Anethole inhibits human U87 Glioma cell proliferation by inducing apoptosis via the PI3K/AKT pathway

Ahmed Abdullah Al Awadh; Elhashimi Eltayb Hassan; Omer Mohamed Shoaib; Osman A.E. Elnoubi; Saadalnour Abusail Mustafa; Yasir Mohammed Althayrayan; Majed Ahmed Althayrayan; Mohammed Merae Alshahrani

doi:10.1371/journal.pone.0336975

Peer Review History

Original SubmissionFebruary 26, 2025
31 Mar 2025 Decision Letter - Zahra Lorigooini, Editor Dear Dr. Alshahrani, 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. ============================== ACADEMIC EDITOR: After reviewing the manuscript, I would like to suggest several revisions that will improve its clarity, consistency, and overall alignment with the journal's guidelines. Adherence to Journal Guidelines: It appears that some sections of the manuscript do not fully comply with the formatting and citation guidelines provided by the journal. Kindly refer to the journal’s style guide and make necessary adjustments, particularly in relation to: Correct formatting of the references. Consistency in the use of terminology and abbreviations. Proper structuring of headings and subheadings. Language and Clarity: The manuscript would benefit from a thorough review of the language and grammatical structure to enhance clarity. I recommend having the text reviewed for grammatical and syntactical accuracy. Methodology Section: It would be helpful to provide additional details regarding the source and purity of anethole, the solvents used, and any quality control measures taken during its preparation and administration. This will improve reproducibility and transparency. Figures and Tables: The figures and tables should be clearly labeled, and all axes should be properly defined. I suggest ensuring that figure captions fully describe what is presented, and that they are referenced correctly within the text. Supplementary Data: If any supplementary data or additional experiments have been conducted, please include them as supplementary files or specify their omission and provide an explanation. I would greatly appreciate it if you could make these revisions and resubmit the manuscript. Please also ensure that the manuscript is reviewed for consistency in the presentation of the data, particularly concerning statistical analyses and experimental results. ============================== Please submit your revised manuscript by May 15 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. 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, Zahra Lorigooini 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. 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. [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? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: No Reviewer #2: Yes ****** Reviewer #1: Abstract: "Colony formation was notably inhibited in a dose-dependently." → Grammatical error. Correct it to: "Colony formation was notably inhibited in a dose-dependent manner." • How many replicates were performed for IC50 determination? Mention explicitly. • Western blot results should be summarized more concisely. Introduction: • Page 2, Paragraph 2: "Gliomas are essentially the most prevalent and therefore malignant primary brain tumors..." → Unclear phrasing. Gliomas are not "therefore malignant"; glioblastomas are. Reword for clarity. • Page 3, Last Paragraph: "These findings underscore the versatility of anethole as an anticancer molecule..." → Mention gaps in knowledge regarding anethole in gliomas. • The introduction should provide a clearer rationale for targeting PI3K/Akt in glioblastoma. Materials and Methods: • Page 4, "CCK-8 Assay": What was the exact duration of anethole treatment? Specify. Cite the references • Page 5, "AO/EB Staining": How were apoptotic vs. necrotic cells quantified? State whether manual counting or software analysis was used. Cite the reference • Page 6, "Molecular Docking": Why was the docking performed with AutoDock instead of newer methods like AutoDock Vina or Schrödinger? Justify. • Page 7, "Western Blot Analysis": Were the densitometry results normalized to loading controls? Mention which software was used. Results: • Page 8, Figure 1: What is the sample size (n) and standard deviation (±SD) for IC50 values? Ensure reproducibility. • Page 9, "Anethole Triggers Apoptosis": Clarify if apoptosis was confirmed via flow cytometry or just AO/EB staining. • Page 10, "Molecular Docking Results": Docking energy (-9.32 kcal/mol) is strong, but was in vitro validation performed to confirm target binding? Discussion: • Page 12, Paragraph 1: How does anethole compare to existing PI3K inhibitors like Wortmannin or LY294002? A comparative discussion is needed. • Page 13, "Potential Clinical