PONE-D-23-35455Mitochondrial Dysfunction and Cell Death Induced by Gallic Acid in Toona Sinensis Leaf Extracts through MEK/ERK Signaling in Glioblastoma CellsPLOS ONE

Dear Dr. Tsai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please note that the reviewers have raised several points that should be addressed in full. Specifically, the technical points raised by reviewer 1 should be taken into account and be appropriately addressed. It would be important to evaluate the effect in some additional glioma cell lunes and recommendable to generated intracranial xenografts.

Please submit your revised manuscript by Mar 10 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Guillermo Velasco, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please amend the manuscript submission data (via Edit Submission) to include author Hui-Yuan Su and Aij-Lie Kwan.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript the authors explore the therapeutic potential of Toona sinensis leaf extracts (TSL) against glioblastoma multiforme (GBM), focusing on gallic acid as its primary active component. The main findings reveal that TSL exerts significant cytotoxic effects on GBM cells, primarily through inducing apoptosis and cell cycle arrest. The study demonstrates that TSL treatment leads to increased production of ROS within the cells, contributing to mitochondrial dysfunction which in turn lead GBM cells apoptosis. They found that this effect is linked to the ability of TSL to inhibit MEK/ERK signaling pathway, highlighting a crucial molecular target for GBM treatment. Furthermore, the study observes a dose-dependent increase in proapoptotic proteins like Bax and Puma, coupled with a decrease in the antiapoptotic protein Bcl-2, underlining the intrinsic mitochondrial-mediated apoptotic pathways activated by TSL.

Overall, the study is interesting, since it includes a number of different molecular biochemical approaches which display the potential of TSL as a promising candidate for GBM treatment and it offers a novel treatment approach based on natural products.

However, despite the potential interest and scientific merit of the manuscript, there are some areas that could be improved to enhance the robustness of the research presented.

-One of the main weaknesses of the MS is its reliance on in vitro GBM cell line models. While cellular models may offer initial insights, they may not fully capture the complex dynamics of in vivo tumor environments. Therefore, the significance of the work could be enhanced by incorporating orthotopic animal models to evaluate the efficacy of TSL in a more physiologically relevant setting. Such experiments would provide crucial data on the therapeutic potential and safety profile of TSL in a living organism. If animal studies are not possible (justify why), the authors should clearly mention this limitation in their discussion and include in future objective the realization of these in vivo studies and how they could be performed.

-The focus of the study is on two GBM cell lines, who display similar effects to TSL treatment. However, it would be interesting expand the cell lines to ensure a more comprehensive biological assessment regarding the efficacy of the extracts.

-To ascertain whether TSL exhibits preferential activity towards tumor cells over normal cells, it is essential to include at least one non-tumoral cell line in the study. Such an analysis would be particularly relevant in the context of targeted cancer therapies, as it could provide insights into the specificity of the cytotoxic effects of TSL.

- I understand that the main objective of the study is to evaluate the efficacy of TSL extracts as its whole and do not focusing on the separate compounds. However, it would be important if, in addition to gallic acids, the authors discuss what other compounds of TSL could be responsible for its cytotoxic effect. In this regard, making an introductory table where they report the main components of this extract could be useful. Also, please report and discuss potential synergies between them. This consideration would be particularly valuable in developing treatments that exploit the full therapeutic potential of TSL's complex mixture of bioactive compounds.

- Figure 3A: Why the authors observed only a partial modification in cell cycle in both cell lines at the highest concentration, whereas the viability of the cells at that concentration (80 ug/mL) was totally affected by TSL? I do not understand why almost half of cells still is in G0/G1 phase. please discuss that.

- The authors show that mitochondrial and cellular ROS are drastically increased after treatment. It would be important to see if this increased oxidative stress is responsible, at least partly, for cell death. This could be easily verified by treating cells with antioxidant, as NAC, to rescue cell death/apoptosis after TSL treatment. This assay would increase the mechanistic value of the study, which is actually poor.

- The applicability of gallic acid in preclinical models is quite limited due to its bioavailability. Please include and mention in the text some key references related to application of NPs to encapsulate gallic acid, which are related to this study: https://doi.org/10.1002/iub.2436, doi:10.3389/fnut.2022.963413; this discussion would be useful to expand the scope and application of your study.

