Radioprotective effect of the anti-diabetic drug metformin

Silvia Siteni; Summer Barron; Krishna Luitel; Jerry W. Shay

doi:10.1371/journal.pone.0307598

Peer Review History

Original SubmissionApril 1, 2024
8 May 2024 Decision Letter - Hanbing Li, Editor PONE-D-24-13104RADIOPROTECTIVE EFFECT OF THE ANTI-DIABETIC DRUG METFORMINPLOS ONE Dear Dr. Shay, 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 Jun 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. 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Additional Editor comments: Thank you for submitting your manuscript for consideration for publication in PLOS ONE. Please address the comments raised by the reviewers and submit the revised version of your manuscript later. [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In the current manuscript, the authors have repurposed metformin for its radioprotective effects, which are currently being used as a first-line treatment for managing type 2 diabetes. The authors utilized various in vitro and in vivo assays to explore its beneficial effects, including its potential use in space missions using galactic cosmic ray simulation (GCRsim). Additionally, the authors studied the possible mechanisms of radioprotection by metformin. In my opinion, the manuscript is well-written and organized. However, I have several queries that the authors need to address in their revised version of the manuscript: 1. In the determination of in vitro concentrations, the authors tested concentrations of 0.5, 1, 2.5, and 5 mM of metformin, ultimately deciding to use the lowest concentration of 0.5 mM. Why did they not explore even lower concentrations than 0.5 mM? 2. For the in vivo study, the authors administered 200 mg/kg b.wt of metformin. How did the authors determine this concentration? 3. What is the LD50/30 dose for 129/sv mice? I ask this because researchers generally use sub-lethal doses for mechanistic studies and slightly higher doses than the LD50/30 for survival studies. In this study, the authors used 7.5 Gy for mechanistic studies and 10 Gy for survival studies. Reviewer #2: The manuscript presents a comprehensive study investigating the potential radioprotective effects of metformin and its underlying mechanisms. Here's a review based on the provided criteria: Technical Soundness and Data Support for Conclusions: The manuscript appears technically sound, with a detailed exploration of the effects of metformin on various cellular processes, particularly in response to ionizing radiation. The authors provide data on AMPK activation, changes in antioxidant enzyme expression, DNA repair kinetics, and survival outcomes in both in vitro and in vivo models. The findings suggest that metformin pre-administration confers radioprotection by enhancing DNA repair mechanisms and mitigating cellular damage induced by ionizing radiation. However, while the data presented support the conclusions drawn, there are some areas where further clarification or additional experiments may strengthen the claims. For instance, it would be beneficial to include more detailed analyses of the molecular pathways involved in metformin-mediated radioprotection, such as downstream targets of AMPK activation and the interplay between different cellular processes. Presentation and Language: The manuscript is generally well-presented and written in standard English, making it intelligible to readers. The authors provide clear descriptions of experimental procedures, results, and interpretations, facilitating understanding of the study's findings. However, there are sections where the text could be streamlined for clarity and conciseness. Additionally, the manuscript could benefit from improved organization and structure to enhance readability and flow. For example, grouping related findings together and providing more explicit transitions between sections would help guide the reader through the study's complex data and analyses. Overall, the manuscript presents valuable insights into the potential radioprotective effects of metformin and contributes to our understanding of its mechanisms of action. With some revisions to address the points mentioned above, it has the potential to make a significant contribution to the field of radiobiology and could be considered for publication in a scientific journal. ****** 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: Nur Fariesha Md Hashim ******** [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.0307598.r001
Revision 1
22 Jun 2024 Author Response Review Comments to the Author We want to thank the editor and the reviewers for taking the time to read our paper and for the relevant questions. Our responses to the initial review comments are detailed in the revised manuscript and summarized below. Reviewer #1: In the current manuscript, the authors have repurposed metformin for its radioprotective effects, which are currently being used as a first-line treatment for managing type 2 diabetes. The authors utilized various in vitro and in vivo assays to explore its beneficial effects, including its potential use in space missions using galactic cosmic ray simulation (GCRsim). Additionally, the authors studied the possible mechanisms of radioprotection by metformin. In my opinion, the manuscript is well-written and organized. However, I have several queries that the authors need to address in their revised version of the manuscript: 1. In the determination of in vitro concentrations, the authors tested concentrations of 0.5, 1, 2.5, and 5 mM of metformin, ultimately deciding to use the lowest concentration of 0.5 mM. Why did they not explore even lower concentrations than 0.5 mM? Thank you very much for pointing out an important question. We did test lower concentrations of metformin and we focused on the lower concentrations of metformin able to induce the activation of SOD1. We added the data in the revised submission (S1 Fig B). 