Application": While anethole has brain permeability potential, is there direct evidence that it crosses the blood-brain barrier (BBB)? Cite relevant studies. • Page 14, "Limitations": The study lacks animal model validation. Suggest including a statement on future in vivo studies. Conclusion: • The conclusion should briefly highlight future directions (e.g., combinational therapies, BBB permeability studies). Reviewer #2: The authors need to compare the binding constants of anethole with PI3K and JAK2 calculated by MD simulations with experimental data by performing ITC/ BLI/SPR studies if possible which will further supports the mechanism of binding. ****** 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: Jesil Mathew A Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0336975.r001
Revision 1
15 Apr 2025 Author Response Dear editor, We thank the reviewers for their thoughtful and constructive comments, which have significantly improved the quality and clarity of our manuscript. We have addressed each point below and made corresponding changes to the manuscript as indicated. Reviewer #1 Abstract 1. “Colony formation was notably inhibited in a dose-dependently.” → Grammatical error. ✔ Corrected to “Colony formation was notably inhibited in a dose-dependent manner” in the abstract. 2. How many replicates were performed for IC₅₀ determination? Mention explicitly. ✔ We now state that all IC₅₀ values were calculated based on three independent experiments (n=3), with data expressed as mean ± standard deviation (SD). 3. Western blot results should be summarized more concisely. ✔ The Western blot summary in the abstract has been streamlined to focus on the major findings: induction of Bax and reduction of Bcl-2, and suppression of PI3K/Akt phosphorylation. Introduction 4. Page 2, Paragraph 2: Unclear phrasing regarding glioma malignancy. ✔ Rephrased the sentence to: “Gliomas are essentially the most prevalent and primary brain tumors, constituting about 80% of all malignant intracranial neoplasms [1].” 5. Page 3: "These findings underscore..." → Mention gaps in knowledge regarding anethole in gliomas. ✔ Revised to note that although anethole shows promise in other cancers, its role in glioma remains unexplored and warrants targeted investigation. This has included in detail 6. Provide a clearer rationale for targeting PI3K/Akt in glioblastoma. ✔ We have added supporting context and references explaining that the PI3K/Akt pathway is frequently dysregulated in glioblastoma and is associated with tumor progression, making it a rational therapeutic target. Materials and Methods 7. Page 4, “CCK-8 Assay”: Specify exact duration of anethole treatment. ✔ The duration has been specified as 24 hours, in line with Contant et al., 2021 (now cited in the revised manuscript). 8. Page 5, “AO/EB Staining”: Clarify quantification method and cite reference. ✔ We now state that apoptotic, necrotic, and viable cells were quantified using manual cell counting under a fluorescence microscope based on morphological and staining criteria, referencing Ribble et al., 2005. 9. Page 6, “Molecular Docking”: Justify use of AutoDock over newer methods. ✔ We have included a rationale noting that AutoDock remains a validated and widely used tool, particularly suitable for flexible ligand docking and preliminary screening of natural products due to its robust scoring functions and availability. 10. Page 7, “Western Blot”: Were densitometry results normalized? ✔ Yes. We now clarify that ImageJ software (NIH) was used for densitometry, and expression levels were normalized to β-actin as the loading control. Results 11. Page 8, Figure 1: Include sample size and SD for IC₅₀. ✔ This information has been added: IC₅₀ values were derived from three independent experiments and are expressed as mean ± SD. 12. Page 9, Apoptosis: Was flow cytometry used? ✔ No flow cytometry was used. We clarify that apoptosis was assessed only via AO/EB staining in this study. 13. Page 10, Docking: Was in vitro validation of target binding performed? ✔ We now acknowledge that in vitro validation (e.g., via SPR or ITC) was not performed and have highlighted this as a limitation and future direction in the discussion. ________________________________________ Discussion 14. Compare anethole with existing PI3K inhibitors like Wortmannin and LY294002. ✔ We have added a paragraph discussing that while anethole demonstrates moderate affinity for PI3K, it may offer advantages over conventional inhibitors due to lower toxicity and broader multi-target effects. We also reference the known drawbacks of Wortmannin and LY294002 (e.g., instability, cytotoxicity). 