In the MS there are some sentences that should be improved for scientific clarity. The English should be revised by mother tongue. Here some suggestions to improve the readability of some sentences, but pay attention to revise the whole text:

- The sentence: “In this study, we evidenced that gallic acid was the major effective components in for aqueous extracts in TSL treatment and it could penetrate across the BBB” could be improved as: “In this study, we established that gallic acid is the primary active component in TSL aqueous extracts, demonstrating its ability to penetrate the BBB”.

- The sentence: “For traditional GBM chemotherapy, temozolomide is the most widely used and essential agent, which, through the formation of O6-methylguanine, mismatches with thymine in subsequent DNA replication cycles to induce cell cycle arrest in G2/M and then exerts a cytotoxic effect." Should be improved as: "Temozolomide, the most used chemotherapeutic for GBM therapy, acts by forming O6-methylguanine, which disrupts DNA replication by mismatching with thymine, leading to cell cycle arrest in the G2/M phase and subsequent cytotoxic effects."

- The sentence: “Our data revealed that TSL treatment induced the levels of proapoptotic Bax and Puma and decreased the antiapoptotic Bcl-2 expression level through the mitochondrial-related intrinsic pathway” should be implemented as: “Our findings indicate that TSL treatment upregulates proapoptotic proteins Bax and Puma while downregulating the antiapoptotic protein Bcl-2, signaling through the mitochondrial-related intrinsic apoptosis pathway.”

- The sentence: “Next, U0126 was used as an inhibitor of MAPK/ERK kinase. The results demonstrated that p-ERK was more suppressed, and apoptosis was obviously induced." Should be changes as “Upon employing U0126, a specific inhibitor of the MAPK/ERK kinase, we observed enhanced suppression of phosphorylated ERK and a more pronounced induction of apoptosis.”

- The sentence: “Finally, our results demonstrated that TSL treatment induced apoptosis through antioxidant effect via the MEK/ERK pathway." could be improved as "Ultimately, our results demonstrate that TSL induces apoptosis through an antioxidant mechanism mediated by the inhibition of the MEK/ERK signaling pathway."

Reviewer #2: General comments

This manuscript presents significant findings constituting a breakthrough in the current understanding of the molecular mechanisms underlying the antitumor effects of T. sinensis leaf extracts (TSL), particularly rich in Gallic acid, against glioblastoma multiforme (GBM). GBM is an exceedingly aggressive tumor with a bleak clinical prognosis and limited therapeutic options.

The authors propose an intriguing hypothesis suggesting that TSL treatment inhibits MEK/ERK activation, leading to an augmentation in mitochondrial reactive oxygen species (ROS) production and a reduction in ATP production. Consequently, this induces mitochondrial dysfunction, resulting in the upregulation of proapoptotic proteins (Bax and Puma) and the downregulation of the antiapoptotic protein (Bcl-2), along with G2/M cell cycle arrest. The obtained results demonstrate that TSL induces a reductive environment preceding G2/M arrest and apoptosis, playing a role in oxidative stress that ultimately leads to cell death.

The manuscript's strengths lie in its meticulously designed and objective experimental strategy, yielding robust results. It is noteworthy that, for the first time, the therapeutic potential of TSL targeting the MEK/ERK signaling pathway is highlighted, establishing a novel avenue for this plant with a well-established therapeutic history in traditional Chinese medicine.

However, certain information and statements require further elaboration. Below are some suggestions, queries, and corrections to enhance the clarity and completeness of the manuscript.

Minor comments

1. Title:

I recommend excluding the term "Gallic acid" from the manuscript's title. This suggestion arises from the fact that over 90% of the results and discussions presented in the manuscript are centered around the TSL extract. Moreover, no experimental tests were conducted directly with this specific compound to establish a causal link between its presence and the observed effects. Rather, the effects discussed are likely influenced by the diverse array of compounds inherent in a plant extract.

2. Abstract

The abstract provides a precise overview of the research and its outcomes; however, I would like to propose to the authors the inclusion of information regarding the concentrations and time of TSL treatment within the text. Additionally, a succinct conclusion aligned with the results and discussions would further enhance the comprehensiveness of the manuscript.

