for normality and homogeneity of variance, followed by one-way ANOVA and Tukey’s multiple comparison tests where applicable. All statistical results, including test assumptions, p-values, F-values, and significance levels, are provided in detail in the Supplementary Materials.

Specific comments

Comment:
Why didn’t the authors use a non-tumor cell line (e.g., BEAS cells) to validate treatment specificity? In this sense, the selectivity index is important.

Response:
We thank the reviewer for raising this important point. We fully agree that comparative studies involving non-tumor cell lines and the calculation of selectivity indices are essential when the primary objective is to experimentally evaluate treatment selectivity. However, the objective of the present study is fundamentally different and therefore required a distinct experimental design.

The primary aim of this work was to investigate the redox-driven apoptotic mechanism induced by cold atmospheric plasma, with particular emphasis on the time-resolved dynamics of reactive oxygen and nitrogen species (RONS) under biologically relevant conditions. This study is explicitly grounded in the mechanistic framework proposed by Bauer and co-workers, in which plasma-induced apoptosis in cancer cells is explained by a cascade involving long-lived RONS, secondary singlet oxygen generation, partial inactivation of membrane-associated catalase, and subsequent ROS amplification loops.

Importantly, in Bauer’s mechanistic studies, tumor selectivity is not experimentally assessed through parallel viability measurements in normal cell lines. Instead, selectivity emerges conceptually from intrinsic biochemical differences between malignant and non-malignant cells, such as differences in surface catalase activity, NOX-mediated ROS production, and redox signaling pathways. Consequently, these studies focus on elucidating the reaction cascades leading to apoptosis within tumor cells rather than on comparative survival analyses between tumor and normal cells.

In line with this mechanistic approach, our experimental design was centered on resolving the temporal evolution of RONS under IC50 conditions, which represent a critical transition point at which apoptotic signaling becomes dominant in cancer cells. The IC50 was used here as a functional mechanistic reference point, not as a metric of selectivity. Introducing a non-tumor cell line would have shifted the scope of the study toward comparative cytotoxicity without providing additional mechanistic insight into the redox pathways under investigation.

For this reason, we deliberately restricted our experiments to a single tumor cell line and avoided making claims regarding selectivity or safety toward normal cells. These limitations are now explicitly acknowledged in the revised manuscript, and the importance of future studies incorporating non-tumor cell models to experimentally validate selectivity has been clearly stated in the limitations and outlook section.

Statistical analysis

Comment:
It is important that the authors perform the corresponding statistical analyses of the variables analyzed.

Response:
In response to this comment, a comprehensive statistical analysis has been conducted for all relevant figures highlighted by the reviewer. Data distributions were evaluated for normality and homogeneity of variance prior to hypothesis testing. One-way analysis of variance (ANOVA) was performed where appropriate, followed by Tukey’s post hoc multiple comparison test. Detailed statistical tables, including test assumptions, p-values, F-values, and significance levels, are provided in the Supplementary Materials. References to these analyses have been added to the main text where relevant.

Response to detailed comments:

bstract

Comment:
L.17. Adjust the abstract to less than 200 words, in accordance with the author guidelines.

Response:
The abstract has been carefully shortened in the revised manuscript to comply with the journal guidelines and is now below 200 words, while preserving all key concepts and essential information.

Comment:
Puntualizar el objetivo del manuscrito.

Response:
The objective of the manuscript has been explicitly clarified and emphasized in the revised abstract.

Comment:
L.18. Include the meaning of CAP.

Response:
The abbreviation CAP has been defined as “cold atmospheric plasma” at its first occurrence in the revised manuscript. Similar abbreviations have been consistently defined throughout the text.

Comment:
Include the keywords.

Response:
Keywords have been added to the revised manuscript.

Introduction

Comment:
L.41. An introduction typically concludes with the manuscript's objective. Please highlight the manuscript's objective. The introduction does not include results derived from the authors' experiments.

Response:
We thank the reviewer for this comment. In the revised manuscript, the final paragraph of the Introduction has been completely rewritten to clearly state the objective and novelty of the study, while strictly avoiding the inclusion of any experimental results.

Comment:
L.42. Include the meaning of CAP.

Response:
This abbreviation has been defined at its first occurrence in the revised manuscript.

Comment:
L.49. Include the meaning of RONS.

Response:
RONS has been explicitly defined as “reactive oxygen and nitrogen species” in the revised manuscript.

Comment:
L.81. Check the meaning of the sentence.

Response:
The sentence has been revised to ensure clarity and correct interpretation.

Materials and Methods

Comment:
L.168. Include brand, model, state, country.

Response:
The brand, model, and manufacturer information (including state and country) for the microscope camera have been added to the revised manuscript.

Comment:
L.178. This wavelength corresponds to the visible region, not the UV region.

Response:
This typographical error has been corrected. In addition, to improve manuscript coherence and reduce length, the section related to DCFH validity and emission spectroscopy has been transferred to the Supplementary Materials, where the corrected spectroscopic descriptions are now provided.

Comment:
L.257. Check the meaning of this sentence.

Response:
The sentence has been rewritten to accurately convey the intended meaning.

Comment:
L.263–264. Sentences generally do not begin with numbers.

Response:
This issue has been corrected in the revised manuscript.

Comment:
L.283. Include the meaning of DMSO.

Response:
DMSO has been defined at its first occurrence in the revised manuscript.