15. BBB permeability: Is there direct evidence for anethole crossing the BBB? ✔ We clarify that while anethole is lipophilic and theoretically capable of BBB penetration, direct experimental evidence is lacking. We reference Yu et al., 2011 and recommend further pharmacokinetic studies. 16. Limitations: No in vivo validation. Suggest adding statement. ✔ We have included a clear statement acknowledging the lack of in vivo data and recommending future studies using animal models to validate the therapeutic potential and pharmacodynamics of anethole. Conclusion 17. Highlight future directions (e.g., combinational therapy, BBB studies). ✔ The conclusion now includes suggestions for combinational therapy with current chemotherapeutics, investigation of BBB permeability, and in vivo validation as future directions. Reviewer #2 1. Compare MD-predicted binding constants with experimental data (ITC, BLI, SPR). ✔ We appreciate this important suggestion. In the discussion, we have noted that while molecular docking predicted strong binding to PI3K, experimental confirmation using ITC, BLI, or SPR is necessary to validate these findings. This limitation has been acknowledged, and we propose these methods as next steps in validating the binding mechanism. Response to Editor’s Comments We thank the Editor for the valuable suggestions aimed at enhancing the clarity, consistency, and compliance of our manuscript with PLOS ONE guidelines. Below, we provide point-by-point responses to each of the recommendations and describe the revisions made accordingly: 1. Adherence to Journal Guidelines • Correct formatting of the references Response: The entire reference list has been reformatted to align with the PLOS ONE style, as per the journal’s formatting requirements. • Consistency in the use of terminology and abbreviations Response: All abbreviations and scientific terms have been reviewed for consistent usage throughout the manuscript. A list of abbreviations has also been provided where necessary. • Proper structuring of headings and subheadings Response: The manuscript has been revised to ensure uniform formatting of all section headings and subheadings in accordance with the journal’s structure. 2. Language and Clarity • Grammatical and syntactical accuracy Response: The manuscript has undergone a detailed language review to improve clarity, correct grammatical issues, and enhance the overall readability of the text. 3. Methodology Section • Additional details on source, purity, solvents, and quality control of anethole Response: Anethole (99% purity) was obtained from Sigma-Aldrich (Oakville, ON, Canada) and dissolved in methanol to prepare a 3 mM stock solution. It was subsequently used at different concentrations for the experiments 4. Figures and Tables • Clear labeling, defined axes, and detailed captions Response: All figures and tables have been reviewed and revised. Axes are now clearly labeled, units are defined, and figure captions have been expanded to fully describe the contents. Each figure and table is now properly referenced within the manuscript. 5. Supplementary Data ________________________________________ 6. Consistency in Presentation of Data • Statistical analysis and experimental result presentation Response: The manuscript has been carefully reviewed to ensure consistent reporting of sample size (n), standard deviations (SD), and p-values. All statistical analyses are now clearly defined in both the Methods and Results sections. 7. Original Images for Blot/Gel Data • Compliance with blot and gel image requirements Response: As per PLOS ONE’s latest guidelines, the original, uncropped, and unadjusted image data underlying all Western blot figures have been included as Supporting Information files. We have also ensured that the preparation of these images and figure panels adheres to the specific requirements detailed in the journal’s policy. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0336975.r002
4 Jul 2025 Decision Letter - Zahra Lorigooini, Editor Dear Dr. Alshahrani, 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 Aug 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. 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 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, Zahra Lorigooini 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. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #3: I Don't Know ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #3: Yes ****** Reviewer #3: Although the authors have been responsive, the following high-level suggestions may further strengthen the study: Add a Note on Flow Cytometry as Future Work While the authors clarified they did not use flow cytometry, suggesting its inclusion in future validation would demonstrate deeper awareness of methodological robustness in apoptosis detection. Clarify Lipophilicity Measurement The statement that anethole is “lipophilic” and may cross the BBB is speculative. Including a brief reference to LogP or in silico BBB permeability prediction (e.g., via SwissADME) would lend more support. Densitometry Quantification – Loading Control Uniformity Western blot images (if reviewed) should show uniform β-actin levels across conditions. While the use of ImageJ is stated, it's critical that raw blot bands (provided as supplementary) confirm integrity. ****** 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: Dr. Majid Asadi-Samani ******** [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 https://doi.org/10.1371/journal.pone.0336975.r003
Revision 2
10 Jul 2025 Author Response Dear Editor, We thank you for the consideration of our manuscript. We also thank the reviewers for the suggestions for further strengthening of our manuscript and we have incorporated all suggestions of the respected reviewer. Reviewer #3: Although the authors have been responsive, the following high-level suggestions may further strengthen the study: Add a Note on Flow Cytometry as Future Work While the authors clarified they did not use flow cytometry, suggesting its inclusion in future validation would demonstrate deeper awareness of methodological robustness in apoptosis detection. Response: We appreciate this insightful recommendation. We have revised the Discussion section to acknowledge the value of incorporating flow cytometry (e.g., Annexin V/PI staining) in future studies for quantitative apoptosis detection. Clarify Lipophilicity Measurement The statement that anethole is “lipophilic” and may cross the BBB is speculative. Including a brief reference to LogP or in silico BBB permeability prediction (e.g., via SwissADME) would lend more support. Response: We agree with the reviewer and have now included in silico SwissADME predictions in the Results and Discussion sections. These predictions show a consensus LogP of 2.79 and classify anethole as BBB permeant, supporting its CNS bioavailability (Supplementary Figure S1 and S2). Densitometry Quantification – Loading Control Uniformity Western blot images (if reviewed) should show uniform β-actin levels across conditions. While the use of ImageJ is stated, it's critical that raw blot bands (provided as supplementary) confirm integrity. Response: We fully agree and have included densitometery quantification in Figure 2C, 2D, 5D-G. The uncropped raw Western blot images as Supplementary file, clearly showing consistent β-actin expression across all experimental conditions. This addition supports the integrity of protein loading and quantification. Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_2.docx https://doi.org/10.1371/journal.pone.0336975.r004
22 Sep 2025 Decision Letter - Yasmina Abd‐Elhakim, Editor Dear Dr. Alshahrani, 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 Nov 06 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. 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 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, Yasmina Abd‐Elhakim Academic Editor PLOS ONE Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. 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.] Reviewer's Responses to Questions Comments to the Author Reviewer #4: (No Response) Reviewer #5: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #4: No Reviewer #5: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #4: Yes Reviewer #5: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #4: Yes Reviewer #5: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #4: Yes Reviewer #5: Yes ****** Reviewer #4: This manuscript studies the anticancer cell effect of anethole in glioma cell lines and normal human astrocytes. Anethole has been broadly described in several cancer cell types, by different groups and being reviewed, showing antiproliferative and antimetastatic effect on neoplastic cells. As described by others, anethole induces apoptosis, cell cycle arrest, autophagy, antioxidant GHS, reduction of ROS, and metalloproteinases, etc., by interfering in different signaling pathways. The manuscript by Al Alwadh et al., demonstrates that anethole exerts the same effect in glioma cells as in other cancer cells, inducing apoptosis and reduction of PI3K/AKT phosphorylation. The mayor hypothesis and aim of this manuscript is to show the blocking effect of anethole in the PI3K/AKT pathway. for that, the authors analyzed in silico prediction of PI3K and anethole interaction. Although this is an interesting subject, the work should be completed by correcting some errors and adding some data as follows indicated: Main concerns: 1- The most interesting data is the predictable