3. Keywords

I recommend that the authors consider adding the term "apoptosis" to the list of keywords.

4. Introduction

In my view, the introduction is clear, concise, and poses valid research questions. However, I suggest incorporating at least one paragraph specifically discussing the MEK/ERK signaling pathway, considering its significance as a molecular target in this manuscript. This addition would further enhance the context and relevance of the study.

5. Methodology

The methods and study design are suitable for addressing the research hypothesis. However, I would like to offer the following suggestions:

a. Include the concentration range used in the description of all the tests carried out.

b. Indicate in the text why 48 hours was chosen for most of the tests.

c. Include a brief description of the colony assay method.

Statistical Analysis

The statistical analysis employed is commonly used for in vitro tests involving cell culture. I would like to suggest that the authors provide information regarding the experimental "n" for the tests.

6. Results

I would like to provide a few comments and suggestions regarding the presented results:

a. For item 3.1, I believe the most appropriate title would be "Characterization of Phenolics in TSL and Prediction of BBB Permeation."

b. Is Figure C truly necessary? It appears to convey essentially the same information as figure B, albeit with the structure of gallic acid.

c. Figure 1 D. It would also be interesting to present the BBB score of the other 4 components of the extract. Perhaps a table with the score for each of the active ingredients of interest. I think this information is essential because the other compounds of interest in TSL could also cross the BBB and potentiate the pharmacological effect of gallic acid.

d. Figure 2: I believe it would be crucial to include a table presenting the IC50 values of TSL (perhaps as supplementary material) for the three time points and in the two cell types. Merely stating that the cytotoxicity was dose-dependent lacks the necessary quantitative robustness as a result. Providing the IC50 values is essential for obtaining a quantitative understanding of the concentration- and dose-dependent cytotoxicity of TSL.

e. Please specify the content of each column in the graph in Figure 2B.

f. The resolution of the microscopy in Figure 2C seems to differ between the two cell types, with A172 cells exhibiting greater resolution than U251 cells (????).

g. Figure 3: it would be interesting to comment in the text that the G2/M percentages of 29.87% and 31.27% refer to the higher concentration of TSL (80 µg).

h. Continuing with Figure 3, it is crucial to note that the S phase also exhibited a concentration-dependent increase.

i. Figure 4: It is essential to generate a graph illustrating the quantification of the Western blot bands, as evaluating data solely from the electrophoresis images can be challenging. Alternatively, if the numbers below the bands represent these measurements, kindly include them in the figure caption. Additionally, please clarify the absence of data presentation in Figure 4A.

j. The complexes I to IV are integral components of the electron transport chain (ETC) and they are part da oxidative phosphorylation (OXPHOS). These complexes primarily facilitate electron transport, with complexes I and II being particularly prone to generating reactive oxygen species (ROS) through electron dissipation. Oxidative phosphorylation specifically pertains to complex V, also known as the ATP synthase pump. Kindly consider revising the text to accurately reflect this distinction.

k. On page 16, in the last sentence of item 3.6: Unfortunately, I am having difficulty understanding the statement, "Due to the above findings, we found ERK activation is required for apoptosis induction by TSL treatment in GBM cells." Could it be that it should be "inhibition" instead of "activation"?

l. It is intriguing that the content of "early apoptosis" (Fig 6B) for U251 cells is nearly identical between TSL and TSL+ inhibitor. Could the combination of TSL with the inhibitor not result in a doubling effect, as observed with A172 cells? I am interested in understanding the authors' perspective on this matter.

m. I believe it would be beneficial to delve deeper into the observation that "early apoptosis" is linked to the intrinsic pathway, aligning well with the results obtained for Bax, Bcl-2, PUMA, ERK/ERK2, etc. In contrast, the extrinsic pathway appears to be more closely associated with DNA damage, PARP fragmentation, and the occurrence of "later apoptosis."

n. I think it would be interesting to standardise the text MEK/ERK signalling pathway or MAPK signalling pathway (items 3.6 and 3.7)

7. Discussion

I would like to share the following observations for the authors to consider:

a. Page 18, line 13: Unfortunately, I couldn't understand the sentence "Figure 8. Model for TSL treatment in GBM cells."

b. Page 18, line 15: Please correct "OXPHOS complexes" to "ECT complexes"

c. c. Important note: Typically, PARP cleavage is associated with the extrinsic pathway rather than the intrinsic pathway and is more aligned with "late apoptosis." It would be valuable for the authors to contemplate this emphasis on page 18 of the discussion.