Comment:
L.302–303. Sentences generally do not begin with numbers.

Response:
This issue has been corrected.

Comment:
L.321. Change “complete” to “completely”.

Response:
The sentence has been rephrased in the revised manuscript to eliminate ambiguity and ensure grammatical correctness.

Comment:
L.361–365. This part is not needed in this section.

Response:
As suggested, this content has been removed from the original section and relocated to a more appropriate section of the manuscript.

Comment:
L.366–368. Did the numerical variables meet the assumptions of normality and homogeneity of variance? Why were ANOVA and Tukey tests not performed?

Response:
In the revised manuscript, all relevant datasets have been subjected to statistical evaluation, including tests for normality and homogeneity of variance. One-way ANOVA followed by Tukey’s multiple comparison test has been performed where appropriate. Full statistical results are provided in the Supplementary Materials.

Results

Comment:
L.369. This is the results section only.

Response:
The section title and structure have been corrected to ensure that this section presents results only.

Comment:
L.379–380. It was not 12 kV, 7 kHz but 11 kV, 7 kHz. Why do some regimes increase viability?

Response:
The voltage and frequency labeling has been corrected in the revised manuscript. To avoid ambiguity, the corresponding voltage and frequency values are now explicitly indicated alongside each label in the figure.

Regarding the observed increase in viability at certain plasma regimes, it should be noted that plasma–cell interactions are inherently complex and depend on multiple interrelated parameters, including device-specific characteristics, discharge chemistry, and cellular redox signaling pathways. In handmade plasma jet systems, identical voltage and frequency values can lead to different biological outcomes across devices. Explaining regime-dependent increases in viability would require extensive additional diagnostics of plasma chemistry and intracellular signaling pathways, which is beyond the defined scope of the present mechanistic study. Similar observations have been reported in the literature, where such effects are documented without assigning a single universal explanation.

Comment:
L.394, L.433. Why was statistical analysis not performed for this variable?

Response:
Statistical analyses for the indicated figures have now been performed and are fully reported in the Supplementary Materials.

Comment:
L.441. Important! Perform the statistical analysis of the data in Figure 5c.

Response:
A full statistical analysis of the data presented in Figure 5c has been conducted and included in the Supplementary Materials. In the revised manuscript, this figure has been transferred to the Supplementary section.

Comment:
Figure 6. Please indicate if this series of images corresponds to 13 kV, −6 kHz.

Response:
This information has been added to the revised manuscript.

Comment:
Figure 7 is not referenced in the text.

Response:
This issue resulted from a figure renumbering error in the previous version. In the revised manuscript, the figure (now Figure 6) is correctly referenced in the text.

Comment:
L.504. Standardize the way figures are referenced.

Response:
Figure referencing has been standardized throughout the revised manuscript.

 

Discussion and Conclusion

Comment:
L.585. Separate the conclusion from the discussion.

Response:
The Conclusion has been separated from the Discussion and is now presented as an independent section. In addition, sections addressing limitations and future perspectives have been added.

Comment:
L.590. This must be verified with non-tumor cell lines and the selectivity index calculated.

Response:
This point has been addressed in detail in the general response to the reviewer and is now explicitly discussed in the limitations section of the revised manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Although the authors have considered most of the comments made in the first round of reviews, the comment on statistical analysis was not addressed in the manuscript, since when performing an ANOVA followed by a Tukey test, letters must be placed in the experimental groups to define significant differences. It is not necessary to include the full analysis as shown in the appendix.

L.74. The objective must be written in the past tense, and what was done must also be written in the past tense.
Figure 3. Figures are usually placed after they have been discussed in the text.
L.401-402. It is not necessary to include the entire statistical analysis; instead, letters should be placed in the figure to denote the significance between treatments.
Figure 9. And the letters corresponding to the statistical analysis?
L.598. The authors cannot speak of selectivity without a non-tumor cell model.

Comments for author File: Comments.pdf

Author Response

General comment: Although the authors have considered most of the comments made in the first round of reviews, the comment on statistical analysis was not addressed in the manuscript, since when performing an ANOVA followed by a Tukey test, letters must be placed in the experimental groups to define significant differences. It is not necessary to include the full analysis as shown in the appendix.

Answser: As rightly asked by the reviewers the statistical analysis has been removed from the supplementary data while letters have been added on the graphs (Fig.6, Fig.9 and supplementary Fig s1).

Detailed comments:

L.74. The objective must be written in the past tense, and what was done must also be written in the past tense.

The past tense has now been used following the reviewer query in the last paragraph of the introduction section dealing with the objectives of this work.

Figure 3. Figures are usually placed after they have been discussed in the text.

Figure3 was shifted right after its discussion in the manuscript in section 3 Results A) MTT-assay.

L.401-402. It is not necessary to include the entire statistical analysis; instead, letters should be placed in the figure to denote the significance between treatments.

Supplementary section B was suppressed together with its previous indication in the manuscript.

Figure 9. And the letters corresponding to the statistical analysis?

Figure 9 was updated with statistic analysis.

L.598. The authors cannot speak of selectivity without a non-tumor cell model.

The sentence was changed for:

“Our findings provide experimental validation of Bauer’s redox model of plasma mode of action [25, 27], while also establishing a predictive framework based on oxygen dy-namics recorded within the first 20 minutes post-CAP exposure”

Selectivity was changed for mode of action.