interaction of anethole and PI3k, some aspects in this regard should be considered. First if the effect of anethole takes place through interaction with PI3K and its inhibition, why does it not affect Normal human astrocytes (NHA)? The authors demonstrate, as previously described in cancer cells, reduction of PI3K and AKT phosphorylation only in one line of cancer cells. Then, the effect of anethole in NHA cells, as new data, would be nice to add in. Second, independent of the result about the effect in NHA, Anethole's stronger effect on cancer cells needs discussion. Could this be due to increased diffusion through cancer cell membranes? It would be interesting to discuss this, as increased anethole affinity to PI3K in cancer cells is unlikely. Finaly, AKT can take place through an independent mechanism as well, and it will be interesting to show either some downstream signal, or upstream signal that demonstrate PI3K direct inhibition. 2- In discussion section, it is indicated “In addition, anethole has also been shown to elevate ROS accretion in cancerous cells, promoting oxidative stress and apoptosis [8]. ROS accumulation has been estimated to impair mitochondrial membrane potential, thereby activating intrinsic apoptotic sequences as described in breast cancer cells [26]”, the reference 8 showed that anethole reduces ROS and increased GSH antioxidant. Therefore, this paragraph and the argument should be changed accordingly. 3- Regarding the treatment with anethole, the most limiting aspect is that, even when anethole can diffuse through the BBB, due to low solubility in water and high elimination when orally administrated, the effect in gliomas looks limited. Some discussion also in this area should be nice to discuss. 4- The figure legends need to indicate clearly whether the graphs show the media plus SD of an independent out of three experiments, or the media plus SD of three independent experiments. 5- The AO/EB analysis is used to indicate early and late apoptosis, the quantification of cells in these two statuses should be indicated in the graph. On the other hand, it would be convenient to show cell cycles of treated cells, also in NHA cells. 6- In general, it is indicated that experiments were performed three times, but whether the graph represents one out three or the media plus SD of three independent experiments need to be indicated. 7- The apoptosis assay needs to be performed with normal NHA cells, to consistently confirm the specific effect of anethole in gliomas. Also, the 8- The relative expression of p-PI3K and p-AKT needs to be calculated according to the total amount of PI3K and AKT protein, rather than to actine, and needs to be indicated in the Figure 5 legend. Minor concerns: 1- Material, please indicate the precedence of: cell lines, NHA cells, CCK-8 assay, and al used material in the material and methods section. 2- Please, indicate the meaning of acronyms first time used in the text 3- Indication of the antibody clone used, and the company of precedence needs to be included in the material and methods section. 4- Correct the sentence at page 35- “Cells were subsequently lysing was done using RIPA buffer”, 5- Either Figures 5a and 5b have been mixed up, or the text in the legends and in the 'Results' section (3.4) is incorrect. 6- The images in Figures 3, 4, 5a and 5b should be clearer. Reviewer #5: Based on the authors’ responses to the previous reviewers’ comments, I believe the manuscript is acceptable after some minor revisions, as outlined below. 1- Mechanistic Depth and Specificity: The study compellingly shows that anethole inhibits the PI3K/AKT pathway and induces apoptosis. However, to strengthen the claim that apoptosis is directly caused by PI3K/AKT inhibition (and not a parallel event), a rescue experiment would be highly valuable. Could the pro-apoptotic and anti-proliferative effects of anethole be reversed by introducing a constitutively active form of AKT (e.g., via plasmid transfection) into the U87 cells prior to anethole treatment? If such an experiment is beyond the scope of this revision, explicitly stating this as a key limitation and a requirement for future validation would strengthen the manuscript's conclusions. Where to place this: In the Discussion (to frame the findings) and/or the Conclusion (as a future direction). 2- The figures (e.g., Figure 2B, 5C) show representative Western blot bands, but the molecular weight markers are not visible. Including a lane with markers in the main figure or the supplementary uncropped blots is essential for verifying the identity of the protein bands. Figure 5A and 5B captions are swapped. Figure 5A describes the JAK2 interaction, while the image is labeled for PI3K, and vice versa. This must be corrected. 