8. Conclusion

After obtaining such remarkable results, wouldn't it be pertinent for the authors to provide a concluding section to the manuscript?

9. Reference

a. On page 11, reference [37] is mentioned at the end of item 2.3, following the previous reference [28]. Similarly, on page 12, reference [38] is cited at the end of item 2.5, with the preceding reference being [28]. Please verify that there hasn't been a referencing error in these instances.

b. Regrettably, I couldn't find references [37], [38], [39], and [40] cited in the list of references at the end of the manuscript.

10. Language

I would recommend the authors to thoroughly revise the text, focusing on both the English language usage and the phrasing of sentences that appear somewhat ambiguous in the current context.

Ultimately, I support the publication of this manuscript; however, it should undergo a comprehensive review considering all the previously mentioned comments before moving forward.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: Yes:  Rozangela Curi Pedrosa

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PONE-D-23-35455R1Mitochondrial Dysfunction and Cell Death Induced by Toona Sinensis Leaf Extracts through MEK/ERK Signaling in Glioblastoma CellsPLOS ONE

Dear Dr. Tsai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please note that the concerns raised by the reviewers have been incompletely addressed and that to be re-considered the authors need to address at least the following points via the performance of additional experiments:

1. The mechanistic connection between the different events delineated in the manuscript is still not solid (the use of the MEK inhibitor suggest an additive effect with the extract and does not support the idea that MEK/ERK is downstream of TSL extracts). The authors should show that reactivation of the MEK/ERK pathway prevents the effect of TSL on apoptosis (and ROS production) and analyze whether ROS scavengers can prevent the effect of TSL on glioma cells. As it is the manuscript does not cogently support the scheme of Figure 8.
2. In the absence of a more detailed characterization of additional GBM cell lines exhibiting different features, the request of performing in vivo analyses is even more important to provide with physio-pathological relevance to their in vitro results.  If the authors had previously employed orthotopic xenografts they should use them in this case too, to corroborate the anticancer activity of the extract in a more relevant model and to validate their claim regarding the passage of the BBB.

Please submit your revised manuscript by Jul 05 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Guillermo Velasco, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments:

The authors have incompletely answered the comments raised by the reviewers.

Specifically:

1. The mechanistic connection between the different events delineated in the manuscript is still not solid (the use of the MEK inhibitor suggest an additive effect with the extract and does not support the idea that MEK/ERK is downstream of TSL extracts). The authors should show that reactivation of the MEK/ERK pathway prevents the effect of TSL on apoptosis (and ROS production) and analyze whether ROS scavengers can prevent the effect of TSL on glioma cells. As it is the manuscript does not cogently support the scheme of Figure 8.

2. The authors should perform some of the experiments indicated by reviewer 1. Specifically, in the absence of a more detailed characterization of additional GBM cell lines exhibiting different features, the request of performing in vivo analyses is even more important to provide with physio-pathological relevance to their in vitro results. If the authors had previously employed orthotopic xenografts they should use them in this case too, to corroborate the anticancer activity of the extract in a more relevant model and to validate their claim regarding the passage of the BBB.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I recognize that the authors have taken into account most of my concerns and have provided detailed explanations regarding the utility of using new models and cell lines to validate the effects of the extracts in vivo. However, I noted that they did not include any additional experiments in this revision, such as introducing a non-tumoral cell line for comparative analysis. While I acknowledge the potential logistical and resource challenges they might face in organizing additional experiments at this time, incorporating at least some of these elements could have strengthened the manuscript significantly. I trust the editor to consider this perspective in making the final decision on the manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PONE-D-23-35455R2Mitochondrial Dysfunction and Cell Death Induced by Toona Sinensis Leaf Extracts through MEK/ERK Signaling in Glioblastoma CellsPLOS ONE

Dear Dr. Tsai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Specifically the authors have only partially answered the questions raised in the editorial report. The contribution of the ERK pathway on the mechanisms of action still need to be reinforced. Likewise, the in vivo confirmation of their observations is still necessary to cogently support their findnings.

