PONE-D-23-40260Achillea fragrantissima (Forssk.) Sch.Bip instigates the ROS/FADD/c-PARP expression: An actuation of apoptosis in breast cancer cellPLOS ONE

Dear Dr. ALASMARI,

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 22 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,

Ajay Kumar, PhD

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. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files.

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

  

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

5. 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.

[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: No

Reviewer #2: Yes

Reviewer #3: Partly

********** 

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: No

********** 

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

Reviewer #3: 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: 1. Revise the scientific name of the plant throughout the entire manuscript.

2. Ensure that all gene names are formatted in italics.

3. Authors should reference the supporting papers for the materials and methods section and specify the number of cells used in various in vitro assays.

4. Clearly indicate the specific part of the plant utilized in this study.

5. Rewrite the heading "Determination of Live/Dead assay (AO/EtBr)."

6. Consider replacing "produces" with "induces" in the sentence "The 24-hour experimental time revealed that A. Fragrantissima induces 50% cell death at 20 µg/mL in MCF-7 cells."

7. Based on the results, it appears that the selected plant extract is toxic to normal cells at very low concentrations. If the IC50 value exceeds 100 µg/mL for the normal cell line (HEK293), it can be assumed that the extract is safe for normal cells.

8. Question the need for performing a cell viability experiment if the cell viability has already been assessed through the MTT assay. Consider excluding this experiment from the article.

9. Elaborate on the results section of 3.4, as the material and method for this experiment are missing.

10. To validate cellular integrity, Hoechst or DAPI staining will be used. The colony formation assay only validates the cell migration ability of cancer cells.

11. Authors should provide macroscopic and microscopic images of colonies in the colony formation experiment section.

12. Check the statistical significance in Figure 1d. Authors have marked **** in the image panel.

13. Include a scale bar in Figure 3d.

14. Enhance the language to improve readability and understanding.

15. Avoid using abbreviations in the abstract.

16. In the abstract section, write the name of the plant, Achillea fragrantissima (A. fragrantissima), in the proper format, as well as throughout the entire manuscript.

17. The introductory part should include a brief overview of the current status, overall incidence rates, and epidemiology of breast cancer to make the article more impactful.

18. Address grammatical errors and spelling mistakes throughout the entire article, including line 102, 111, 115, 121, 167, 207, 271.

19. Characterize the ethanolic extract of A. fragrantissima and include information about various bioactive compounds present in the extract.

20. Ensure consistency between the result section 3.1 and Figure 1b, as the normal cells HEK-293 are mentioned as HEK-293T in Figure 1b.

21. Provide the relevance of the present results with previous research to enhance the impact of the article.

22. Specify the criteria for selecting the time intervals in Figures 1d, 3a, and 4.

23. Clearly distinguish between gene and protein for better understanding.

24. Highlight the "toxicity and pharmacokinetic studies" of the ethanolic extract of A. fragrantissima in the article.

25. Improve image quality for better visualization of cell morphology.

26. Ensure that the concentration unit in Figure 1a, b, and c is in μg/mL.

27. In Figure 7, correct the mention of CYSC protein to CYCS in the graphical part.

28. Elaborate on the concluding parts of the article.

Reviewer #2: 1. The manuscript contains several grammatical errors.

2. Inclusion of other breast cancer cell lines alongside MCF-7 will improve the manuscript.

3. In the section 3.1.A, in place of using HEK cells as a control, it is advised to use MCF-10A cells, which is more appropriate control for breast cancer studies.

4. Vehicle control (e.g., ethanol alone) is missing in the ethanol-extracted compound experiment. Further, inclusion of positive control in cell death experiments, such staurosporine or doxycycline induced cell death etc. will be useful.

5. In case of dual staining experiments, apart from merged images, images from the individual channels must be added.

6. Lack of Hoechst 33342 based nucleus staining in the control samples need to be explained.

7. In the mRNA expression analysis, both, PARP and caspase 8 are downregulated. However, in the western-blot based experiments, both are increased. What is the reason behind this discrepancy?

8. In case of cytochrome c release experiment, it is not clear whether the whole cell extract was used or only mitochondrial extract was used.

Reviewer #3: Abdulrahman ALASMARI evaluated the efficacy of Achillea fragrantissima against the breast cancer cell (MCF-7) and claims the possible involvement of ROS/FADD/c-PARP expression for induced apoptotic network genes (Caspase-3, Caspase-8, Caspase-9, Cytochrome c, BCL-2, BID, BAX, PARP, PTEN, PI3K, and Akt).

The manuscript is well designed and scientifically sound, however data analysis should be improved. Discussion part is poorly explained.

I offer the following comments in hope to assist with improvement:

1. Author used ethanol extract of A. Fragrantissima in treating MCF-7 cells. Did they used any appropriate vehicle control for experiment? Include this in manuscript.

2. A. Fragrantissima has shown the decreased cell viability against normal cells - HEK-293 T cells (fig 1). How the result was correlated?

3. I wonder to see the image of DCFH-DA (ROS) and Hoechst-33342 (DNA Fragmentation) stained cells. No cellular expression has been observed in control/MCF-7 cells (Fig 2).

4. The quantitative analysis is poorly defined and need to be elaborated (such as percentage change or modulation-% increase/decrease etc.) for all data.

5. Manuscript title is not conclusive; it could be improved.

********** 

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: Akhilesh Kumar

Reviewer #3: Yes: Akhilendra Kumar Maurya

**********

[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.

Achillea fragrantissima (Forssk.) Sch.Bip instigates the ROS/FADD/c-PARP expression: An actuation of apoptosis in breast cancer cell

PONE-D-23-40260R1

Dear Dr. ALASMARI,

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,

Ajay Kumar, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have adequately addressed all the comments asked by the reviewers and have accordingly revised the manuscript. Hence, the manuscript can be accepted for publication in its present form.

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 #2: All comments have been addressed

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 #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: 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 #2: 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 #2: Yes

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 #1: The manuscript has undergone a comprehensive review process. All the comments, suggestions, are addressed. Recommended for publication.

Reviewer #2: (No Response)

Reviewer #3: Authors incorporated all the suggestions and concern; and addressed properly in manuscript for its betterment. I recommend my acceptance 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 #1: No

Reviewer #2: Yes: Akhilesh Kumar

Reviewer #3: Yes: Akhilendra Kumar Maurya

**********