3- Statistical Analysis Description: The Methods section (2.9) states that experiments were performed in triplicate and data is presented as mean ± SD. For the colony formation assay, it would be helpful to specify how many technical replicates (wells) and biological replicates (independent experiments) were performed, as this assay typically has fewer replicates. 4- Language and Flow Minor Revisions: The manuscript is well-written but would benefit from a final proofread for minor grammatical redundancies. For example, in the Introduction (page 33): "...impede growth via cell cycle arrest in prostate cancer cells via arresting cell cycle." The phrase "via arresting cell cycle" is redundant and can be removed. ******** what does this mean? ). If published, this will include your full peer review and any attached files. [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". 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Revision 3
15 Oct 2025 Author Response Dear Editor We sincerely thank you and the anonymous reviewers for their constructive comments and valuable suggestions, which have significantly improved the quality and clarity of our manuscript. We have carefully addressed all concerns point by point, as detailed below. All textual changes have been incorporated into the revised version of manuscript and have been highlighted in red. Reviewer #4 This manuscript studies the anticancer cell effect of anethole in glioma cell lines and normal human astrocytes. Anethole has been broadly described in several cancer cell types, by different groups and being reviewed, showing antiproliferative and antimetastatic effect on neoplastic cells. As described by others, anethole induces apoptosis, cell cycle arrest, autophagy, antioxidant GHS, reduction of ROS, and metalloproteinases, etc., by interfering in different signaling pathways. The manuscript by Al Alwadh et al., demonstrates that anethole exerts the same effect in glioma cells as in other cancer cells, inducing apoptosis and reduction of PI3K/AKT phosphorylation. The mayor hypothesis and aim of this manuscript is to show the blocking effect of anethole in the PI3K/AKT pathway. for that, the authors analyzed in silico prediction of PI3K and anethole interaction. Although this is an interesting subject, the work should be completed by correcting some errors and adding some data as follows indicated: Main concerns 1- The most interesting data is the predictable interaction of anethole and PI3k, some aspects in this regard should be considered. First if the effect of anethole takes place through interaction with PI3K and its inhibition, why does it not affect Normal human astrocytes (NHA)? The authors demonstrate, as previously described in cancer cells, reduction of PI3K and AKT phosphorylation only in one line of cancer cells. Then, the effect of anethole in NHA cells, as new data, would be nice to add in. Second, independent of the result about the effect in NHA, Anethole's stronger effect on cancer cells needs discussion. Could this be due to increased diffusion through cancer cell membranes? It would be interesting to discuss this, as increased anethole affinity to PI3K in cancer cells is unlikely. Finaly, AKT can take place through an independent mechanism as well, and it will be interesting to show either some downstream signal, or upstream signal that demonstrate PI3K direct inhibition. Response: We thank the reviewer for this insightful comment. While we acknowledge that inclusion of NHA apoptosis and phosphorylation data would strengthen the study, new wet-lab experiments are beyond the scope of this revision. To address this point, we have added a detailed explanation in the Discussion section discussing (i) why normal astrocytes show limited PI3K/AKT response, (ii) the potential role of altered membrane permeability and PI3K/AKT hyperactivation in glioma cells as the cause of selective sensitivity, and (iii) possible AKT-independent mechanisms requiring future validation. This clarification provides a mechanistic rationale for cancer-selective action of anethole and outlines future directions for in vivo and molecular validation. 2- In discussion section, it is indicated “In addition, anethole has also been shown to elevate ROS accretion in cancerous cells, promoting oxidative stress and apoptosis [8]. ROS accumulation has been estimated to impair mitochondrial membrane potential, thereby activating intrinsic apoptotic sequences as described in breast cancer cells [26]”, the reference 8 showed that anethole reduces ROS and increased GSH antioxidant. Therefore, this paragraph and the argument should be changed accordingly. Response: We appreciate this correction. The cited paragraph has been revised in the Discussion section to accurately reflect the findings of reference [8], clarifying that anethole predominantly reduces ROS levels and enhances GSH antioxidant capacity, rather than increasing ROS. The interpretation was rewritten accordingly. 