Please submit your revised manuscript by Sep 19 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . IN this way I strongly suggest not re-sending a revised version of the manuscript before having completed all the experiments in full. We can provide with additional time but we will not reconsider a new version of the manuscript that has not solve all the issues indicated in this editorial report. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Guillermo Velasco, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments:

The authors have partially answered the questions raised in the editorial report and have shown that NAC can prevent the effect of TSL in glioma cells. In contrast, the use of tBHQ as a way of activating ERK is less sound as this compound in quite unspecific and can also act as antioxidant. Likewise, the results in Figure 6f do not support that this treatment enhances ERK activity over the level of the control at least on a A172 cells. Overall, in my opinion is not completely clear yet that the connection with the ERK pathway is as depicted in Figure 8 and therefore this point is only partially solved.

Regarding the in vivo experiments, I perfectly understand the situation of the authors. However, and unfortunately, the soundness of the results cannot depend on the capacity of the authors to carry out the experiments and therefore these experiments are still required to strengthen the conclusions of the work.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

The authors have partially answered the questions raised in the editorial report and have shown that NAC can prevent the effect of TSL in glioma cells. In contrast, the use of tBHQ as a way of activating ERK is less sound as this compound in quite unspecific and can also act as antioxidant. Likewise, the results in Figure 6f do not support that this treatment enhances ERK activity over the level of the control at least on a A172 cells. Overall, in my opinion is not completely clear yet that the connection with the ERK pathway is as depicted in Figure 8 and therefore this point is only partially solved.

Regarding the in vivo experiments, I perfectly understand the situation of the authors. However, and unfortunately, the soundness of the results cannot depend on the capacity of the authors to carry out the experiments and therefore these experiments are still required to strengthen the conclusions of the work.

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study focused in cell lines due the unavailability of the authors to perform in vivo study. However, the authors included new data showing that the reactivation of the MEK/ERK pathway can prevent the effect of TSL on apoptosis and ROS production. They provided new figures summarizing this data in the revised manuscript. They addressed mostly of the reviewer comments.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PONE-D-23-35455R3Mitochondrial Dysfunction and Cell Death Induced by Toona Sinensis Leaf Extracts through MEK/ERK Signaling in Glioblastoma CellsPLOS ONE

Dear Dr. Tsai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Specifically the authors should modify the scheme included in the manuscript to attenuate the function attributed to ERK (for example by including a discontinue narrower line. The authors should also modify the discussion to mention the limitations of the experimental approach that they undertook and also to indicate that although it is likely that inhibition of ERK plays a role on the effect of TSL it cannot be ruled out that other mechanisms may also cooperate in producing the effects on glioma cells including regulation of ROS production, mitochondrial activity and induction of apoptosis.

 The authors should provide additional information regarding the in vivo experiment (as indicated in the report) and/or repeat it in a convincing manner to cogently suppoort the idea that the TSL exctract has anticancer activity in vivo.

Please submit your revised manuscript by Jan 18 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Guillermo Velasco, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments:

The authors have performed additional experiments and have been able to strength a bit more their conclusions. Hower the work still has significant weaknesses. Firstly, the IKVAV peptide used by the authors to stimulate ERK is not a specific stimulator of this kinase (as it has also been described to activate AKT). Probably the ideal experiment would consist in expressing constitutively active form of ERK or MEK to analyze whether this is interfering with the effect of the TSL extract.

Overall, my impression is that the authors have not been able to cogently support the idea that the effect of TSL is primarily based on the stimulation of ERK. Therefore, the authors should modify the scheme included in the manuscript to attenuate the function attributed to ERK (for example by including a discontinue narrower line to indicate that this is one of the possible mechanisms involved in the effect that they observe but that it cannot be ruled out that there could be others that could also play an important role in the effects induced by this treatment in glioma cells). The authors should also modify the discussion to mention the limitations of the experimental approach that they undertook and also to indicate that although it is likely that inhibition of ERK plays a role on the effect of TSL it cannot be ruled out that other mechanisms may also cooperate in producing the effects on glioma cells including regulation of ROS production, mitochondrial activity and induction of apoptosis.

