PONE-D-23-32383Scutellarin combined with lidocaine attenuates growth of human glioma cells associated with down-regulation of epidermal growth factor receptorPLOS ONE

Dear Dr. He,

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: 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: In this manuscript, the authors tell a story about the effects of Scutellarin-lidocaine on human glioma cells. The results of this work indicated that Scutellarin-lidocaine could repress the proliferation and promote the apoptosis of U251 and LN229 cells. However, there are some problems.

1. Scutellarin and lidocaine have been widely reported in diseases, including various tumors. The authors should cite more references and make more discussions to reveal why such a combination was designed.

2. Where is SCU from (the manufacturer and product No.), how about its purity? BP? AP? SP? or UP? Would the impurities in the sample affect the cells?

3. It's not strange that SCU and lidocaine could affect the proliferation, migration and apoptosis of glioma cells. In many other reports, both SCU and lidocaine could also affect the properties of other tumor cells and normal cells, e.g., in our previous work, SCU and lidocaine could affect vascular endothelial cells, Müller cells and fibroblast cells via regulating STAT3 associated axes. Therefore, my suggestion is to use some normal cells as blank, to investigate their differences with the same drug doses.

4. Actually, EdU assay is to investigate the DNA synthesis but not proliferation, and PCNA could be used as a proliferation biomarker.

5. Fig 2 seems confusing, in some cases, low concentration of SCU could even promote the proliferation of U251, and at 400 uM, the addition of lidocaine did not promote the effects of SCU. Therefore, a normal blank cell is necessary here.

6. In Figure 4d, the authors should carefully check the apoptosis figures, which reveal similar contours. After scanning by some software, several ones seem like to be the same one at different time.

7. The most important problem of this work is the absent of mechanism. The authors indicated that Scutellarin-lidocaine could downregulate EGFR, however, what's the direct target of SCU and what's the target of lidocaine? The section between Scutellarin-lidocaine and EGFR is still a black box. Logically, the authors should at least predict the target of SCU via some online tools, e.g., SwissTargetPrediction, screen some ones and confirm them by Biacore, nano-ITC, IP or in vivo navigation.

In the recent stage, the manuscript is not an integrated story.

Reviewer #2: Scutellarin combined with lidocaine attenuates growth of human glioma cells

associated with down-regulation of epidermal growth factor receptor

Comments

In the current article, Xiu et al., had found that a combination of Scutellarin and lidocaine attenuates growth of human glioma cells by down-regulation of epidermal growth factor receptor. Overall, the study has several strengths, including the use of multiple assays to assess various aspects of glioma cell behaviour, such as proliferation, migration, and apoptosis. Additionally, investigating the combination of two compounds, scutellarin and lidocaine, adds novelty to the research. However, there are some considerations and areas for improvement in the study design and future research:

Major Suggestions:

• Can you clarify the rationale behind the choice of cell lines used in the study?

• While the general cell culture conditions are provided, it would be beneficial to mention the passage number of the cells used. Continuous passaging can lead to genetic drift and altered cellular behaviour, affecting experimental reproducibility.

• The choice of CCK-8 assay for cell viability analysis is common and suitable. However, the duration of drug intervention (48 hours) seems arbitrary and lacks justification. Providing a rationale for this time frame would enhance the clarity and reproducibility of the experiment.

• I recommend that the authors conduct cytotoxicity analysis on normal astrocytes to confirm the toxic impact of the two compounds.

• Additionally, the effect of these drugs should be compared with the standard drug for glioma viz., Temozolomide through in vitro assays to validate their efficacy.

• For the plate clone formation assay, it would be helpful to include information on the criteria used for defining a clone (e.g., minimum number of cells required) and how the counting of clones was performed to ensure consistency and reproducibility.

• Considering the heterogeneity of Glioma, incorporating 3D culture models such as spheroid or organoid cultures would enhance the relevance and translatability of the findings to in vivo settings.

• The study proposes a combination therapy approach of Scutellarin and lidocaine against Glioma. Have similar combination therapies been explored in clinical trials or preclinical studies for GBM, and what are the challenges associated with translating this approach to clinical settings?

• While the study implicates the downregulation of EGFR protein as a potential mechanism for the observed effects, additional experiments has to be conducted to further elucidate the molecular pathways involved. In that case, which downstream signalling cascades will the authors study and why?

Minor Suggestions:

• Check for grammatical accuracy and maintain a consistent writing style.

Overall, the paper has a solid foundation, but refining and expanding on certain aspects will contribute to a more comprehensive and reader-friendly manuscript.

Reviewer #3: He et al attempted to explore the role of Scutellarin and lidocaine in glioma growth. Both compound inhibited glioma proliferation. They found interesting results. This study open the window for glioma therapeutic approach.

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Reviewer #1: Yes: Jun Shao

Reviewer #2: Yes: Daisy Precilla S

Reviewer #3: Yes: Mehdi Hayat Shahi

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PONE-D-23-32383R1Scutellarin combined with lidocaine exerts antineoplastic effect in human glioma associated with repression of epidermal growth factor receptor signalingPLOS ONE

Dear Dr. He,

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 by one of the reviewers during the review process. Specifically, check your figures, provide data on purity of scutellarin and clarify your manuscript on minor concerns.  Please submit your revised manuscript by Oct 25 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,

Surinder K. Batra

Academic Editor

PLOS ONE

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Reviewer #1: 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 #3: Yes

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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

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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: 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: 1. CCK8 assay is only used to test the DNA synthesis or duplication, but not cell proliferation, it should be clarified in the whole manuscript.

2. In the authors' response, "scutellarin (CAS No. 27740-01-8) was purchased from MedChemExpress Company

(MCE). Its purity has reached 98.56%, which could eliminate the influence of the impurities on cells." Could a 98.56% purity eliminate the influence of the impurities on cells?

3. Could the authors carefully check the apoptosis figures, several sub-images showed the similar contours.

Reviewer #3: Combination of Scutellarin and Lidocaine as antineoplastic potential drug for glioma. This is significance study and add new knowledge in glioma tumour therapeutic approach.

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Reviewer #1: No

Reviewer #3: Yes: Mehdi Hayat Shahi

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Scutellarin combined with lidocaine exerts antineoplastic effect in human glioma associated with repression of epidermal growth factor receptor signaling

PONE-D-23-32383R2

Dear Dr. He,

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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Comments to the Author

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Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: 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: Partly

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: N/A

Reviewer #3: Yes

Reviewer #4: 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 Response)

Reviewer #3: Yes

Reviewer #4: 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: (No Response)

Reviewer #3: Yes

Reviewer #4: 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: 1. Usually, to investigate the cell proliferation, simply CCK-8 assay is not enough, and it should be used together with other protocols, e.g. Edu, PCNA or MTT assays.

2. Usually, for pharmaceutical tests, the compounds should be further purified via RP-HPLC, TLC or other protocols if conditions permit. Or the impurities should be carefully characterized.

Reviewer #3: (No Response)

Reviewer #4: Please change the figure 1 and 2 Y-axis labeling should be changed from inhibite ratio to inhibition ratio.

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Reviewer #1: No

Reviewer #3: Yes: MEHDI HAYAT SHAHI

Reviewer #4: No

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