PONE-D-24-17168Elucidating the Impact of Hypericum alpestre Extract and L-NAME on the PI3K/Akt Signaling Pathway in A549 Lung Adenocarcinoma CellsPLOS ONE

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PLOS ONE

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Additional Editor Comments:

The authors have effectively tackled the scientific query, offering ample background information and pertinent methodological details. The manuscript is well-crafted to enhance reader comprehension, however the authors should conduct essential and crucial experiments as suggested by the reviewers including the incorporation of additional lung cancer cell lines and performing confirmatory or supplementary western blots and other experiments as recommended.

Regarding the drug extract, kindly conduct a hemolytic test with the HA extract and please analyze the purity of the HA extract using HPLC.

In a study published in Pharmacol Rep 2012;64(3):511-20 by Jana Kopincova, that both short term and long term (acute and chronic) L-NAME treatment can affect blood pressure and vascular reactivity by reducing the availability of nitric oxide (NO). Interestingly, lower doses of L-NAME may trigger the production of NO through feedback mechanisms when used over an extended period. This phenomenon should be further discussed to provide a clearer understanding

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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: Partly

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: Yes

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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: No

Reviewer #3: Yes

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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: The authors are trying to uncover the anti-cancer effect of natural product HA extract, which is a promising strategy for cancer therapeutics targeting PI3/AKT signaling. How ever some critical experiments need to be addressed for successful publication

Please find my comments below

1. Authors needs to mention what dose the compounds are used and how they came up to the dose by showing viability assay with Ic50 values. If combination of HA and LA are cytotoxic to cells needs to be checked by a viability assay and colony formation ability as they are important indicators or cancer cell proliferation.

2. Authors can adopt wound healing assay with/with out treatment to show growth inhibition

3. I would recommend performing western blots as-well showing reduction in the PI3/AKT phosphorylation. I would like to know if the effects are temporary and if you do washout studies after treatment, will the cell recover? Also, a commercially available AKT /PI3 K inhibitor should be used as control.

4. At least 1 or two downstream signaling targets of PI3 /AKT is necessary to show ceasing of signaling pathway

5. Have you thought about looking the other arm of PI3 /AKT, which is the RAS/RAF pathway as a compensatory mechanism. Will the HA extract/combination affect RAS/RAF pathway effects, please mention your thoughts in the discussion

6. It would be nice to see schematic representation emphasizing where the compounds are targeting and downstream effects, would certainly enhance the publication quality

7. Please mention how Apoptosis rate was calculated. Visually Fig 3 B has more caspase activity (even though 3 cells), however 5-Fu has low apoptosis compared to other treatments, please elaborate the quantification

Reviewer #2: I recommend the inclusion of an additional cell line such as, Calu3 or NCI-H727 to validate the results obtained in A549 cells. The effect of HA, LN and combination of HA+LN was in A549 cells was reported, however, the docking studies have revealed the active ingredients within these extracts to be specific in the inhibitory effect on PI3K/AKT pathway. Is it possible to check the inhibitory effects of these active ingredients in these cells rather than the whole extracts? 5-FU is being used at a relatively higher concentration of 40uM in cells. Have the authors titrated the dose prior to its use in the assays? In Fig1 E and F, there is no data corresponding to LN, hence, the combined effect of HALN that is presented is left to speculation than a mere conclusion supported by evidence. Hence, effect of LN should also be included. How is the synergy observed upon treatment with HALN of Cox2, when the individual effects tend to be opposite? There is no observed synergy of HALN treatment on MMP2.

Overall, I recommend the inclusion of an additional cell line to validate the results and address the comments above.

Reviewer #3: The authors have done good job in addressing the scientific question. They have provided sufficient background inormation on the study, relevant details in the method section and detailed results significance. Manuscript is nicely written. For making the current study more comprehensible for the readers, authors are suggested to improve the quality of their figures, throughout the manuscript. This will enhance the clarity and overall significance of the study.

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Reviewer #1: Yes:  Sreeja C Sekhar

Reviewer #2: No

Reviewer #3: No

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Elucidating the Impact of Hypericum alpestre Extract and L-NAME on the PI3K/Akt Signaling Pathway in A549 Lung Adenocarcinoma and MDA-MB-231 Triple-negative Breast Cancer Cells

PONE-D-24-17168R1

Dear Dr. Nikolay Avtandilyan,

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.

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Kind regards,

Vino Cheriyan, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have revised the manuscript satisfactorily, responded to the majority of the comments and manuscript has been modified wherever necessary, I suggest that this work be published.

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

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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 #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

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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

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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: Authors have addressed all concerns raised previously and I recommend this manuscript for publication.

Reviewer #2: The authors have addressed all of the comments raised in the initial review. The authors have clarified the questions and revised the manuscript at places as required.

Reviewer #3: Manuscript is revised satisfactorally. Most of the comments have been addressed by the authors. Recommended to accept.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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