Likewise, I appreciate the effort made by the authors in performing the in vivo experiment. However, the results are inconclusive. Firstly, the volume reached by the tumors after 70 days of inoculation is very small (the maximum size of the tumors is less than 150 mm3) and measurements of tumors with such an small volume are subjected to major error. Also, the authors used a limited number of animals per experimental condition which also contribute to enhance the error. Moreover, authors did not represent the tumor size of each tumor with a dot but just the mean +/- SD). Of note, in most cases measurement of subcutaneous xenografts is started when they reach 200 or 250 mm3. Also, the vehicle treated-tumors did not grow until day 60, so there is only a significant difference in one of the experimental points /0 days). Why the authors did not continue with the experiment until seeing whether the observed differences were larger? Also, the pictures of the animals are unclear in the photographs provided (the animals are not at their correct proportions, two control animals are missing, the red arrows do not allow seeing the actual tumors that in any case look flat and probably injected too deep on the animals and not subcutaneously which again increases the error). Therefore, unfortunately this experiment is - at least apparently - not correctly performed/analyzed. The authors should therefore provide additional information regarding this experiment to clarify this point and/or repeat it in a convincing manner.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have thoroughly and adequately responded to all reviewer and editor observations, including additional experiments and new data that reinforce the link between TSL and the MEK/ERK pathway in the context of glioblastoma. The inclusion of in vivo experiments (Fig. 8) significantly enhances the study's robustness, supporting the conclusions on TSL's anti-tumor effect and demonstrating its potential as a therapeutic strategy for glioblastoma.

This reviewer appreciates the effort of the authors to include such relevant experiment.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: Yes:  Marco Cordani

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PONE-D-23-35455R4Mitochondrial Dysfunction and Cell Death Induced by Toona Sinensis Leaf Extracts through MEK/ERK Signaling in Glioblastoma CellsPLOS ONE

Dear Dr. Tsai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Mar 12 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Javier S Castresana

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

The present manuscript has been reviewed by various reviewers and one academic editor. Now, a new reviewer and a new academic editor have been included. The new reviewer is worried about the statistics used in the in vivo experiment. Please, check carefully what you did to improve the in vivo experiment after the successive reviews, and carefully study what the new reviewer suggests now you to do. And give us an answer about it. Maybe, as you can see, a new experiment or statistical method mighth be needed.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have made significant changes in the last revision, including clarifying the role of the MEK/ERK pathway by modifying the schematic and discussion to highlight its potential contribution alongside other mechanisms such as ROS production and mitochondrial dysfunction. They also addressed concerns about experimental limitations and provided additional data points for transparency, improving the robustness and clarity of their findings. These updates substantially enhance the manuscript's scientific rigor and presentation. Their study deserves to be published as it.

Reviewer #3: Previous reviewers addressed the small tumor volume in the xenograft experiments. This may difficult measuring and showing the tumors. The author has included a figure with the individual tumors that show the removed tumors, and their volumes are clearly visible now. The vehicle treated group is the only one that shows appreciable tumor growth in some of the mice, while the TSL treated group doesnt show growth at all. However, the growth observed in the vehicle+treated group may be due to the growth of individual outliers in that group, and not due a general difference. It would be advisable to include an statistic method that identifies the presence or lack of outliers, and refer it in the text.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #3: Yes:  Javier de la Rosa

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

<p>Mitochondrial Dysfunction and Cell Death Induced by Toona Sinensis Leaf Extracts through MEK/ERK Signaling in Glioblastoma Cells

PONE-D-23-35455R5

Dear Dr. Cheng Yu Tsai ,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Javier S Castresana

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: The author has performed a solid statistics analysis regarding their xenograft assay. This analysis proves that the observed differences between the treated and control groups are not caused by big outliers in the control group, rather, is a general effect caused by the treatment. The author has included this statistical test in the discussion of the results. Altogether, the xenograft experiment is now conclusive, leaving no room to doubts. The mansuscript is ready for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #3: Yes:  Francisco Javier de la Rosa Fernandez-Pacheco

**********