2. For the in vivo study, the authors administered 200 mg/kg b.wt of metformin. How did the authors determine this concentration? Thank you for the question. We explained our choice in the material and methods section (p. 11, Drug preparation). Briefly, we evaluated the usual metformin administration in humans (between 1000 and 2000 mg per day). We used the body surface area (BSA) normalization method [53] according to the FDA guidelines [54] and we found a minimal dosage of 250 mg/kg. However, since our aim was to study the radioprotective effects of metformin in healthy wild type mice (and potentially in very healthy human beings such as astronauts) we decreased the concentration to 200 mg/kg. We explained this better in the revised manuscript 3. What is the LD50/30 dose for 129/sv mice? I ask this because researchers generally use sub-lethal doses for mechanistic studies and slightly higher doses than the LD50/30 for survival studies. In this study, the authors used 7.5 Gy for mechanistic studies and 10 Gy for survival studies. According to previous studies in our lab and others, wild type mice are sensitive to 7.5 Gy of total body x-rays. However, when we did a pilot test, our mice were not lethally affected by 7.5 Gy, but most wild type mice died after two weeks when irradiated once with 10 Gy x-rays. We used the sublethal dose of 7.5 Gy to better investigate the DNA repair kinetics and the lethal dose of 10 Gy to evaluate the overall survival in the presence or absence of metformin. Reviewer #2: The manuscript presents a comprehensive study investigating the potential radioprotective effects of metformin and its underlying mechanisms. Here's a review based on the provided criteria: Technical Soundness and Data Support for Conclusions: The manuscript appears technically sound, with a detailed exploration of the effects of metformin on various cellular processes, particularly in response to ionizing radiation. The authors provide data on AMPK activation, changes in antioxidant enzyme expression, DNA repair kinetics, and survival outcomes in both in vitro and in vivo models. The findings suggest that metformin pre-administration confers radioprotection by enhancing DNA repair mechanisms and mitigating cellular damage induced by ionizing radiation. However, while the data presented support the conclusions drawn, there are some areas where further clarification or additional experiments may strengthen the claims. For instance, it would be beneficial to include more detailed analyses of the molecular pathways involved in metformin-mediated radioprotection, such as downstream targets of AMPK activation and the interplay between different cellular processes. Thank you for appreciating our work and paper. We agree with the reviewer about including a deeper investigation into the mechanisms to elucidate the role of AMPK effects downstream. We did investigate the main pathways involving the DNA damage response and AMPK. Below is what we discussed in the original draft that addresses this comment: “…we found metformin 0.5 mM increases the phosphorylation of ataxia telangiectasia mutated (ATM) at serine 1981 and p53 expression after 48 hours. ATM is a kinase that acts upstream of p53 and controls a DDR pathway critical to resolving DSBs [45]. However, we did not find any increase in γH2AX foci in vitro or in 53BP1 foci in vivo, but this might be related to the time-points we chose to analyze. Vazquez-Martin et al. [46] reported ATM phosphorylation at serine 1981 and formation of γH2AX foci after metformin exposure in epidermoid carcinoma cells, but not of 53BP1. This is in line with what was reported by Bakkenist & Kastan [47], showing ATM activation is not dependent on direct binding to DNA strand breaks, but may result from changes in the structure of chromatin. Interestingly, the increased expression of p53 might have effects on the cell cycle, inducing a transient arrest in G1 phase, and thus providing time for DNA repair from the effects of IRs.” We did not obtain significant data about CHK1, CHK2, BRCA1, ATR expression and these results are not included in the revised manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0307598.r002
9 Jul 2024 Decision Letter - Hanbing Li, Editor RADIOPROTECTIVE EFFECT OF THE ANTI-DIABETIC DRUG METFORMIN PONE-D-24-13104R1 Dear Dr. Jerry W Shay, 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. 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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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 ****** 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 ****** 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 taken care of all the queries raised by me and accordingly revised the manuscript. Now it can be accepted. Reviewer #2: Thank you for addressing my comments and questions with detailed and thoughtful responses. The inclusion of additional data on lower concentrations of metformin and the rationale for the in vivo dosage selection significantly strengthens the manuscript. Your explanation of the sublethal and lethal doses used in the mouse model studies, and the addition of the data on SOD1 activation, provide greater clarity and support for your experimental design. Furthermore, your elaboration on the downstream targets of AMPK activation and the role of ATM and p53 in the DNA damage response enhances our understanding of the molecular pathways involved in metformin-mediated radioprotection. The revised manuscript is well-organized and provides comprehensive insights into the radioprotective effects of metformin, supporting its potential application in space missions and radioprotection. I recommend acceptance of the manuscript after incorporating the discussed revisions. ****** 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: No ******** https://doi.org/10.1371/journal.pone.0307598.r003
Formally Accepted
12 Jul 2024 Acceptance Letter - Hanbing Li, Editor PONE-D-24-13104R1 PLOS ONE Dear Dr. Shay, 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 If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks 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. 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 Professor Hanbing Li Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0307598.r004

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