3- Regarding the treatment with anethole, the most limiting aspect is that, even when anethole can diffuse through the BBB, due to low solubility in water and high elimination when orally administrated, the effect in gliomas looks limited. Some discussion also in this area should be nice to discuss. Response: We agree with the reviewer’s important observation. Additional discussion has been incorporated in the Discussion section addressing pharmacokinetic limitations of anethole such as low aqueous solubility and high first-pass metabolism. We have also added potential strategies—such as lipid-based nanoformulations, prodrug development, and intranasal delivery—to improve systemic and brain bioavailability. 4- The figure legends need to indicate clearly whether the graphs show the media plus SD of an independent out of three experiments, or the media plus SD of three independent experiments. Response: We have revised all figure legends to explicitly state that “data are presented as mean ± SD of three independent biological experiments,” clarifying that each represents the mean of three independent replicates. 5- The AO/EB analysis is used to indicate early and late apoptosis, the quantification of cells in these two statuses should be indicated in the graph. On the other hand, it would be convenient to show cell cycles of treated cells, also in NHA cells. Response: We appreciate this valuable suggestion. Quantitative analysis of total apoptosis (early + late) was added in the Methods (AO/EB assay) and described in Figure 2 legend. As additional flow cytometry experiments were not performed, we clarified in the Discussion that detailed early/late apoptosis and NHA apoptosis assays will be considered in future work. 6- In general, it is indicated that experiments were performed three times, but whether the graph represents one out three or the media plus SD of three independent experiments need to be indicated. Response: This point has been addressed throughout the Results and Figure legends, which now specify that all data represent the mean ± SD of three independent biological experiments. 7- The apoptosis assay needs to be performed with normal NHA cells, to consistently confirm the specific effect of anethole in gliomas. Also, the Response: We acknowledge the reviewer’s suggestion. While inclusion of NHA apoptosis data would provide valuable confirmation, conducting new experiments exceeds the current revision scope. However, this limitation is now explicitly stated in the Discussion (last paragraph), and future studies will incorporate apoptosis assays on NHA and in vivo models to verify selectivity. 8- The relative expression of p-PI3K and p-AKT needs to be calculated according to the total amount of PI3K and AKT protein, rather than to actine, and needs to be indicated in the Figure 5 legend. Response: We appreciate this important observation. As suggested, the densitometric analysis was recalculated and normalized to the respective total proteins (p-PI3K/PI3K and p-AKT/AKT). This correction has been clearly indicated in both the Methods (Western blot) and Figure 5 legend. Minor concerns 1- Material, please indicate the precedence of: cell lines, NHA cells, CCK-8 assay, and al used material in the material and methods section. Response: The Materials and Methods section was expanded to include the source, catalog numbers, and vendors for all reagents and cell lines (Section 2.1). 2- Please, indicate the meaning of acronyms first time used in the text Response: All acronyms (e.g., NHA, AO/EB, TPSA, BBB) are now defined at first mention throughout the manuscript. 3- Indication of the antibody clone used, and the company of precedence needs to be included in the material and methods section. Response: The Western blot subsection (2.7) now lists the antibody source, catalog number, and manufacturer for each primary antibody. 4- Correct the sentence at page 35- “Cells were subsequently lysing was done using RIPA buffer” Response: The sentence has been corrected to “Cells were lysed using RIPA buffer.” 5- Either Figures 5a and 5b have been mixed up, or the text in the legends and in the 'Results' section (3.4) is incorrect. Response: Thank you for noticing this. The mix-up was corrected—the figure panels and legends now correctly correspond to PI3K (Figure 5A) and JAK2 (Figure 5B). 6- The images in Figures 3, 4, 5a and 5b should be clearer. Response: We increased the resolution of Figures 3–5 Reviewer #5 1- Mechanistic Depth and Specificity: The study compellingly shows that anethole inhibits the PI3K/AKT pathway and induces apoptosis. However, to strengthen the claim that apoptosis is directly caused by PI3K/AKT inhibition (and not a parallel event), a rescue experiment would be highly valuable. Could the pro-apoptotic and anti-proliferative effects of anethole be reversed by introducing a constitutively active form of AKT (e.g., via plasmid transfection) into the U87 cells prior to anethole treatment? If such an experiment is beyond the scope of this revision, explicitly stating this as a key limitation and a requirement for future validation would strengthen the manuscript's conclusions. Where to place this: In the Discussion (to frame the findings) and/or the Conclusion (as a future direction). Response: We appreciate this valuable suggestion. As additional transfection experiments could not be performed at this stage, we have explicitly included this as a key limitation in the Discussion (final paragraph) and highlighted it in the Conclusion as a future research direction. 2- The figures (e.g., Figure 2B, 5C) show representative Western blot bands, but the molecular weight markers are not visible. Including a lane with markers in the main figure or the supplementary uncropped blots is essential for verifying the identity of the protein bands. Figure 5A and 5B captions are swapped. Figure 5A describes the JAK2 interaction, while the image is labeled for PI3K, and vice versa. This must be corrected. Response: We have now included uncropped blots with visible molecular weight markers as supplementary material and corrected the swap between Figure 5A and 5B to align with the text. 3- Statistical Analysis Description: The Methods section (2.9) states that experiments were performed in triplicate and data is presented as mean ± SD. For the colony formation assay, it would be helpful to specify how many technical replicates (wells) and biological replicates (independent experiments) were performed, as this assay typically has fewer replicates. Response: We agree and have updated the Colony Formation Assay (Section 2.4) to indicate that each condition included two technical replicates and three independent biological experiments. 4- Language and Flow Minor Revisions: The manuscript is well-written but would benefit from a final proofread for minor grammatical redundancies. For example, in the Introduction (page 33): "...impede growth via cell cycle arrest in prostate cancer cells via arresting cell cycle." The phrase "via arresting cell cycle" is redundant and can be removed. Response: We thank the reviewer for this observation. The entire manuscript has been carefully proofread to correct redundancies and improve fluency. The specific phrase mentioned has been revised accordingly in the Introduction. We are deeply grateful to both reviewers for their thoughtful input, which helped us strengthen the manuscript. We hope the revisions satisfactorily address all concerns. Sincerely, Dr. Mohammed Merae Alshahrani (Corresponding Author) Department of Clinical Laboratory Sciences Najran University, Saudi Arabia Email: mmalshahrani@nu.edu.sa Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_3.docx https://doi.org/10.1371/journal.pone.0336975.r006
2 Nov 2025 Decision Letter - Yasmina Abd‐Elhakim, Editor Anethole Inhibits Human U87 Glioma Cell Proliferation by Inducing Apoptosis via the PI3K/AKT Pathway PONE-D-25-10072R3 Dear Dr. Alshahrani, 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. For questions related to billing, please contact billing support . 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, Yasmina Abd‐Elhakim Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #5: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #5: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #5: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #5: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #5: Yes ****** Reviewer #5: Thank you for your thorough revisions and detailed point-by-point responses to my comments. I have reviewed the updated manuscript and find that you have addressed all of my concerns satisfactorily. The manuscript is now significantly improved and I recommend it for acceptance. ****** 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 #5: Yes: Gad Elsayed Mohamed Salem ******** https://doi.org/10.1371/journal.pone.0336975.r007
Formally Accepted
Acceptance Letter - Yasmina Abd‐Elhakim, Editor PONE-D-25-10072R3 PLOS ONE Dear Dr. Alshahrani, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Dr. Yasmina Abd‐Elhakim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0336975.r008

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